summary - Netherlands Proteomics Centre

netherlands
centre
npc highlights 11 | April 2010
Featuring cutting edge research projects
and enabling technologies of the
Netherlands Proteomics Centre

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Frontpage
DeltaVision real-time imaging. Shown is a specialized tissue
culture slide mounted on a nanometer-accuracy motorized
stage of a DeltaVision RT microscope. Cells expressing various
fluorescent markers are imaged live at high resolution while
undergoing cell division. Example images are displayed in the
background.
Contents
About
Preface
News headlines
HighLights
6 Geert Kops; Dividing the goods: preventing chromosome segregation errors
10 Rumyana Karlova and Sacco de Vries; The plasma membrane receptor complex perceiving plant steroids
14 Paul Boersema, Vanessa Ding and Albert Heck; In depth profiling of tyrosine phosphorylation in human embryonic
stem cells
18 Arjen Scholten, Reinout Raijmakers and Albert Heck; Finding new targets of small molecules by chemical proteomics
22 Barbara Möller and Dolf Weijers; Cell-cell communication in plant embryos
Interviews: Paola Picotti; Proteomics for the analysis of cellular networks
Jonathan Sweedler; Understanding neuropeptide complexity in the brain
Valorisation within the Netherlands Proteomics Centre
Bioinformatics
NPC Top Publications
NPC Research Hotels
Martin Schuurmans, Combining the best existing forces
11
| NPC Highlights 11 | April 2010
>
About
The Netherlands Proteomics Centre (NPC) is a strategic collaboration of research groups from six
universities, four academic medical centres and several research institutes and biotech companies.
With a scientific program addressing key areas of proteomics in several projects, and specialised
‘research hotels’, the NPC performs high-quality research and knowledge transfer in an
international context. The NPC is part of the Netherlands Genomics Initiative.
In NPC Highlights researchers present progress and results from NPC projects of the scientific program
and the research hotels. NPC Highlights is published by the Netherlands Proteomics Centre.
Netherlands Proteomics Centre
Coordination and editing
Padualaan 8
Marian van Opstal
NL 3584 CH Utrecht
Bèta Communicaties, The Hague
t +31 30 253 4564
e info@npc.genomics.nl
Lay-out
w www.netherlandsproteomicscentre.nl
Frans Koeman
F.Koeman DTP-Services, Zoetermeer
Editorial Board
Albert Heck, Scientific Director NPC
Photography frontpage
Werner Most, Managing Director NPC
Bas van Breukelen
Bert Poolman, member Executive Board NPC
Hermen Overkleeft, member Executive Board NPC Printing
Rob Liskamp, member Executive Board NPC
Bestenzet BV, Zoetermeer
Martje Ebberink, Communications NPC
To subscribe to NPC Highlights please
send an e-mail with your full name, organisation
© 2010 Copyright Netherlands Proteomics Centre and address to info@npc.genomics.nl

Welcome This edition of NPC Highlights
features a number of selected research
projects from the NPC scientific program.
Some of them were presented at the NPC
Progress Meeting 2010 held on February 16.
The meeting was once again a very well
attended and lively event, and included a
poster session and presentations by the keynote speakers Paola
Picotti, Institute of Molecular Systems Biology, ETH Zurich, and
Jonathan Sweedler, Chemistry Department, University of Illinois.
Interviews with these inspiring scientists as well as information
about the first NPC poster prize winners are included in this
issue.
With the start of the renewed program in 2009, the NPC has also
intensified and extended some complementary initiatives, in
particularly: the bioinformatics-proteomics interface program
in collaboration with NBIC; the valorisation program including
dedicated funding for proof-of-concept studies; personal
coaching and more public-oriented educational activities like our
contributions to the science days in NEMO and the very successful
DNA labs on the road for high schools. More information about
these initiatives is also presented in this issue of NPC Highlights.
Finally, I would like to point out that you are always welcome
at the NPC office for further information or any relevant
contribution to this proteomics community. Please feel free to
visit our website at www.netherlandsproteomicscentre.nl or get
in contact with our public relations officer, Martje Ebberink, at
info@npc.genomics.nl
|
Albert Heck, scientific director NPC
preface

News Headlines
Inspiring NPC Progress
Meeting 2010 well attended
Pink Ribbon and KWF grant
for Arzu Umar
The Netherlands
Proteomics
Centre (NPC)
held its annual
Progress
Meeting on
February 16.
The event drew
an audience
of around
200, consisting of scientists from inside and outside the NPC
program, as well as delegates from several biotech companies.
The international speakers, Jonathan Sweedler and
Paola Picotti, and the NPC researchers put their marks on
this event by highlighting successes achieved and challenges
still looming. Congratulations to Lara Fornai (FOM Institute
AMOLF), Remon van Geel (Nijmegen Centre for Molecular Life
Sciences) and Nikolai Mischerikow (Utrecht University) who
were awarded a travel grant of e 1,000 for the best poster at
the NPC meeting.
| NPC Highlights 11 | April 2010
NPC researcher Arzu Umar and Madelaine
Tilanus-Linthorst (Erasmus MC) have been
awarded a Pink Ribbon grant of € 125,000.
Umar and Tilanus will use their grant to
identify protein markers for the early
detection of breast cancer using a tissue
proteomics approach. The results of this project will aid in the
development of a non-invasive diagnostic test for the early
detection of breast cancer.
NPC researchers Arzu Umar, John Foekens and Theo Luider
(Erasmus MC) have received a grant of € 534,000 from the KWF
Kankerbestrijding (Dutch foundation for cancer research). The
aim of the project is to identify a predictive protein profile for
tamoxifen therapy resistance in patients with advanced breast
cancer. The results of this project will provide a predictive tool
for tamoxifen therapy resistance as well as pinpoint any key proteins
that could serve as targets for new therapy development.
Uncovering stem cell
differentiation
Signal proteins for plant
stem cells discovered
Wageningen biochemist Dolf Weijers and
his German colleagues have discovered
how stem cells in a plant embryo are
formed. The cells communicate with
one another via the transportation of
a protein. The research results have
been recently published in Nature (10
March 2010). Unlike animals, plants
produce new organs — leaves, roots and
flowers — throughout their entire life. This task is undertaken
by the meristems, growth tips in which stem cells are located.
Meristems are located in the young plant embryo. Weijers
studied the formation of root meristems in the embryo of the
model plant Arabidopsis thaliana. The process begins with the
programming of one cell as the ‘hypophysis’ which regulates
stem cells in the roots. It is known that the formation of the
hypophysis is controlled by the gene activator called Monopteros.
However, it was hitherto unknown how this activator regulates
hypophysis formation. Weijers received a VIDI grant in 2006 from
NWO for this research and a NPC grant (awarded to Sacco de
Vries). More information about this research has been described
by Weijers in the highlight article in this issue (see pages 21-24).
Researchers of NPC (Utrecht University, Hubrecht Institute
and Leiden University Medical Centre) have reported on a
significant advance in the understanding of stem cell differentiation
using proteomics technology. The results are published
in the high profile journal Cell Stem Cell, shedding light on
how we can manipulate and programme stem cells to specific
fates, creating a source of tissue replacement for regenerative
medicine purposes.
Biomarker discovery for
head and neck cancer
Annually, some 2,500 people in the
Netherlands are diagnosed with
head and neck cancer. In 20-40% of
patients, the tumour returns after
surgery. Up to now, it was impossible
to predict among which patients this
could occur. In her PhD study, NPCresearcher
Tieneke Schaaij-Visser
(UU) used proteomics techniques to compare hundreds of
proteins of normal and (pre)cancer tissues of patients. She has
managed to identify a number of proteins (biomarkers), with
which one can effectively predict which treated patients have
a significant risk of a recurring tumour.

awards honours news
Albert Heck visiting
Professor ETH Zurich
ETH Zurich has appointed Albert Heck, scientific director of
the Netherlands Proteomics Centre, as visiting professor of
Systems Biology. Starting 1 March 2010, Heck will for five
months be resident of the Institute for Molecular Systems
Biology, led by Ruedi Aebersold. Heck will use his time in
Zurich to further study the opportunities of this rapidly
evolving scientific field. Heck’s visiting professorship is made
possible by the ‘Distinguished Visiting Scientist Stipend’ of the
Netherlands Genomics Initiative (NGI).
Jacques Neefjes wins
ERC Advanced Grant
In its second competition for established
top researchers, the European
Research Council (ERC) has selected
research leaders to perform their
pioneering research throughout
Europe. Grants are awarded to
exceptional research leaders who
have distinguished themselves in
their originality and significance of
their research contributions. NPC
project leader Jacques Neefjes (NKI) has won the prestigious
ERC Advanced Grant (€ 2,1 million).
Neefjes is interested to obtain more insight in how the immune
system is being controlled. Hereto Neefjes focuses on a
certain group of molecules, MHC class II, which are important
for the production of antibodies and stimulation of killer-cells.
They are involved in immune defense against infections and
cancer, but also play a role in auto-immunity upon transplantation.
Neefjes tested many human proteins and identified
about 300 proteins that are someway involved in this system.
Neefjes will use his grant to sort out how these proteins are
interconnected. Moreover he will explore the effects of
various chemical compounds.
DGMS Award 2010 for
Albert Heck
Albert Heck, scientific director of the Netherlands
Proteomics Centre, has been awarded the DGMS Award 2010
‘Massenspektrometrie in den Biowissenschaften’ for his
scientific contributions in mass spectrometry. Heck received
his award at the annual (43rd) mass spectrometry conference
in Hall (7-11 March 2010). The DGMS award is an initiative of
the German Mass Spectrometry Society and has been awarded
since 2009 to those who have provided an excellent scientific
contribution to mass spectrometry.
NPC participates in DNA-labs
on the road
The DNA-labs on the road are a great
hit among high schools, teachers and
students. Hundreds of teachers and
thousands of students can talk about
the success of this educational project. But still the DNA-labs
continue in developing educational material and giving insights
in modern genomics research.
On March 12 the Centre for Society and Genomics organized
the ‘DNA-lab dag’, a national conference on genomics for high
school teachers and technical assistants. Keynote speakers
were forensic geneticist Prof. Dr. Peter de Knijff (genetics
and human evolution) and Prof. Dr. Jan Hoeijmakers (DNA and
ageing). Also workshops on genomics were organised, including
two workshops of the Netherlands Proteomics Centre.
NPC researchers receive
STW Grant for characterization
of antibodies
Technology Foundation
STW has awarded NPC
researchers Albert Heck
and Esther van Duijn
(Utrecht University) a
STW Grant of € 992,000.
Heck and Van Duijn
will use their grant to characterize antibodies for therapeutic
applications. In collaboration with MS Vision, a unique mass
spectrometer will be built dedicated to the analysis of intact
antibodies. Dutch biotechnology company Merus also takes part
in the project, ensuring access to complex mixtures of antibodies
with improved clinical efficacy.
Geert Kops receives
KWF Grant
NPC theme leader Geert Kops (UMCU)
has received a grant of € 500,000 from
the KWF (Dutch foundation for cancer
research) for his research on a protein
(bubr1) which plays an important role
in chromosome division. Mutations of
this protein causes a rare disease called
‘mosaic variegated aneuploidy’ (MVA). Patients with this
disease have all kinds of developmental disorders, develop
cancer at a young age and also die young. Kops and his
colleagues showed that mutations in the bubr1 gene cause
aneuploidy. They will use the grant to find the precise function
of the protein and how mutations disturb its function.
|

Geert Kops
Dividing the goods:
preventing chromosome
segregation errors
| NPC Highlights 11 | April 2010
Cell division requires duplication of the genome and the
subsequent physical separation of the two duplicates. This
separation occurs during the strictly regulated process of
mitosis. One of the critical enzymatic activities in mitosis is
carried out by the kinase Mps1. Inhibiting Mps1 is sufficient
to kill cells, making it a potential target for novel anti-cancer
strategies. We have generated a unique cellular model
system that allows in-depth examination of the roles of
Mps1 activity in mitosis.
Each and every time a cell divides, the whole complement
of chromosomes needs to be distributed equally amongst the
daughter cells during the process known as mitosis. Errors in
chromosome segregation are devastating to the developing
organism, almost always leading to early embryonic death.
Similar errors in cells in the adult can contribute to tumour
formation in various ways and indeed, whole chromosome
aneuploidy is a hallmark of human cancers. To prevent errors
in chromosome segregation, cells have evolved highly regulated
networks of processes that monitor mitosis at various
stages. The Mps1 kinase is a central player in many of these
processes. Our NPCII-funded research aims at understanding
how Mps1 regulates the chromosome segregation machinery.
Error-prevantion machinery Mitosis is a dramatic process.
In the span of a mere hour, cellular morphology and most
structures in the cell change to accommodate chromosome
segregation. During this time, the chromosomes condense, the
nuclear envelope breaks down, most tubulin is re-assembled
into a highly dynamic mitotic spindle, the cell rounds up and
all chromosomes attach to spindle microtubules migrate to
the cell equator and are subsequently pulled apart. Essential
for error-free chromosome segregation is chromosome
bi-orientation, meaning the attachment of one copy of a pair
of duplicated chromosomes to microtubules from one side of
the cell and attachment of the other copy to microtubules
from the opposing side (see Figure 1).
Two processes ensure that this configuration is achieved for
all chromosomes before the cell starts the segregation process
in mitotic anaphase. The first of these is the attachment
error-correction machinery. In early mitosis, many errors in
chromosome attachment that cannot result in bi-orientation
are made, and the Aurora B kinase corrects such errors [1].
The second process is the mitotic checkpoint. This checkpoint
ensures that the segregation phase is not initiated until
all chromosomes are fully and stably attached [2]. On the
molecular level, the checkpoint is engaged by chromosomes

What this research is about:
Cell division checkpoint possible
target for anti-cancer drug
In order to get from a fertilized egg-cell to a full human being, trillions and trillions of cell
divisions are needed. All of them have to be carried out perfectly, since mistakes in the distribution
of chromosomes between the two daughter cells lead to cell death and miscarriage.
“It is very important to an organism that every cell division is error-free. We want to discover
how that can be possible,” says Geert Kops of the Department of Physiological Chemistry and
Cancer Genomics Centre at the University Medical Centre Utrecht.
Cell division is a complex and dynamic process that is heavily controlled by molecular checkpoints.
These checkpoints monitor if the duplicated chromosomes are properly connected
before the process of cell division can go into its next phase. If they are not, the cell will
receive more time to achieve proper chromosome connection. To elucidate this mechanism,
Kops also looks at tumour cells. These cells make mistakes during cell division because their
checkpoints do not efficiently see the faulty connections. Kops: “Errors seem a formula for
success for these cells. But there is a limit to the amount of mistakes that can be tolerated
without compromising viability. If we can inactivate the checkpoint by some drug, the tumour
cell will make so many mistakes that it will soon die.”
In this article the author describes the research on the key drugable protein in the checkpoint
machinery. “If this protein is switched off the cell invariably dies. With proteomics we want
to find out how this checkpoint protein exactly works and which other proteins it talks to. For
instance, protein modifications cause the switches in its activity and location,” says Kops. “So
if we understand how this works we can design better strategies for the development of new
anti-cancer treatments aimed at inhibiting this protein. That is the ambition lurking on the
horizon.”
|
NPC Research Theme T1: Cancer Proteomics
that have made no connections to spindle microtubules. In
such a case, the unattached chromosomes themselves produce
an inhibitor of the anaphase promoting complex/cyclosome
(APC/C), an E3 ubiquitin ligase that directs destruction of
proteins to initiate anaphase. Thus, as long as unattached
chromosomes persist, the checkpoint will not allow cells to
proceed with chromosome segregation.
bi-orientation
checkpoint
It has been proposed that the error-correction and mitotic
checkpoint machineries are dysfunctional in cancers, causing
whole chromosome aneuploidy [3]. In addition, we have shown
that full but not partial inactivation of the mitotic checkpoint
induces cell death by causing massive chromosome segregation
errors [4, 5]. In collaboration with René Medema of the
Laboratory of Experimental Oncology at the University Medical
Center Utrecht, our lab has recently shown that such levels of
chromosome mis-segregations can also be reached by partial
inhibition of multiple pathways in mitosis [5]. Importantly,
tumour cells proved to be more sensitive to such combinatorial
prometaphase
metaphase
Figure 1 | In early mitosis (prometaphase), the mitotic checkpoint prevents
cell-cycle progression while chromosomes attempt to attach with each copy
to opposite poles (bi-orientation). Once productive attachment of every
chromosome is reached and chromosomes have bi-oriented (metaphase), the
checkpoint is satisfied and chromosome segregation is allowed.

accept modified forms of ATP or ATP-like compounds [7]. One
such form, a bulky version of the Src inhibitor PP1, inhibits
these engineered kinases. Another is a bulky APT that carries
a tagged phosphate that, when transferred to a substrate, can
be affinity purified [8]. Figure 3 shows two human cell lines
that express the engineered form of Mps1 as their sole source
of Mps1 protein. Addition of the ATP analog 23dMB-PP1 inhibited
Mps1 activity and caused severe chromosome segregation
errors. Careful timing studies showed that full enzymatic
inhibition can be achieved within 5 minutes of addition of this
ATP analog.
Figure 2 | Mps1 facilitates the correction of faulty chromosome-to-microtubule
attachments by Aurora B kinase. Mps1 directly phosphorylates Borealin
to promote activation of Aurora B thus enhancing error-correction.
targeting than immortalized normal human cells. Organismal
studies are underway to examine if this distinction between
cancerous and non-cancerous cells will allow specific tumour
cell killing in an animal.
In collaboration with Albert Heck and Shabaz Mohammed of
the Biomolecular Mass Spectrometry lab at Utrecht University,
these cell lines are currently being used to find Mps1 substrates
with two approaches (see Figure 3). In the first, phosphor-proteins
in whole cell lysates of Mps1-inhibited cells will
be quantitatively compared to those of uninhibited cells using
stable isotope labelling in cell culture (SILAC) and dimethyl
labelling, depending on the cell line.
In the other approach, recombinant engineered Mps1 protein
will be added to fractioned cell lysates together with tagged
Dual function protein Not surprisingly, many enzymes
partake in the mitotic processes that coordinate and regulate
chromosome bi-orientation and the mitotic checkpoint.
A
B
| NPC Highlights 11 | April 2010 | Geert Kops
Mps1 is a dual specificity kinase that is critical for correcting
improper chromosome-microtubule attachments as well as for
ensuring mitotic checkpoint activity. In addition, Mps1 is one
of the most promising targets in the anti-cancer strategy that
induces the high frequency of chromosome segregation errors.
It is not understood how activity of Mps1 is regulated or how
it orchestrates the various Mps1-dependent processes. Thus
far, only one functionally relevant substrate of Mps1 has been
found. Our lab showed in 2008 that Mps1 phosphorylates
Borealin, an activator of Aurora B, and thereby facilitates
attachment error-correction [6] (see Figure 2). The Mps1-dependent
phosphorylation sites on Borealin were identified by
proteomics of in vitro phosphorylated protein, and inactivating
mutation of these sites led to significant problems in attachment
error-correction. Strikingly, mutation of these sites
to mimic phosphorylation fully rescues Mps1 deficiency in relation
to error-correction and effectively bypassed the need for
Mps1 in this process. These experiments proved that Borealin
is a relevant functional substrate of Mps1 in the error-correction
process. The identity of substrates of Mps1 in the mitotic
checkpoint or other mitotic processes, however, is unknown.
C
Chemical genetics To investigate the role of Mps1 in mitosis
in more detail, our lab generated a cellular system that allows
specific inhibition of Mps1 enzymatic activity and can also be
used to chemically tag direct cellular substrates. This system,
termed chemical genetics, was pioneered by Kevan Shokat
(UCSF) and makes use of engineered kinase mutants that can
Figure 3 | Chemical genetic inhibition of Mps1.
A. Two human cell lines expressing LAP-tagged Mps1 variants, either analogsensitive
(as) or wild-type (WT), instead of endogenous Mps1.
B. Inhibition of Mps1-as activity (pT676) in mitosis (M) by 23dMB-PP1.
C. Two proteomics approaches for identifying cellular substrates of Mps1
using chemical genetics. Green represents acitve kinase, red inactive kinase.

ATP analog. Subsequent affinity purification and proteomic
analyses will reveal potential relevant direct Mps1 substrates.
Uncovering networks Tight spatio-temporal control of the
processes that govern chromosome segregation ensures chromosomal
stability. This control is exerted, amongst others,
by various protein kinases. Our lab aims to understand the
signalling networks that are influenced by a group of kinases
that is activated at unattached kinetochores and is pivotal for
chromosome bi-orientation and the mitotic cell-cycle checkpoint.
A powerful method to uncover these networks is the use
of chemical genetics combined with quantitative phospho-proteomics.
With chemical genetics, a cell line is generated that
lacks expression of endogenous kinase but instead expresses
an engineered form that is amenable to inhibition by bulky,
ATP-like compounds.
Using NPC funding, we have established such cell lines to
study the Mps1 kinase. Our initial results have shown that
Mps1 can be enzymatically inhibited with bulky PP1 analogs
within minutes, leading to absence of all known Mps1
functions in mitosis. Using quantitative phospho-proteomics
methods, these cells will be used to identify Mps1-controlled
pathways and direct Mps1 substrates. The same methods will
be applied to other kinases within this functional subfamily.
Ultimately, these studies will facilitate the description of
the signalling networks that ensure error-free chromosome
segregation.
References
1 Vader, G., Medema, R.H., and Lens, S.M. (2006) The
chromosomal passenger complex: guiding Aurora-B through
mitosis. J Cell Biol 173, 833-837.
2 Musacchio, A., and Salmon, E.D. (2007) The spindle-assembly
checkpoint in space and time. Nat Rev Mol Cell Biol 8,
379-393.
3 Kops, G.J., Weaver, B.A., and Cleveland, D.W. (2005) On
the road to cancer: aneuploidy and the mitotic checkpoint.
Nat Rev Cancer 5, 773-785.
4 Kops, G.J., Foltz, D.R., and Cleveland, D.W. (2004)
Lethality to human cancer cells through massive chromosome
loss by inhibition of the mitotic checkpoint. Proc Natl
Acad Sci USA 101, 8699-8704.
5 Janssen, A., Kops, G.J., and Medema, R.H. (2009) Elevating
the frequency of chromosome missegregation as a strategy
to kill tumor cells. Proc Natl Acad Sci USA 106, 19108-
19113.
6 Jelluma, N. et al. (2008) Mps1 phosphorylates Borealin to
control Aurora B activity and chromosome alignment. Cell
132, 233-246.
7 Bishop, A.C., Buzko, O., and Shokat, K.M. (2001) Magic bullets
for protein kinases. Trends Cell Biol 11, 167-172.
8 Blethrow, J.D. et al. (2008). Covalent capture of kinasespecific
phosphopeptides reveals Cdk1-cyclin B substrates.
Proc Natl Acad Sci USA 105, 1442-1447.
Author
|
Geert Kops
Summary
From conception onwards, many trillions of cell divisions generate
a human individual. Each cell division requires duplication
of the genome and the subsequent physical separation of
the two duplicates. This separation occurs during the process
of mitosis, after the replicated genomes in the form of distinct
chromosomes attach to fibres of the spindle apparatus
coming from opposite directions and align on the cell equator.
Shortly after all chromosomes have aligned in this fashion,
each chromosome copy is dragged to opposite sides of the cell
after which the cell cleaves in the middle. Mitosis is highly
dynamic and strictly regulated in space and time. Amongst
summary
Contact
Dr. Geert Kops
Department of Physiological Chemistry and
Cancer Genomics Centre
UMC Utrecht
Universiteitsweg 100
3584 CG Utrecht
T +31 88 755 51 63
g.j.p.l.kops@umcutrecht.nl
http://ruummc.med.uu.nl/research/signalkops.html
the critical enzymatic activities that exert this control is the
Mps1 kinase. Depleting Mps1 protein is sufficient to kill cells
by causing massive chromosome segregation errors. Mps1
has thus been proposed as a potential target in anti-cancer
strategies that aim at elevating the frequency of chromosome
segregation errors. We have generated a cellular model system
that allows specific, penetrant and temporally controlled inhibition
of the enzymatic activity of Mps1. These cells are being
used to uncover the roles of Mps1 in mitosis and to identify its
direct substrates using quantitative phospho-proteomics. They
furthermore present the best cellular model system to date
for investigations into the effects of penetrance and duration
of Mps1 inhibition on cell viability.

Rumyana Karlova and
Sacco de Vries
The plasma membrane
receptor complex perceiving
plant steroids
10 | NPC Highlights 11 | April 2010
Just like in humans, steroids play an important role in
the development of a plant. Large protein complexes
located in the plasma membrane act as receptors for these
steroids. Researchers of Wageningen University have
isolated such a plasma membrane receptor complex by
co-immunoprecipitation and determined the composition
by mass spectrometry. This forms the basis for a model to
explain how steroid signalling in plant cells works.
Animals as well as plants use steroid hormones as important
signalling molecules. Plant steroids or brassinosteroids are
perceived by a hetero-oligomeric plasma membrane receptor
complex. This membrane complex is instrumental in transmitting
the steroid signal to a wide range of different targets that
control cell elongation, cell division, plant fertility and help
defend against plant pathogens. It is not clear how all these
different effects are integrated.
To help find additional components we have isolated the receptor
complex from Arabidopsis seedlings by co-immunoprecipitation
with one of the GFP-tagged receptors. Identification
of the associated proteins was then done by LC-MS. To verify
the interactions several independent methods have been used.
Biochemical interaction using phosphoproteomics was performed
on isolated proteins. Furthermore, genetic interactions
were established using intermediate strength mutant alleles
and cellular interaction was determined using fluorescence
microspectroscopy of differentially tagged proteins. Only
when all these criteria were met an interactor was considered
to be ‘real’.
Our current NPC2 project focuses on one of the receptorinteracting
proteins, surprisingly a transcription regulator of
the MADS-box class. The aim of this project is to determine
the spatial dynamics of this direct membrane-to-nucleus
shortcut and how that operates functionally in plant cells.
Plant hormones Steroid hormones are potent molecules that
are important long-distance regulators in the animal body.
Natural steroids are synthesized starting from cholesterol and
well-known examples are testosterone, estradiol or corticosterone.
They are synthesized in gonads or in adrenal glands,
are lipid-soluble and are perceived in the cytoplasm of target
cells by soluble receptors that act as transcriptional regulators.
Steroids are involved in a large number of processes as
diverse as development, inflammation and fertility.
Similar molecules, called brassinosteroids, exist in the model
plant Arabidopsis Initially they were isolated from Brassica
pollen, hence their name. They are synthesized from the

What this research is about:
How plant hormones can influence
plant development
Sooner or later we all depend on sufficient plant growth. That could be in the form of biomass for
the production of biofuels or for our own nutrition. “That’s why we are interested in the genetic
circuits that determine how a plant develops. Plant hormones, such as steroids, play an important
role in plant growth,” explains Sacco de Vries, professor of Biochemistry in the department of
Agrotechnology & Food sciences at Wageningen University. “By adapting genetic routes you can influence
the height and yield of a crop. Of course there is a limit to what you can change.”
De Vries and his research group study a protein complex of the model plant Arabidopsis that is
involved in growth but also affects the defence system against plant pathogens. “When this protein
complex is triggered by the presence of steroids, it influences both processes. In humans this works
in the same way. When we are busy fighting infections there is no energy left for growth or vice
versa.”
The big question is how you can breed plants that have high resistance to disease and at the same
time produce a large amount of the desired products.
“Simple solutions do not exist,” says De Vries. “If you change one pathway, it could have an effect
on other cell processes. To find the right balance we need to improve our knowledge on a fundamental
level.”
In this article the authors describe their approach to determine the existence, location and function
of their protein complex, using a proteomics-based analysis in combination with biochemical and genetic
techniques. “The combination of techniques is essential. We found proteins we would not have
found by using the classical techniques. Furthermore we even manage to visualise the protein-protein
interactions and location of the protein complex in the membrane by fluorescent labels,” says
De Vries. “Our next project is to elucidate the three-dimensional structure of the protein complex.”
| 11
NPC Research Theme T2: Proteome Biology of Plants
steroid campestanol, are also lipid-soluble and are found to
consist of about 70 different molecules. They occur naturally
in plants, especially in flowers, and constitute a normal element
of our daily diet, although the concentrations are probably
too low to have any physiological effect on animal steroid
perception. In contrast to animal cells, they are not perceived
by nuclear receptors but by a plasma membrane located
receptor called brassinosteroid-insensitive-1 or BRI1 [1].
While the BRI1 receptor was identified in a genetic screen
aimed at identifying mutants insensitive to the ligand, it was
also found as a mutant that behaved in the dark as if it was
in the light, indicating a close connection between brassinosteroids
and light control [2]. Based on the phenotypes of
the plants, roles in male fertility, plant resistance, immunity
and in morphogenesis have been reported [2]. At the cellular
level, the action of brassinosteroids is thought to be primarily
in the control of cell expansion, the reason why strong bri1
mutations result in miniature plants (see Figure 1). An open
question therefore is how this single ligand-receptor combination
is able to control all of these diverse pathways.
Figure 1 | A wild-type (back)
and bri1 mutant (front) plants.

Figure 2 | FRET-FLIM employing BRI1-CFP and SERK3-YFP using a transient
assay in protoplasts. A τ of less than 2.1 (green in the false-colour image)
indicates that the two proteins are in close proximity to form a PM-located
hetero-oligomer.
12 | NPC Highlights 11 | April 2010 | Sacco de Vries
Research from several groups including ours [3, 4] has indicated
that the BRI1 receptor is part of a complex that also
contains members of another class of PM-located receptors,
the Somatic Embryogenesis Receptor-like Kinases (SERKs),
that as BRI1 itself are LRR type single pass TM, dual specificity
type of receptor kinases capable of transphosphorylating both
Ser/Thr as well as Tyr residues [5, 6]. Most likely the SERKs
do not bind ligands themselves and are therefore more likely
co-receptors. There is some evidence that the active configuration
is a tetramer [4]. We have used a proteomics approach
to isolate the membrane receptor complex that is involved in
brassinolide perception starting from tagged versions of the
SERK receptors.
Here we will describe how we used additional lines of
evidence using genetics, biochemistry and direct interaction
studies using fluorescence microscopy to verify
the configuration of the initially determined receptor
complex. The main research question in our group is what
the precise biochemical role is of the non-ligand-binding
co-receptors. Based on genetic and biochemical evidence
it was recently proposed that they act as ‘signalling output
enhancers’ by increasing the phosphorylation status
after activation of the main BRI1 receptor. There is also
some evidence that suggests that the precise combination
of co-receptors with the main receptor serves to increase
specificity or to link brassinosteroid pathways with other
pathways [7].
Members of the family The expression pattern of the SERK1-
GFP gene assessed in fluorescent Arabidopsis plants appeared
highly complex [8]. The SERK1 protein complex was isolated
by using this GFP-tagged version under the control of its
own promoter after re-introduction into a wild type plant
[4]. We could identify the third member of the SERK family,
SERK3 or BAK1, identified as a partner of BRI1 by others. BRI1
was also detected, strengthening the idea that SERK1 was
indeed a novel co-receptor in the main brassinolide pathway.
Biochemical interactions using pull-down and in vitro transphosphorylation
assays confirmed the interactions between
SERK1, SERK3 and BRI1 [5]. However, null mutations of the
SERK1 gene yielded completely wild-type looking plants with
no obvious defects. When combining an intermediate strength
mutant allele of BRI1 with alleles of SERK3 and SERK1 the BRI1
phenotype became much more pronounced [9], providing formal
genetic proof for the interactions. Finally, we could show
that fluorescently-tagged SERK1 and BRI1 receptors indeed
co-localize at the PM and by using FLIM (see Figure 2). FRET
was shown between both proteins [10]. This way, biochemical,
genetic and cellular interaction indeed all confirmed
the original observation made of the membrane complex [4].
Current work is directed in trying to quantify the proportion of
the receptors that are in complex in vivo using a variety of different
approaches and to establish the structures of the SERK
receptors (see Figure 3).
Our new project within the NPCII program is directed towards
a third interacting protein of the SERK1 receptor complex, the
transcriptional regulator AGL15. This protein is a member of
the large family of MADS box proteins under study in the group
of Gerco Angenent. We have also verified the interactions
at both biochemical, genetic and cellular level and are in
the process of looking at the precise cell types in the intact
plant where this interaction is taking place because in plant
research it is quite unusual to find a transcriptional regulator
directly interacting with a PM receptor kinase in this project.
Investigating mutants The main conclusion from the work is
that plant membrane receptor complexes can be isolated
Figure 3 | Comparative modelling of the SERK1 kinase domain is helpful in
elucidating its final structure using X-ray diffraction. Red: ATP binding site,
yellow: activation loop and blue: C-terminal tail.
Model drawn by Matije van den Toorn (Wageningen UR),

using a single-step affinity purification and in sufficient
quantity to enable identification of its components by mass
spectrometry. More recently we have modified the original
procedure fairly extensively to the point where we can start
to compare the receptor complexes associated with different
members of the gene family and also to investigate how
receptor complexes look like from mutant backgrounds that
miss one or two of the complex components.
This work should provide a basis for answering basic questions
concerning the replacement of the now missing proteins by
close homologs and/or the stability of the remaining complexes.
In a model species such as Arabidopsis, where research is
dominated by forward and reverse genetics as the main tools
for identifying interactors, the isolation of protein complexes
offers an alternative approach to identify proteins that would
otherwise have been missed. It is now being incorporated in
the research activities of many laboratories.
One thing that has become clear from our work is that other
lines of evidence such as from genetic and cell-biological
experiments are required to confirm the protein interactions
found.
8 Kwaaitaal, M. and de Vries, S.C. (2007) The SERK1 gene is
expressed in procambium and immature vascular cells. J
Exp Bot 58, 2887-2896.
9 Albrecht, C. et al. (2008) The Arabidopsis SERK proteins
serve brassinolide-dependent and independent signaling
pathways. Plant Physiol 148: 611-619.
10 Hink, M.A. et al. (2008) Fluorescence Fluctuation Analysis
of AtSERK1 Oligomerization. Biophysical J 1052-1062.
Research team
1 2 3
4
5
References
1 Li, J. and Chory, J. (1997) A putative leucine-rich repeat
receptor kinase involved in brassinosteroid signal transduction.
Cell 90, 929–938.
2 Clouse, S.D. and Sasse, J.M. (1998) BRASSINOSTEROIDS:
Essential Regulators of Plant Growth and Development.
Annu Rev Plant Physiol Plant Mol Biol 49, 427-451.
3 Nam, K.H. and Li, J. (2002) BRI1/BAK1, a receptor kinase
pair mediating brassinosteroid signaling. Cell 110, 203-212.
4 Karlova, R. et al. (2006) The Arabidopsis Somatic
Embryogenesis Receptor-like Kinase 1 protein complex includes
Brassinosteroid-insensitive1. Plant Cell 18, 626-638.
5 Karlova, R. et al. (2009) Identification of in vitro phosphorylation
sites in the Arabidopsis thaliana Somatic
Embryogenesis Receptor-like Kinase family. Proteomics 8,
368-379.
6 Wang, X. et al. (2008) Sequential transphosphorylation
of the BRI1/BAK1 receptor kinase complex impacts early
events in brassinosteroid signaling. Dev. Cell 15, 220–235.
7 Chinchilla, D. et al. (2009) One for all: the receptor-associated
kinase BAK1. Trends Plant Sci 14: 535-541.
Summary
Plant steroids have an important role in plant development
and are perceived by a plasma membrane receptor complex.
This complex consists of a main, ligand-binding receptor involved
in the entire spectrum of cellular processes affected by
steroids and a number of co-receptors that are thought to be
non ligand-binding. We have isolated such a plasma membrane
receptor complex by co-immunoprecipitation using one of the
co-receptors fused to GFP. The composition of the complex
summary
6 7 8
Esther van Loenen (1), Cathy Albrecht (2), Jan Willem Borst (3),
Na Li (4), Rumyana Karlova (5),
Sjef Boeren (6), Walter van Dongen (7) and Sacco de Vries (8).
Contact
Prof. Sacco C. de Vries
Laboratory for Biochemistry
Wageningen University and Research centre
Dreijenlaan 3
6703 HA Wageningen
T +31 317 483 866
sacco.devries@wur.nl
was then determined by mass spectrometry, followed by
extensive biochemical, genetic and cell biological experiments
for confirmation.
Apart from other related plasma membrane receptors,
proteins acting as adaptors such as a member of the 14-3-3
family were found. The most surprising result however was
the presence of the transcriptional regulator AGL15 as part of
the membrane receptor complex. Current work is therefore
aimed to establish a comprehensive cellular model to explain
how steroid signalling in plant cells uses a plasma membrane
receptor directly activating a transcription factor.
| 13

Paul Boersema, Vanessa Ding
and Albert Heck
In depth profiling of tyrosine
phosphorylation in human
embryonic stem cells
14 | NPC Highlights 11 | April 2010
Human stem cells are inclined to differentiate easily
during culturing; thus losing their ability to transform into
other cell types. Fibroblast growth factor 2 plays a role
in preventing this process by activating a receptor that
transmits the signal by phosphorylating tyrosine residues of
proteins. But the events following signal transmission that
lead to the maintenance of the pluripotency of stem cells
remain unclear. Using a very sensitive proteomics set-up,
the tyrosine phosphorylation sites were studied in detail
and a comprehensive picture of tyrosine phosphorylation
was obtained.
tency. Culturing conditions have been optimized by the use of
additives to induce self-renewal rather than differentiation, but
how exactly the added factors are involved in the maintenance
of the pluripotency state remains unclear.
Stem cells are used more and more to study embryonic development
in vitro. Also, these pluripotent cells promise therapeutic
applications for diseases that are currently difficult to treat.
Human ESCs can be derived from an early stage of an embryo.
These cells are isolated and can then be grown and expanded
for more efficient use and to limit the number of embryos that
are required.
However, embryonic stem cells show a tendency to proliferate
and differentiate easily during growth. Their culturing therefore
requires special attention. Any stimulation, such as stress,
may result in the development of these stem cells into specific
cell types after which they will largely lose their ability to
transform into other cell types. It requires precise fine tuning
of growth conditions to keep these cells in a state of pluripo-
Several cellular signalling pathways have been shown to be
implicated with hESC self-renewal. Fibroblast growth factor 2
(FGF-2) is a crucial growth factor in the maintenance of the
pluripotent state of human embryonic stem cells (hESCs). FGFs
bind receptor tyrosine kinases, i.e. they phosphorylate tyrosine
residues in proteins. Their main task is the transmission of the
signal from the stimulus by phosphorylating tyrosine residues
on specific down-stream proteins. Inhibition of the FGF receptor
causes the down-regulation of several proteins that are
considered markers of pluripotency. FGF-2, on the other hand,
is known to activate FGF receptors, which have been shown
to turn on several signalling pathways. Therefore, the growth
factor FGF-2 is widely used for the long-term culture of hESCs.
However, the downstream signalling events following FGF-2
stimulation and its link to hESC self-renewal and the maintenance
of pluripotency remain to be determined.

What this research is about:
Preventing stem cells specialising
too early
Stem cells have the ability to differentiate into a range of specialized cell types. They promise therapeutic
applications for diseases that are currently difficult to treat such as Alzheimer’s disease because of this
property. Human stem cells are isolated from embryos and then cultured and grown. During the culturing,
however, the stem cells have a tendency to differentiate into specialized cells prematurely, after which
they lose their ability to transform into other cell types.
The growth factor FGF-2 is added during the culturing of stem cells, explains researcher Paul Boersema. “If
you leave this factor out, the stem cells start to specialize. But we do not exactly understand how FGF‐2
operates in this process.” The growth factor stimulates a receptor on the cell membrane, which in turn
starts a signalling process in the cell. The signal is transmitted through phosphorylation on a tyrosine residue
of certain proteins. “Some of the proteins which are phosphorylated by the FGF-2 receptor are known,”
explains Boersema, “but most of these are present in such a low concentration that they are hard to study.”
Boersema has therefore designed a process to isolate these phosphorylated tyrosine peptides by binding
them to a bead containing an antibody. Subsequently the beads are separated, the peptides eluted and
analyzed using LC-MS. Boersema cooperated with researchers from the Singapore Bioprocessing Technology
Institute who showed him the details of the enrichment of tyrosine phosphorylated peptides, while the
Utrecht group instructed the Singaporeans on the LC-MS methods, including stable isotope labelling
techniques to implement quantitative module.
The cooperation resulted in the discovery of many proteins that were formerly not known to play a role
in FGF receptor signalling. Several of these proteins are known to have a role in cytoskeletal dependent
processes in adult cells. “This may mean that they also play a role in the differentiation of stem cells,” says
Boersema. “Eventually it may enable us to find a suitable growth factor to regulate specialization of stem
cells.”
| 15
NPC Enabling Technologies E3: New Mass Spectrometric Tools in Proteomics
In this collaborative study, the effect of the growth factor FGF‐2
on hESCs was investigated. NPC researchers at the Utrecht
University collaborated with researchers from the Bioprocessing
Technology Institute, Biopolis in Singapore, to study the effect
of FGF-2 on hESCs by profiling tyrosine phosphorylation upon
FGF-2 stimulation. The experience of the Utrecht group with
sensitive LC-MS and stable isotope dimethyl labelling for quantitative
proteomics was combined with the know-how of the
Singapore group about culturing hESCs and performing
immunoprecipitation of tyrosine phosphorylated peptides.
Figure 1 | Immunoprecipitation with immobilized antibodies against tyrosine
phosphorylation can be performed before digestion at the protein level or
after digestion at the peptide level.
Isolating phosphorylated peptides We focused on the profiling
of tyrosine phosphorylation upon FGF-2 stimulation. As the
target proteins that are involved in this signalling are largely
unknown, we set out to identify them by an LC-MS based
proteomics approach. The large scale analysis of cellular phosphorylation
— and particularly, tyrosine phosphorylation — by
LC-MS is challenging because of the relatively low abundance of
phosphorylation. The overwhelming background of non-modified
peptides causes phosphopeptides to remain undetected when
no proper precautions are taken.

New discoveries To quantitatively profile to what extent individual
tyrosine sites are phosphorylated upon FGF-2, we had to
incorporate a stable isotope labelling method in the workflow.
The milligrams of sample required for an efficient immunoprecipitation
of tyrosine phosphorylated peptides make most
labelling approaches cost-prohibitive. For that reason, we chose
to implement stable isotope dimethyl labelling as it uses rather
cheap reagents and can thus be used for the labelling of large
amounts of sample. hESCs were stimulated with FGF-2 for 0,
5 or 15 minutes respectively. The proteins from these samples
were digested and differentially labelled with the dimethyl
labels (see Figure 2). From the mixture of the three different
samples, tyrosine phosphorylated peptides were selectively
enriched by immunoprecipitation. LC-MS analysis on these
enriched peptides then allowed us to identify and quantify 316
unique tyrosine phosphorylated peptides [2].
Of these peptides, 138 showed different levels of increased
tyrosine phosphorylation (Figure 3), while for most of the
other peptides the phosphorylation levels did not significantly
change. An increase in tyrosine phosphorylation was, not
surprisingly, found on peptides from FGF receptors and some of
their known downstream targets. Furthermore, an increase in
Figure 2 | The proteomics workflow to study the effect of FGF-2 on the tyrosine phosphorylation was found on many proteins that are
tyrosine phosphoproteome of hESCs. hESCs are stimulated with FGF-2 for not known to be involved in FGF signalling. These proteins may
0, 5 or 15 min after which the cells are lysed and the proteins digested.
be new players in the FGF pathway or may shed some light on
The different samples are differentially labelled with stable isotope
new effectors of FGF-2. For example, several receptor tyrosine
dimethyl labels after which the mixture of these peptides is enrich for
tyrosine phosphorylated peptides by immobilized antibodies against tyrosine kinases other than FGF receptors were found to be increased
phosphorylation. The enriched peptides are then analyzed by nano-LC-MS. in tyrosine phosphorylation. As FGF-2 has no known specificity
for these receptors, transactivation of these receptors or some
16 | NPC Highlights 11 | April 2010 | Paul Boersema other effect may be the cause of their activation. To confirm
the phosphorylation of these receptor tyrosine kinases we used
Various techniques have been developed to enrich for phosphopeptides
prior to LC-MS analysis, for example, approaches based
a human phospho-RTK array. In this array, antibodies against
on titanium dioxide (TiO 2 ) or immobilized metal affinity chromatography
(IMAC). TiO 2 and IMAC are specifically suited for the
analysis of serine and threonine phosphorylation. The even
lower abundance of tyrosine phosphorylation requires a different
approach. Antibodies have been generated that specifically
bind phosphorylated tyrosine residues. By immobilizing these
antibodies on agarose beads, tyrosine phosphorylated peptides
or proteins can be immunoprecipitated and a high degree of
enrichment can be obtained. Performing the precipitation
step at peptide level can be advantageous. As the end product
that is analyzed by LC-MS is a peptide mixture, performing the
precipitation at peptide level reduces the number of nonmodified
peptides that can arise from non-modified stretches
of the proteins or from non-modified proteins that happen to
bind tyrosine phosphorylated proteins [1] (see Figure 1). The
precipitated tyrosine phosphorylated peptides can be identified
by LC-MS.
We have been utilizing a very sensitive set-up in which relatively
narrow (i.e. 50 mm inner diameter) but long (up to 40 cm)
LC columns allow for efficient separation of the compounds also
when longer gradients of up to three hours are used. Using this
approach, we could generate a library of 735 unique tyrosine
phosphorylation sites from hESCs that were stimulated with
FGF-2 for 0, 1, 5, 15 and 60 minutes, respectively [2]. These
Figure 3 | Clustering of different tyrosine phosphorylation profiles upon
sites only partially overlapped with sites that were detected FGF‐2 stimulation. All tyrosine phosphorylated peptides from the FGF receptors
and several of their known substrates are found in cluster 3 and before and that have been deposited in public repositories.
4.

42 of the human RTKs are immobilized. After proteins from the
sample are bound to the antibodies, a phosphotyrosine antibody
that is conjugated to horseradish peroxidase is used to detect
tyrosine phosphorylation levels. In this way, the activation of
insulin receptor, insulin growth factor 1 receptor, ephrin type A
receptors 1 and 2 and vascular endothelial growth factor receptor
2 were confirmed.
Amongst the other proteins on which tyrosine phosphorylation
was shown to be increased were Src family kinase proteins and
other proteins that, in adult cells, are known to be involved in
cytoskeletal depending processes. Interestingly, changes to cytoskeletal
dependent processes have been known to play major
roles during hESC differentiation.
References
1 Boersema, P. J. et al. (2009) In depth qualitative and quantitative
profiling of tyrosine phosphorylation using a combination
of phosphopeptide immuno-affinity purification
and stable isotope dimethyl labeling. Mol Cell Proteomics
Jan;9(1):84-99
2 Ding, V.M.Y et al. (2010) Tyrosine phosphorylation profiling
in FGF-2 stimulated human embryonic stem cells.
Manuscript in preparation
Successful approach The combination of stable isotope
dimethyl labelling with selective immunoprecipitation of tyrosine
phosphorylated peptides and sensitive LC-MS allows for a
comprehensive analysis of the immediate tyrosine phosphorylation
events following the stimulation of receptor tyrosine kinases
such as the EGF and FGF receptors [1,2]. We have been able
to identify relatively high numbers of tyrosine phosphorylation
sites in single LC-MS runs. The immunoprecipitation of tyrosine
phosphorylated peptides has thereby increased the specificity
of the approach, while the (nano-) LC-MS approach enabled the
sensitive detection of also lower abundant phosphopeptides.
Finally, stable isotope dimethyl labelling allowed MS based
quantitation while working with large amounts of sample.
Research team
In the study of the effect of the growth factor FGF-2 on hESCs
we successfully applied the new approach. Several known substrates
of FGF receptors were shown to be increased in tyrosine
phosphorylation upon FGF-2 stimulation, but also proteins that
have not been associated with FGF receptor signalling were
found with increased tyrosine phosphorylation. A significant
amount of these proteins have been known, in adult cells, to
be involved in cytoskeletal dependent processes. These processes
are important in the differentiation of embryonic cells.
Therefore, the current data would suggest that FGF-2 is one of
the regulators of these processes which, in turn, may be the
reason for FGF-2 being required for long-term culture of hESC.
Summary
To keep stem cells in their pluripotent state, specific growth
conditions are required. One of the additives often used is the
growth factor FGF-2. In this collaborative study between NPC
researchers in Utrecht and researchers of the Bioprocessing
Technology Institute in Singapore, the role of FGF-2 in the
maintenance of the pluripotency state of human embryonic
stem cells was investigated. FGF-2 stimulates FGF receptors
that, in turn, phosphorylate several proteins at specific tyrosine
residues. Using a combination of stable isotope dimethyl
labelling for MS based quantitation, immunoprecipitation
with antibodies against tyrosine phosphorylation to enrich
summary
From left to right: Simone Lemeer, Vanessa Ding, Paul
Boersema, Leong Yan Foong
Contact
Prof. Albert Heck
Biomolecular Mass Spectrometry and Proteomics Group
Utrecht University
Padualaan 8,
3584 CH Utrecht, The Netherlands.
T +31 30 253 6797
a.j.r.heck@uu.nl
http://bioms.chem.uu.nl
for tyrosine phosphorylated peptides, and sensitive LC-MS
analysis we have been able to identify relatively high numbers
of tyrosine phosphorylation sites and thereby obtain a rather
comprehensive picture of tyrosine phosphorylation upon a
stimulus. Several hundreds of tyrosine phosphorylation events
upon FGF-2 stimulation could thus be monitored. Several
known targets of FGF receptors were identified, but also some
proteins that have not been associated with FGF receptor
signalling showed an increase in tyrosine phosphorylation.
Several of these proteins are known to have a role in cytoskeletal
dependent processes in adult cells. Furthermore, several
other receptors were shown to be activated upon FGF-2,
which could be confirmed by another phospho-array. Exactly
how these receptors are activated remains to be elucidated.
| 17

Arjen Scholten, Reinout
Raijmakers and Albert Heck
Finding new targets of small
molecules by chemical
proteomics
18 | NPC Highlights 11 | April 2010
Chemical proteomics allows the study of proteins in tissue
or cells by utilizing their affinity for small molecules such as
signalling molecules or drugs. Using a combination of pull-downs
with immobilized compounds and the use of modified
molecules and quantitative mass spectrometric approaches,
a detailed overview can be generated of the specificity
certain proteins have for such molecules. This may lead to
new screening tools or drug target discovery strategies.
In every aspect of life, small molecules, like metabolites and
signalling molecules but also many of the drugs that are used in
the clinic, exert their effects by interacting with proteins. They
can act as substrates, agonists, antagonists or as co-factors. The
interaction of small molecules of both natural and synthetic origin
can be studied by ‘chemical proteomics’; a multidisciplinary
research area that integrates biochemistry and cell biology with
organic synthesis and mass spectrometry [1].
In chemical proteomics, the small molecules of interest are
often engineered to be used as a ‘bait’ to specifically enrich
their protein targets from a complex proteome such as cell or
tissue lysates. This approach is useful in determining which
proteins are targeted by these small molecules. The technique
has proven its worth in the elucidation of drug targets as well
as targets of endogenous signalling molecules. Here we show
an example of both of these to highlight the unique strengths
of chemical proteomics.
In the first example we use immobilized cAMP analogues to
screen the distinct intracellular localization profiles of type I
and type II cAMP-dependent protein kinase through probing
their differential interaction with A-kinase anchoring proteins.
In the second example we used immobilized versions of the
phosphodiesterase-5 inhibitor sildenafil, the active compound
of Viagra (see Figure 1). Chemical proteomics methods
allowed the identification of novel protein targets of sildenafil
outside the family of phosphodiesterases and optimization of
these interactions by using close analogues of sildenafil.
Endogenous signalling molecules The second messenger
cAMP (3’-5’-cyclic-adenosine monophosphate) is a ubiquitous
signalling molecule involved in the translation of extracellular
signals into intracellular responses. It is implicated in a large
number of key cellular processes. The main target of cAMP
is the cAMP-dependent protein kinase (PKA), a hetero-tetrameric
protein that consists of two regulatory (PKA-R) and two

What this research is about:
Fishing for proteins with small
molecule bait
Hundreds of proteins have been isolated, characterized and sequenced using proteomics techniques.
But lately, researchers are reaching the limit: the concentration in which new, uncharacterized
proteins are present in cells or tissue is too low to identify them using standard proteomics methods.
Chemical proteomics is a new research area that solves this problem. Small molecules having an
affinity for the proteins to be studied, are coupled to a solid carrier, mostly small beads. Cell or tissue
lysate is added to this immobilized ‘bait’ and specific proteins bind to it.
These proteins can then be easily separated from the solution with a so-called pull-down experiment.
In this way, the bound proteins are isolated and may then be characterized by, for example, high
resolution mass spectrometry. “The beauty of this method is that you can study proteins that are
present in (very) low concentrations in tissue or cells without modifying them. This offers great new
possibilities,” says Arjen Scholten, who developed this technology for the signalling molecule cAMP
during his PhD research.
In one of the studies described here, Scholten and colleagues used cAMP and its derivatives to study a
large and diverse family of kinase anchoring proteins in rat heart tissue. It is not well known how and
to which type of kinases these AKAPs bind. “We showed that, using the pull-down method, it is possible
to screen the binding properties of many of these AKAPs directly in their natural environment,” says
Scholten.
The same method was used to study, in collaboration with Pfizer, the binding characteristics of
derivates of the well known drug sildenafil, or Viagra. Using the pull-down method, Scholten and
Raijmakers discovered the drug not only has an affinity for phosphodiesterase-5, its target, but also for
another protein. The researchers showed that certain derivatives of sildenafil can even have a higher
affinity for this second protein. “In this way, sildenafil can become a starting point for the development
of drugs against novel targets.”
| 19
NPC Enabling Technologies E2: Chemical Approaches to Proteome Biology
catalytic subunits (PKA-C). To accommodate all the different
tasks of cAMP and PKA within a single cell efficient spatial and
temporal regulation of its activity is required.
Figure 1 | Sildenafil is the active compound of the drug Viagra, most often
used to treat erectile dysfunction. Sildenafil is an inhibitor of phosphodiesterase-5.
http://en.wikipedia.org
This is acquired through interaction with the large and diverse
family of A-kinase anchoring proteins (AKAPs). AKAPs tether
PKA, together with other signalling proteins (phosphatases,
phosphodiesterases etc.), to distinct loci within the cell close
to PKA’s substrates to ensure tight control over the required
signalling paths. There are two main isoforms of PKA-R, type I
and type II. Both have specific preferences for different
AKAPs. For many AKAPs their preference is currently unknown
and the existence of type I specific AKAPs is predicted,
however not established [2]. Furthermore, due to their low
concentrations in cells and tissues, it is a challenge to study
AKAPs using traditional proteomics methods.

NH
NH
NH 2
N
N
NH
NH 2
NH
HN
HN
N N
N N
O
O
O
O
OH O P OH
O O P OH
H 3C
O
O
8-AHA-2’OMe-cAMP (C8-OCH3) 8AHA-cAMP (C8)
Protein Isolation
C
C
N
N
Protein Digestion
Light labeling
Heavy labeling
LC-MS/MS
PKA RIIα
PKA RIα
m/z
m/z
Figure 2 | Experimental setup of the comparison of the cellular protein
interactors of two cAMP analogues by stable isotope labelling and
quantitative mass spectrometry.
On the basis of these observed ratios, six AKAPs behaved as
perfectly PKA-RII interacting proteins, while the other six have
ratios that indicate interaction with both PKA-RI and PKA-RII.
Of these, AKAP1 and AKAP10 are well described so-called dual
specific AKAPs, explaining their intermediate ratios. These
analyses confirmed the known specificity of several AKAPs
and the specificity of AKAP14, AKAP2 and AKAP12 could be
established.
Interactions of sildenafil As described above, the same
technology can be used to determine which proteins interact
with specific drugs, like sildenafil. Sildenafil — sold under the
brand name Viagra — is an inhibitor of phosphodiesterase-5
(PDE-5). PDE proteins form a family of enzymes that hydrolyze
cyclic nucleotides, with PDE-5 specifically targeting cGMP.
PDE-5 is an interesting pharmaceutical target as its inhibition
enhances the activity of the nitric oxide-cGMP pathway, which
is involved in, amongst others, penile erection. Several potent
PDE-5 inhibitors, including sildenafil, are now widely used for
the treatment of erectile dysfunction. These inhibitors have a
track record of being safe and well-tolerated and are considered
to have a very good specificity for their target protein,
PDE-5. Whether these inhibitors can interact with any other
cellular protein targets, is largely unknown.
To identify other potential targets of sildenafil, we generated
several compounds that are structurally related to sildenafil
20 | NPC Highlights 11 | April 2010
| Arjen Scholten
Figure 3 | Analysis of the interaction of different proteins (including PDE-5,
Screening binding specificities We recently developed a
chemical proteomics method based on immobilized cAMP to
isolate and identify a large set of cAMP binding proteins, such
as several PKA-Rs, PDEs, EPACs and more than ten different
AKAPs [3]. In the present study, this method was adapted to
screen a large variety of AKAPs for their binding preference
towards PKA-RI and/or PKA-RII, in cell and tissue extracts [4].
To do so, two immobilized cAMP analogues were used in
parallel for the isolation of target proteins: 8-AHA-cAMP and
2’-OMe-8AHA-cAMP (see Figure 2).
The former interacts equally well with PKA-RI and PKA-RII,
whereas the latter has a three- to four-fold higher affinity
for PKA-RI. As the 2’-OMe beads will effectively isolate more
PKA-RI, also more of its associated AKAPs will present in the
corresponding pull-downs. By comparing the protein enrichment
patterns of both cAMP analogues, using dimethyl stable
isotope labelling and quantitative mass spectrometry, the
binding specificities of AKAPs can be elucidated by comparing
their enrichment behaviour to that of the different PKA
isoforms.
As a proof of concept, we applied this screening method to
two different cell lysates (HEK293 and RCC10) and two different
rat tissues (lung and testis), from which we isolated
and quantified twelve different AKAPs. As expected, the
enrichment pattern of PKA-RI and PKA-RII is distinguishable by
their relative efficiency of recovery from the two pull-downs.
PEBP-2) with several immobilized analogues of the drug sildenafil by label
free quantitative mass spectrometry and confirmation by western blotting.

and that could be immobilized on beads. Using these compounds,
we then performed pull-downs from various rat tissue
samples, followed by (semi-) quantitative LC-MS/MS to identify
the interacting proteins and to compare their affinity for
the different sildenafil analogues. Using this method, we were
able to identify several proteins that could interact directly
with the immobilized sildenafil, albeit with lower affinity than
the main target, PDE-5 (see Figure 3) [5].
Some of these proteins were other members of the PDE
family, but also the family of phosphatidylethanolamine binding
proteins (PEBPs), which includes the abundantly present
Raf-kinase inhibitory protein (RKIP), was found to interact
with sildenafil analogues [6]. Interestingly, some of the
sildenafil analogues that were used in this experiment had a
very different affinity for the various identified proteins, with
one of them showing significantly increased specificity for the
PEBP family of proteins (see Figure 3). This shows that chemical
proteomics, particularly in combination with (small) drug
libraries can be used to identify novel targets for (derivatives
of) existing drugs [7].
3 Scholten, A. et al. (2006) Analysis of the cGMP/cAMP
interactome using a chemical proteomics approach in
mammalian heart tissue validates sphingosine kinase type
1-interacting protein as a genuine and highly abundant
AKAP. J Proteome Res 5, 1435-1447.
4 Aye, T. T. et al. (2009) Selectivity in enrichment of cAMPdependent
protein kinase regulatory subunits type I and
type II and their interactors using modified cAMP affinity
resins. Mol Cell Proteomics 8, 1016-1028.
5 Dadvar, P. et al. (2009) A chemical proteomics based enrichment
technique targeting the interactome of the PDE5
inhibitor PF-4540124. Mol Biosyst 5, 472-482.
6 Dadvar, P. et al. (2009) Phosphatidylethanolamine-binding
proteins, including RKIP, exhibit affinity for phosphodiesterase-5
inhibitors. Chembiochem 10, 2654-2662.
7 Dadvar, P. (2009) Probing the drug interactome by chemical
proteomics, PhD thesis.
Research team
Important challenge We have shown that chemical proteomics
is a versatile tool to study the interactions of small molecules
with a proteome that can be applied in many facets of biology.
We applied the technology both to small signalling molecules
like cAMP and to clinically relevant drugs like sildenafil to
identify target proteins. To gain momentum in drug target
discovery and other fields, the development of more chemical
proteomic tools targeting a wider range of protein classes will
be an important challenge for the coming years.
| 21
References
1 Bantscheff, M., Scholten, A., Heck, A. J. (2009) Revealing
promiscuous drug-target interactions by chemical proteomics.
Drug Discov Today 14, 1021-1029.
2 Scholten, A., Aye, T. T., Heck, A. J. (2008) A multi-angular
mass spectrometric view at cyclic nucleotide dependent
protein kinases: in vivo characterization and structure/
function relationships. Mass Spectrom Rev 27, 331-353.
Summary
Chemical proteomics is an emerging research field that allows
the characterization of all proteins interacting with small
molecules using a combination of pull-downs with immobilized
molecules and LC-MS/MS for identification. As we show here,
this can be applied both to small signalling molecules like
cAMP to reveal their cellular targets, and to clinically relevant
drugs like sildenafil to identify targets and potential off-targets.
By extending this strategy to make use of modified molecules
summary
From left to right: Reinout Raijmakers, Albert Heck, Arjen
Scholten.
Contact
Prof. Albert Heck
Biomolecular Mass Spectrometry and Proteomics Group
Utrecht University
Padualaan 8,
3584 CH Utrecht, The Netherlands.
T +31 30 253 6797
a.j.r.heck@uu.nl
http://bioms.chem.uu.nl
and quantitative mass spectrometric approaches, a detailed
overview can be generated of the specificity certain proteins
have for such molecules. This yields valuable information on
drug specificity and on how abundantly present small molecules
in cells can be part of different pathways.
The work was performed at the Biomolecular Mass
Spectrometry and Proteomics group at Utrecht University, in
close collaboration with Pfizer Pharma Therapeutics, Sandwich
Laboratories, United Kingdom (sildenafil) and the Medical
Physiology department of the University Medical Hospital in
Utrecht (cAMP).

Barbara Möller and
Dolf Weijers
Cell-cell communication
in plant embryos
22 | NPC Highlights 11 | April 2010
The production of leaves, roots and flowers by plants
throughout their entire life is carried out by the so-called
meristems, growth tips in which stem cells are first formed.
Meristems are located in the young plant embryo. We
studied the formation of the root meristems in the embryo
of the model plant Arabidopsis thaliana. The process
begins with the programming of one cell as the ‘hypophysis’
which contols stem cells in the roots. It is known that
the formation of the hypophysis is controlled by the gene
activator called MONOPTEROS. However, it was hitherto not
known how this activator regulates hypophysis formation.
Plants start their lives simple, as seedlings with one or two
leaves, a small stem and a root. Later however, the plant body
is further elaborated by the formation of new leaves, stems,
branches, flowers and roots. Hence, the mature plant often
does not even remotely resemble the seedling. Plants owe this
remarkable capacity to generate many new organs to the activity
of meristems, growth tips harbouring the plant equivalent
of stem cells. These stem cells can be active over many
years, and are prevented from differentiation by organizer
cells. In our research group, we are interested in the problem
of how meristems (stem cells and organizer) are first formed
in the young plant embryo.
Like animals plants derive from a single cell, the zygote,
and all cell types and organs, including the meristems,
are formed early during embryogenesis. The meristem
that will give rise to the entire root system (root meristem)
is formed when the embryo of the model plant
Arabidopsis thaliana is approximately 50 cells in size (see
Figure 1). We have previously shown that the transcription
factor MONOPTEROS (MP), upon its activation by the
plant hormone auxin, promotes the formation of the root
meristem organizer (hypophysis). Intriguingly however,
MP does not act in this cell, but promotes its specification
from the adjacent cells. Hence, the formation of
the first meristem involves cell-cell communication.
The signal that activates MP, auxin, is itself transported
from the MP-expressing cells to the hypophysis cell, but
this is alone not sufficient for specification of the cell
as organizer. We therefore postulated the existence of a
hypothetical second MP-dependent signal that is transported
to the hypophysis [1].

What this research is about:
Discovery of essential protein for
early root formation in plants
How do plant embryonic cells know which organs they should develop into Dolf Weijers,
assistant professor at Wageningen University and his group, together with his German
colleagues from the University of Tübingen and the Max Planck Institute, studied how the
early formation of root tissue is controlled. They discovered a mobile transcription factor
which is essential in the process. The results were recently published in Nature.
In contrast to animals, plants produce new organs, in other words stems, leaves, roots
and flowers, throughout their lifetime. Stem cells are located in the growing tips of roots
and are responsible for the growth of new roots. “Relatively little is known about early
embryogenesis in plants and how one cell is programmed to become for example a root
founder cell,” says Weijers.
In earlier studies on embryonic cells in the model organism, Arabidopsis thaliana,
Weijers and his colleagues discovered that for a cell to become the root founder cell, the
plant hormone auxin is required. This hormone is sent by neighboring embryo cells. The
transcription factor MONOPTEROS (MP) promotes the transport of the hormone to the
neighboring cell.
“However, this is not sufficient on its own,” says Weijers, “so we looked for other genes
which are expressed exclusively in the presence of MP.” In a comprehensive survey of all
the genes activated by MP, the researchers identified the gene TMO7. “We discovered that
the protein formed by the TMO7 gene migrates from the location of its synthesis in the
embryo to the root founder cell. So both auxin and TMO7 are mobile factors activated by MP
and required as signals for root formation in the embryo.” The detective work in the plant
researchers’ genetics laboratory does not end here, however. “We will investigate which
other factors are involved and unravel more of the regulatory network that controls plant
embryogenesis,” says Weijers.
| 23
NPC Research Theme T2: Proteome Biology of Plants
Microarray-based approach In a study that was recently
published [2], we have taken a microarray-based approach
in order to find genes, pathways and eventually intercellular
signals that operate downstream of MP in root meristem
formation. Affymetrix genechips, featuring 22,000 of
the approximately 26,000 Arabidopsis genes, were hybridized
with mRNA isolated from normal seedlings, seedlings
lacking MP altogether, or seedlings in which MP had been
inhibited during 1 hour. After statistical analysis, we obtained
a set of about 100 genes that is robustly downregulated
in the absence of MP activity. When analyzing
gene ontology of these 100 genes, we found that genes
encoding transcription factors were strongly over-represented.
This suggests that MP primarily regulates other
transcription factors, and sits at the summit of a transcriptional
network.
To determine which of these genes are controlled by MP during
early embryogenesis, we analyzed the expression patterns of
a subset (including most transcription factor genes) in normal
embryos and in embryos lacking MP. We found 4 genes that
we named TARGET OF MONOPTEROS (TMO), to be expressed
in the sub-domain of the embryo where MP acts. As expected,
their expression depends on MP activity.
Supported by funding from the NPC (Grant no. NPC4.11
awarded to Sacco de Vries), and helped by the group of Gerco
Angenent, we used Chromatin Immunoprecipitation (ChIP) to
show that MP binds in vivo to the promoters of 3 of these 4
TMO genes. Furthermore, functional genetic investigations
showed that TMO5 and TMO7 both mediate MP-dependent
root formation, and that elimination of TMO7 leads to rootless
defects similar to those caused by the loss of MP.

Auxin
Stem cell divisions
TMO5
MP
Auxin
TMO7
Auxin TMO7
Hypophysis fate
Figure 1 | Model for embryonic root initiation.
Left: Arabidopsis thaliana embryo at globular stage consists of a sphere of
embryonic cells and a filament of extra-embryonic cells. The root meristem
forms at the junction of both and involves inner cells of the embryo (pink)
and the uppermost extra-embryonic cell (yellow).
Right: MP is activated by auxin in the embryonic cells (pink, top), and
activates expression of TMO5 and TMO7 genes. TMO5 remains in these cells
and contributes to stem cell divisions. TMO7 protein moves to the adjacent
extra-embryonic cell (yellow, bottom), where it contributes to specifying
this cell as the organizer (hypophysis). To achieve this, MP also promotes
transport of auxin to the future hypophysis.
24 | NPC Highlights 11 | April 2010
TMO5 and TMO7 encode proteins belonging to the same family
of basic Helix-Loop-Helix (bHLH) transcription factors. This
family mediates cell fate specification in other developmental
processes in plants [3], but also in animals [4]. To determine
how these two bHLH factors contribute to MP-dependent root
formation, we analyzed the localization of both proteins.
We made the remarkable discovery that, while TMO5 protein
remains in the cells where it is activated by MP, TMO7 protein
moves to the hypophysis cell (see Figure 1). This transport
depends on the size of the protein, since increasing its size by
adding 3xGFP impairs movement. Therefore, TMO7 represents
a novel intercellular signal molecule in embryonic root
formation. Even though the mechanism of action of this novel
intercellular signal is not known, it appears that its movement
to the hypophysis is critical for its function in root formation.
considered necessary for DNA binding [4]. The protein is localized
both in cytoplasm and nucleus of the cells it is produced
in, and is mostly nuclear in the cells it is transported to [2].
Several scenarios could explain this localization, among which
are either passive diffusion or active transport and blocking
of further movement by nuclear trapping. No matter what the
mechanisms are, we expect that protein-protein interactions
will be critical to achieve limited directional transport. We are
currently in the process of identifying proteins that interact
with a mobile, or with a non-mobile form of Green Fluorescent
Protein (GFP) – tagged TMO7 protein using immunoprecipitation
(IP) followed by nano-Liquid Chromatography-tandem
Mass Spectrometry (nLC-MS/MS). We have previously used this
method, established by the group of Sacco de Vries, to identify
in vivo interactors of- and phosphorylation sites on plant
proteins [5,6]. From this analysis we expect to identify factors
involved in TMO7 transport. Since bHLH transcription factors
likely act as dimers [4], we also expect to find the transcription
factor with which TMO7 interacts in the hypophysis.
2. How is root formation spatially coordinated through the
TMO transcription factors
TMO5 and TMO7 are both direct MP target genes, and activated
in precisely the same cells. Both are required for root formation,
but in different cells. TMO5 is active in the future stem
cells and TMO7 acts in the future organizer cell (hypophysis).
Certainly, different genes will be controlled in each of these
cell types. We now use these factors to determine which genes
and cellular pathways are controlled in both of these cell types
in order to generate a functional meristem (stem cells and
organizer). We will again employ microarray technology to
identify target genes of the TMO5 and TMO7 bHLH transcription
factors.
3. What controls MP activity
In addition to its role in promoting root formation in the embryo,
MP has many other functions. In the embryo, MP controls
formation of the vascular tissues, of embryonic leaves, and
has several other, post-embryonic functions [7]. To accommodate
all these activities, MP is expressed in a rather broad pattern
(see Figure 2). The TMO genes however, are expressed in
much narrower domains (FIG). A crucial question is how MP ac-
| Dolf Weijers
Figure 2 | Expression patterns of MP, TMO5 and TMO7.
Specific questions The identification of a set of genes that is
directly controlled by the key regulatory transcription factor
MP, and the demonstration that TMO7 is a mobile transcription
factor, now allow asking a number of specific questions.
These are all being addressed in our research group, and will
be discussed below.
1. What is the mechanism of transport and action of the
mobile TMO7 transcription factor
TMO7 is a very small transcription factor, only 11 kDa in
size, and lacks the basic region of the bHLH domain that is
MP TMO5 TMO7
During the heart stage of embryogenesis, MP is expressed in a wide pattern
(left). While some the direct target gene TMO5 (middle) is expressed in a
similar pattern, TMO7 (right) is expressed in a small subdomain that corresponds
to the root stem cells. Schemes represent an interpretation of MP
protein and TMO5 and TMO7 mRNA accumulation patterns.

tivates one target gene in a broad pattern, while it activates
another in a very restricted domain. Our working hypothesis
is that MP assembles into different transcription complexes to
activate distinct sets of genes in different cells.
To determine MP transcription complexes, we have adapted
an immunoprecipitation and nLC-MS/MS procedure for isolating
nuclear proteins and their interacting proteins. We now
use this method to isolate MP-GFP protein from embryos and
identify its co-factors.
6 Smith, M. et al. (2009) Cyclophilin40 is required for miRNA activity
in Arabidopsis. Proc Natl Acad Sci USA 106, 5424-5429.
7 Hardtke C.S. et al. (2004) Overlapping and non-redundant
functions of the Arabidopsis auxin response factors
MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4.
Development 131, 1089-1100.
8 Remington D.L. et al. (2004) Contrasting modes of diversification
in the Aux/IAA and ARF gene families. Plant Physiol
135, 1738-1752.
A large gene family In our recent work we have defined a
cell-cell signaling process as being critical for the formation of
the first meristem in the plant embryo, and we have identified
an intercellular communication signal. Interestingly, MP is a
member of a large gene family of AUXIN RESPONSE FACTORs
(ARFs), transcription factors that control gene expression in
response to auxin. Because auxin is itself transported to the
hypophysis, we anticipate that other ARF transcription factors
act in the hypophysis and perhaps in other pattern formation
processes in the plant embryo. We are currently investigating
the developmental role of the other members of the ARF
family in plant embryo development, and hope to contribute
to understanding the molecular basis for the ability of auxin to
trigger many different responses in plant development.
Research team
1 2 3
4
5
6 7 8
References
1 Weijers, D. et al. (2006) Auxin triggers transient, local signalling
for cell specification in Arabidopsis embryogenesis.
Dev Cell 10, 265-270.
2 Schlereth, A.S et al. (2010). MONOPTEROS controls embryonic
root formation by regulating a mobile trancription
factor. Nature, Advance Online Publication doi: :10.1038/
nature08836.
3 Pillitteri, L.J. and Torii, K.U. (2007) Breaking the silence:
three bHLH proteins direct cell-fate decisions during stomatal
development. Bioessays 29, 861-870.
4 Massari, M. E. and Murre, C. (2000) Helix-loop-helix proteins:
regulators of transcription in eukaryotic organisms.
Mol Cell Biol 20, 429–440.
5 Michniewicz, M. et al. (2007) Antagonistic regulation of PIN
phosphorylation by PP2A and PINOID directs auxin flux. Cell
130, 1044-1056.
Summary
Plants make new organs throughout their lives due to the
activity of stem cells located in meristems. Our group is
interested in the problem of how stem cells and meristems are
initated in the young plant embryo. We have previously shown
that root meristem initiation in the plant embryo requires
cell-cell communication between adjacent embryonic and
extra-embryonic cells. Recently, through identifying direct tarsummary
Dolf Weijers (1), Barbara Möller (2), Bert de Rybel (3),
Eike Rademacher (4), Annemarie Lokerse (5), Cristina Llavata
Peris (6), Willy van den Berg (7), Giulinano Giuliani (8)
Contact
Dr. Dolf Weijers
Laboratory of Biochemistry
Wageningen University
Dreijenlaan 3
6703 HA Wageningen
the Netherlands
T +31 317 482 866
dolf.weijers@wur.nl
www.bic.wur.nl/UK/research/Plant+Development/
get genes of the critical transcription factor MONOPTEROS, we
have discovered a novel intercellular signaling molecule. This
molecule, the small transcription factor TMO7, is transported
from the embryonic to the extra-embryonic cells to initiate
the stem cell organizer. Here we discuss these findings and describe
our current and future work aimed at understanding the
cell-cell communication mechanism and the transcriptional
networks underlying the formation of a functional stem cell
population in the plant embryo.
| 25

Proteomics for the analysis of
cellular networks
Interview with keynote speakers
Increasingly more mathematical modelling and simulation is
used to explain and predict behaviour of cellular networks
under different circumstances. “Mathematical modelling
requires good quantitative data and these are not easy to
obtain from complex protein mixtures,” says Paola Picotti. She
therefore designed assays based on a targeted approach to
obtain quantitative data from the yeast proteome.
“Proteins are fascinating structures,” says Paola Picotti, PhD.
“It’s intriguing to find out how they fold and how they perform
their function.” For almost ten years Picotti has been involved
in studying proteins using different spectroscopic and mass
spectrometric techniques. “I started studying single proteins
and I am now involved in studying complete systems of interacting
molecules.” Picotti joined Prof. Ruedi Aebersold’s group
at ETH Zurich in 2006 as a post-doctoral fellow to learn more
about advanced mass spectrometric techniques. She focuses
on the development of targeted proteomic techniques based
on selected reaction monitoring.
26 | NPC Highlights 11 | April 2010
Targeted approach
Obtaining quantitative data from cellular networks, e.g. metabolic
or signalling networks, is difficult because the proteins
are present in very different concentrations and some of
them have a high sequence similarity. The classical method of
shotgun proteomics is not always efficient enough to monitor
all the proteins of interest. It means the digestion of protein
mixtures, separation of proteins by liquid chromatography and
identification of these proteins by tandem mass spectrometry.
Picotti and her colleagues therefore decided to try a targeted
approach. They created a list from all the known proteins
in the network under investigation. They selected proteotypic
peptides (PTPs), peptides that are unique to a protein
from each protein. They then developed highly sensitive and
quantitative MS assays for each PTP based on selected reaction
monitoring (SRM). “It means that you allow only specified
peptides to be measured, based on MS coordinates that you
have selected to be specific to the peptide,” explains Picotti.
The technique works well; proteins with abundances between
1.3 million and less than 50 copies per cell could be detected
in total yeast proteome digests, without the need for fractionation
or enrichment.
Synthetic peptides
The proof-of-principle of the SRM assay was delivered by the
analysis of a real network, yeast carbon metabolism. “It was
really nice to see that we were able to measure varying concentrations
of metabolic proteins within the entire network in
one hour. However the development and validation of all the
Paola Picotti (IMSB, ETH Zurich) is specialised in the field of spectroscopic
and mass spectrometric techniques. She gave a keynote lecture during the
NPC Progress Meeting 2010 in Utrecht on 16 February.
different SRM assays was tedious and very time-consuming,”
remarks Picotti. “Therefore we developed SRM assays on the
basis of crude synthetic proteotypic peptide libraries. Since
the composition of a synthetic library is known exactly and the
concentrations of the peptides are similar, the confidence in
the synthetic-peptide-based SRM assays is much higher. It
allows us to develop assays much faster than before: we
needed 8 hours for 150 different kinases and phosphatases.”
Picotti and her colleagues have now also developed assays for
the complete yeast proteome, a total of 6,500 assays (for all
the open reading frames). The assays will be put onto a public
website, the MRMAtlas (www.mrmatlas.org), so that anyone
can use them for their own applications. They can also be
“Proteins are fascinating structures.
It’s intriguing to find out how they fold and how
they perform their function”
used for trying to measure proteins so far undetected in different
conditions. About 2,000 proteins which have not been
detected yet are coded by the yeast genome.
Challenging
According to Picotti, it’s now time to have fun with SRM: “We
can focus more on the biological instead of the technological
development, which took a lot of time.”
The next project is the human proteome which is very challenging.
“It may be more difficult to find unique peptides and
the dynamic range of protein abundance will be bigger. We
will focus on the first 9,000 proteins already observed by other
researchers.”
Lilian Vermeer

NPC Progress Meeting 2010
Understanding neuropeptide
complexity in the brain
Unravelling the chemistry occurring in the brain that
influences behaviour is a topic that greatly intrigues Jonathan
Sweedler. It is part of his fascination with neuroscience and
more specifically, in understanding the functioning of the
brain. His research group develops and uses new analytical
tools for nanoscale study of neuronal networks to help
understand cell to cell signalling in the brain.
“Neuropeptides are essential molecules which play an important
role in behaviour, learning and memory,” says Jonathan
Sweedler, professor of chemistry at the University of Illinois,
USA. “However neuropeptides are hard to study because they
are heterogeneously distributed, contain a large number of
post-translational modifications, and are active over a wide
range of physiological concentrations. Furthermore they can
quickly degrade after cellular release.”
Fortunately today the sensitivity of detection methods has improved
in order of magnitude. Sweedler: “Whereas in the past
an ‘entire slaughter house’ was needed to isolate enough purified
peptide material to characterize it, today much smaller
quantities are sufficient.” Sweedler’s group has contributed to
the improvement of detection methods by developing many
new peptidomic approaches for assaying nanoliter volume
samples. They have also developed optimal preparation protocols
for small-volume samples. These methods enabled them
to characterize signalling molecules in samples ranging from
single cells to entire brain regions.
Well conserved
“The nice thing about neurotransmitters and neuromodulators
is that they are remarkably well conserved across many different
species,” says Sweedler. “Therefore we can use a range
of animal models to study their neuronal networks, choosing
the most appropriate animal depending on our research
question. We also use several models to develop and test new
techniques.” Consequently Sweedler’s lab is like a zoo: species
ranging from rats, mice, birds to honey bees and sea slugs are
their neuropeptide research models.
“Neuropeptides are essential molecules
which play an important role in
behaviour, learning and memory”
One of Sweedler’s ‘favourite’ animals is the sea slug, Aplysia
californica. “This animal is frequently used in neuroscience
because it has a simple nervous system, consisting of just a
few thousand large, easily-identified neurons. Moreover, this
animal is able to learn and remember what it learns.
Jonathan Sweedler (University of Illinois, USA) gave a keynote lecture
during the NPC Progress Meeting 2010, in Utrecht on 16 February.
Eric Kandel, with critical help from Aplysia, received a Nobel
Prize in 2000 for boosting our understanding of the biological
basis of learning and memory.” Sweedler’s group is involved in
the annotation of neuropeptides from Aplysia and they have
characterized hundreds of unique peptides from whole brains
to single cells in Aplysia.
Molecular mechanisms
To help understand the molecular mechanisms behind an
animal’s circadian rhythm, Sweedler’s group characterized
endogenous neuropeptides from a specific region of the rat
brain by mass spectrometry. This region, the suprachiasmatic
nucleus (SCN), is known to control circadian rhythm, the timekeeping
system responsible for our daily rhythms. In addition
to established circadian neuropeptides, they found peptides
with unknown roles. One of them, called little SAAS, induces a
phase delay in the circadian system after exogenous application.
Therefore, the MS-based discovery of a peptide has led
to new insights into how we maintain our circadian rhythm.
The Sweedler group identified hundreds of peptides in the
honey bee brain, and is now trying to determine which ones
are bioactive. By quantifying peptide level changes during
food selection and foraging, they found that some peptides
were clearly more abundant in the brains of bees after they
had collected either nectar or pollen. “Bees appear predisposed
to collect a specific type of food and this predisposition
is observable in their brain peptide signature,” says Sweedler.
Future
Sweedler realizes that advances in mass spectrometer instrumentation
enable the discovery of unexpected neuropeptides
at an increasing rate. “However this has also created many
new questions. So who knows what we will discover in the
future!”
Lilian Vermeer
| 27

Valorisation within the
Dear NPC Highlights readers,
Recently we introduced the NPC Valorisation Voucher as a new NPC instrument to enable
proof-of-concept studies for valorisation. There appears to be a great need for this funding,
as we have witnessed many applications upon going public. Two NPC Valorisation Vouchers
have now been granted by the board. Apart from the application from Huib Ovaa (NKI) the
board recently approved an application from Bobby Florea (Leiden University). Both awardees
will present their business plans elsewhere on these pages.
I would like to bring to your attention again that the NPC Valorisation Voucher is truly
meant for valorisation plans with a clear market potential. One could for example think of
beginning a start-up business or supporting the out-licensing of patent rights and obtaining
Bas Nagelkerken
the last proof-of-concept to close a deal. This implies that projects that are in a too early
NPC Valorisation Manager
stage cannot yet be eligible for funding from this source. If you have valorisation plans please
contact me to discuss your ideas. Together we can decide whether your plans are suitable for
submitting for a Voucher or if there might be other ways to promote your initiative.
I’m looking forward to receiving many of your interesting proposals this year!
Contact
All the best!
Netherlands Proteomics Centre
+31 30 253 4564
Bas Nagelkerken
nagelkerken@npc.genomics.nl
NPC Valorisation Manager
www.netherlandsproteomicscentre.nl
28 | NPC Highlights 11 | April 2010 | Valorisation Horizon Valorisation Grant
Life Sciences Pre-Seed Grant
The Life Sciences Pre-Seed Grant offers a great opportunity
for researchers associated with a Dutch university or research
institution. Worth up to € 250,000, the Pre-Seed Grant
offers superb prospects for those involved in applied research
and who are looking to exploit their fundamental research
commercially by starting up a new business. The Life Science
Pre-Seed grant can be positioned as a next step to a NPC
Valorisation Voucher. Deadline upcoming call: 5 October 2010.
More information: www.preseedgrant.nl
New Venture Business Plan
Competition
New Venture is aimed at anyone who wants to enterprise
with an innovative idea. An idea is innovative if it provides
satisfaction to a new or an existing need in a new way, and
if it clearly adds something compared to existing products,
services or technologies. Each round, participants can win
prize money with their idea, feasibility study or business plan.
These winnings rise to three prizes of € 25,000, in the third
round. Moreover, the national press devotes considerable
attention to the winners of New Venture. Each round ends
with a festive ceremony.
More information: www.newventure.nl
The Horizon Valorisation Project Grant is a new valorisation
tool available for researchers that have already been awarded
a Horizon Breakthrough project to further support valorisation
of their research results. The grant may be used to validate
research, or perform pre-clinical research or (further) clinical
research to improve the valorisation potential of the research
as performed in the Horizon Breakthrough project. The
maximum grant per Horizon Valorisation project is € 50,000.
The proposals may be submitted throughout the year, but within
three months after the ending of the corresponding Horizon
Breakthrough project. Before applying, the candidate should
contact NGI to receive a personal link to the ProjectNet system.
For detailed information on the programme, visit the websites
of ZonMw (www.zonmw.nl) or NGI (www.genomics.nl) and click
on the Horizon page.
Patent Workshop 2010
The Netherlands Genomics Initiative, the NL Patent Office
and the Programme Office IOP Genomics kindly invite you
to participate in the Patent Workshop 2010. This workshop
will discuss basic patent facts, as well as look at knowledge
protection from a genomics/Life Science point of view.
If you are a PhD student, postdoc or project leader from either
academia or a company wanting to know more about patents,
this is the workshop for you! The workshop takes place on
11 May 2010 at the Patent Office in Rijswijk and is free of charge.
For more information or registration, please contact
Ms. Anjali Raghoenath anjali.raghoenath@agentschapnl.nl

npc valorisation
Netherlands Proteomics Centre
Two NPC Valorisation Vouchers have now been granted
UbiQ: commercial development
of synthetic Nedd8 and SUMO
conjugates
The start-up company UbiQ originates from research
developed in the Chemical Biology group at the Netherlands
Cancer Institute. The UbiQ team is pleased to be the first
recipient of the Netherlands Proteomics Centre Valorisation
Voucher for their project ‘Commercial development of
synthetic Nedd8 and SUMO conjugates’.
The expertise of UbiQ is the development of research
tools and assay reagents based on Ubiquitin and Ubiquitinlike
proteins. These are small proteins that act as posttranslational
modifiers and are involved in regulating various
biologically important processes. To date, the development
of these types of reagents has been hampered by the
complexity of their biological modification. UbiQ has solved
this problem by developing a highly efficient chemical ligation
technology that gives access to these well-defined protein
conjugates. With this proprietary technology they have
developed reagents for profiling the substrate specificity of
deubiquitinating enzymes and the high-throughput screening
of deubiquitinating enzyme inhibitors.
With the help of the NPC Valorisation Voucher UbiQ will
develop (assay) reagents based on the Ubiquitin-like proteins
Chemical Proteomics Reagents
Chemical Proteomics Reagents (CPR) is an entrepreneurial
initiative from the Bio-organic synthesis group at the Leiden
Institute for Chemistry that received an NPC valorisation
voucher of 85,000 Euros. This grant gives the CPR team the
opportunity to perform a market feasibility study in the next
18 months.
CPR has profound expertise in design and synthesis of
activity-based probes (ABP) for functional proteomics studies.
These probes contain an enzyme reactive group, a targeting
sequence and a reporter group. The technique has successfully
been use for profiling the proteasome [1] and cathepsin
protein hydrolases [2] in vitro, in living cells and animals.
During the start-up period, CPR will focus on serving the
scientific community by marketing novel ABPs and improving
functional and chemical proteomics work flows for profiling
protein hydrolases. Interesting enzymatic activities such as
glucosidases and kinases are in the pipe-line.
CPR starts with a team of three: Bobby Florea as executive
officer for assay development, business administration,
marketing and sales, Rian van den Nieuwendijk as technology
officer for synthesis, Q&A, distribution and logistics and
Hermen Overkleeft for advisory, marketing and PR tasks.
The founders expect a turnover of 20-50 k€ in the first 2 years
of operation followed by robust growth rates provided by full
The UbiQ team (from left): Alfred Nijkerk (CEO), Farid El Oualid (COO) and
Huib Ovaa (CSO).
Nedd8 and SUMO. Since they are potentially new drug
targets, there has been an increasing demand for well-defined
Nedd8 and SUMO based (assay) reagents and a large market
potential. It is interesting to draw a comparison with research
into kinases, which has been one of the prime targets of
pharmaceutical industry over the past decade. For a long
time, this field has had the luxury of an array of techniques
based on the availability of phosphorylated peptides. UbiQ will
fill this commercial space for the Ubiquitin, Nedd8 and SUMO
research market.
Contact
Huib Ovaa & Farid El Oualid
+31 20 512 1979; h.ovaa@nki.nl
The CPR team consist of (from left): Bobby Florea, Hermen Overkleeft and
Rian van den Nieuwendijk
spectrum chemical proteomics services for preclinical assays,
target identification, validation and activity quantification.
References
1 Verdoes et al. (2006) A fluorescent broad-spectrum proteasome inhibitor for
labelling proteasomes in vitro and in vivo. Chem & Biol 13, 1217-26.
2 Hillaert et al. (2009) Receptor-mediated targeting of cathepsins in professional
antigen presenting cells. Angew Chem Int Ed Engl 48, 1629-32.
Contact
Bogdan Florea
+31 71 527 4355; b.florea@chem.leidenuniv.nl
| 29

Bioinformatics
High speed light path network
In 2009 SURFnet and NWO successfully organized the
‘Enlighten your research’ competition for the second
time, a competition on dynamic light paths. The goal
was to implement high speed and low latency response
time-dedicated internet connections in the existing
research infrastructure via optical fibres.
Some of the bottlenecks in mass spectrometry based proteomics
are the large datasets and ever increasing size of the
data files. This becomes even more apparent when these data
files need to be transferred from one cluster to the other,
requiring large amounts of time for data transfer (I/O) when
using the current network technologies. Other limitations are
the large numbers of small jobs, where low response time can
result in big delays in processing time. Optical fibres allow
for high speed connections (currently 10 gigabit per second)
and quick response times and therefore would be ideal in this
setup. With the ‘Enlighten your research’ grant it is now possible
to use the so-called dynamic light paths in this network.
Dynamic light paths are not fixed connections between one or
more clusters nodes, but can however be set up ‘on demand’
to provide exclusive fast connections with quick response
30 | NPC Highlights 11 | April 2010 | Bioinformatics
NPC theme leaders Péter Horvatovitch (University of Groningen) and Bas van
Breukelen (Utrecht University) were together one of the three winners of the
‘Enlighten your research competition’. They received the award from Minister
Plasterk at the prize giving ceremony, which was part of the launch event for
GigaPort 3 on February 18, 2010. They also received free access to the light
path network and e 20,000 to integrate light paths in their research.
and tested with the developed tools. At the moment the bioinformatics
group in Groningen, under the supervision of Peter
Horvatovich, are implementing the first high-throughput data
processing services for time alignment of multiple LC-MS chromatograms
using the large computation power of the Dutch
Life Science Cluster (SARA) and the fast connection provided
by the dynamic light paths.
With the help of the dynamic light paths and the Dutch Life
Science Grid we hope and expect to provide high-throughput
and stable data processing services to the Dutch proteomics
community, based on the tools developed in different Dutch
and international bioinformatics laboratories.
times not shared with other users. This in turn allows for an
efficient usage of the available computational and storage
resources where it is most needed.
Implementation
In the coming months the light path connections will be implemented
between five clusters of the Dutch Life Science Grid
located at the Universities of Groningen, Utrecht, Wageningen
and Amsterdam and the Erasmus Medical Centre in Rotterdam
ErasmusMC grid cluster
and data storage
UvA grid cluster
and data storage
Lightpath
Lightpaths
UU grid cluster
and data storage
Lightpath
Lightpaths
RUG grid cluster
and data storage
Lightpath
Lightpath
WUR grid cluster
and data storage
Contact
Dr. Ir. Bas van Breukelen
Biomolecular Mass Spectrometry and Proteomics Group
T +31 30 253 9761
M +31 6 422 225 20
b.vanbreukelen@uu.nl
Task force
The Netherlands Proteomics Centre (NPC) and the Netherlands Bioinformatics
Centre (NBIC) have joined forces in setting up a large task force in
the Gaining Momentum Theme. This task force aims to build a platform
for proteomics based on bioinformatics and to provide tools, workflows
and high-throughput data processing services for the proteomics community.
Several groups throughout the Netherlands contribute to this platform by
creating new (bioinformatics) tools as well as adapting existing tools so
that they can be used as workflow management tools. The idea here is
that all the tools are developed so that they can make use of existing data
standards and be coupled to each other without the need to develop a
plethora of data conversion algorithms. Moreover all the tools will be able
to run as a ‘web service’ which enables other bioinformaticians to couple
these tools without needing to run them locally.
One important part of this platform is the infrastructure which is provided
by NBIC. All the major universities and medical centres have a server (a
cluster node) with multiple CPU’s and storage capacity to host the web
services and which form a grid (BIG GRID and SARA) together.
Impression of the cluster network and light path connections

Top publications with NPC contribution
In this NPC HighLights we provide a short list of
papers that appeared recently in some of the top
journals and to which NPC participants contributed.
With the guarantee of being by far not comprehensive
, this overview shows some elegant
ground-breaking research.
MONOPTEROS controls embryonic root
initiation by regulating a mobile transcription
factor
Schlereth, A., Moller, B., Liu, W., Kientz, M., Flipse, J.,
Rademacher, E.H., Schmid, M., Juergens, G., Weijers D.
Nature (2010) Mar 10
Entwicklungsgenetik, Zentrum für Molekularbiologie der
Pflanzen (ZMBP), Universität Tübingen, Auf der Morgenstelle
3, 72076 Tübingen, Germany. Present address: Syngenta Crop
Protection, CH-4332 Stein, Switzerland.
Acquisition of cell identity in plants relies strongly on
PMID: 20220754
positional information, hence cell–cell communication and
inductive signalling are instrumental for developmental
patterning. During Arabidopsis embryogenesis, an extra-embryonic
cell is specified to become the founder cell of the
primary root meristem, hypophysis, in response to signals from
adjacent embryonic cells. The auxin-dependent transcription
factorMONOPTEROS (MP) drives hypophysis specification by
promoting transport of the hormone auxin from the embryo
to the hypophysis precursor. However, auxin accumulation
is not sufficient for hypophysis specification, indicating that
additional MP-dependent signals are required3. Here we
describe the microarray-based isolation ofMPtarget genes
that mediate signalling fromembryo to hypophysis. Of three
direct transcriptional target genes, TARGET OFMP5 (TMO5)
andTMO7 encode basic helix–loop–helix (bHLH) transcription
factors that are expressed in the hypophysis-adjacent embryo
cells, and are required and partially sufficient for MP-dependent
root initiation. Importantly, the small TMO7 transcription
factor moves from its site of synthesis in the embryo to the
hypophysis precursor, thus representing a novel MP-dependent
intercellular signal in embryonic root specification.
Histone chaperones ASF1 and NAP1
differentially modulate removal of active
histone marks by LID-RPD3 complexes during
NOTCH silencing
Moshkin, Y.M., Kan, T.W., Goodfellow, H., Bezstarosti, K.,
Maeda, R.K., Pilyugin, M., Karch, F., Bray,S.J., Demmers, J.A.,
Verrijzer, C.P.
Mol Cell (2009) Sep 24;35(6):782-93.
Department of Biochemistry, Center for Biomedical Genetics,
Erasmus University Medical Center, P.O.
Box 1738, 3000 DR Rotterdam, The Netherlands
PMID: 19782028
Histone chaperones are involved in a variety of chromatin
transactions. By a proteomics survey, we identified the interaction
networks of histone chaperones ASF1, CAF1, HIRA, and
NAP1. Here, we analyzed the cooperation of H3/H4 chaperone
ASF1 and H2A/H2B chaperone NAP1 with two closely related
silencing complexes: LAF and RLAF. NAP1 binds RPD3 and
LID-associated factors (RLAF) comprising histone deacetylase
RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1,
EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking
RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to
the DNA-binding Su(H)/Hairless complex, which targets the
E(spl) NOTCH-regulated genes. ASF1 facilitates gene-selective
removal of the H3K4me3 mark by LAF but has no effect on
H3 deacetylation. NAP1 directs high nucleosome density near
E(spl) control elements and mediates both H3 deacetylation
and H3K4me3 demethylation by RLAF. We conclude that histone
chaperones ASF1 and NAP1 differentially modulate local
chromatin structure during gene-selective silencing.
Recombination-induced tag exchange to track
old and new proteins
Verzijlbergen, K.F., Menendez-Benito, V., van Welsem, T., van
Deventer, S.J., Lindstrom, D.L., Ovaa, H., Neefjes, J., Gottschling,
D.E., van Leeuwen, F.
Proc Natl Acad Sci U S A (2010) Jan 5;107(1):64-8.
Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX
Amsterdam, The Netherlands.
PMID: 20018668
The dynamic behavior of proteins is critical for cellular
homeostasis. However, analyzing dynamics of proteins and
protein complexes in vivo has been difficult. Here we
describe recombination-induced tag exchange (RITE), a
genetic method that induces a permanent epitope-tag switch
in the coding sequence after a hormone-induced activation
of Cre recombinase. The time-controlled tag switch provides
a unique ability to detect and separate old and new proteins
in time and space, which opens up opportunities to investigate
the dynamic behavior of proteins. We validated the
technology by determining exchange of endogenous histones
in chromatin by biochemical methods and by visualizing and
quantifying replacement of old by new proteasomes in single
cells by microscopy. RITE is widely applicable and allows
probing spatiotemporal changes in protein properties by
multiple methods.
| 31

Nucleotide excision repair-induced H2A ubiquitination
is dependent on MDC1 and RNF8
and reveals a universal DNA damage response
Marteijn, J.A., Bekker-Jensen, S., Mailand, N., Lans, H.,
Schwertman, P., Gourdin, A.M., Dantuma, N.P., Lukas, J.,
32 | NPC Highlights 11 | April 2010 | Abstracts
Vermeulen, W.
J Cell Biol (2009) Sep 21;186(6):835-47.
Department of Genetics, Center for Biomedical Genetics,
Erasmus Medical Center, 3015 GE Rotterdam, Netherlands.
PMID: 19797077
Chromatin modifications are an important component of the of
DNA damage response (DDR) network that safeguard genomic
integrity. Recently, we demonstrated nucleotide excision
repair (NER)-dependent histone H2A ubiquitination at sites
of ultraviolet (UV)-induced DNA damage. In this study, we
show a sustained H2A ubiquitination at damaged DNA, which
requires dynamic ubiquitination by Ubc13 and RNF8. Depletion
of these enzymes causes UV hypersensitivity without affecting
NER, which is indicative of a function for Ubc13 and RNF8 in
the downstream UV-DDR. RNF8 is targeted to damaged DNA
through an interaction with the double-strand break (DSB)-DDR
scaffold protein MDC1, establishing a novel function for MDC1.
RNF8 is recruited to sites of UV damage in a cell cycle-independent
fashion that requires NER-generated, single-stranded
repair intermediates and ataxia telangiectasia-mutated and
Rad3-related protein. Our results reveal a conserved pathway
of DNA damage-induced H2A ubiquitination for both DSBs and
UV lesions, including the recruitment of 53BP1 and Brca1.
Although both lesions are processed by independent repair
pathways and trigger signaling responses by distinct kinases,
they eventually generate the same epigenetic mark, possibly
functioning in DNA damage signal amplification.
Accumulation of ubiquitin conjugates in
a polyglutamine disease model occurs
without global ubiquitin-proteasome system
impairment
Maynard, C.J., Böttcher, C., Ortega, Z., Smith, R., Florea, B.I.,
Díaz-Hernández, M., Brundin, P., Overkleeft, H.S., Li, J.,
Lucas, J.J., Dantuma, N.P.
Proc Natl Acad Sci U S A (2009) Aug 18;106(33):13986-91.
Department of Cell and Molecular Biology, Karolinska
Institutet, S-17177 Stockholm, Sweden
PMID: 19666572
Aggregation-prone proteins have been suggested to overwhelm
and impair the ubiquitin/proteasome system (UPS) in polyglutamine
(polyQ) disorders, such as Huntington’s disease (HD).
Overexpression of an N-terminal fragment of mutant huntingtin
(N-mutHtt), an aggregation-prone polyQ protein responsible for
HD, obstructs the UPS in cellular models. Furthermore, based on
the accumulation of polyubiquitin conjugates in brains of R6/2
mice, which express human N-mutHtt and are one of the most
severe polyQ disorder models, it has been proposed that UPS
dysfunction is a consistent feature of this pathology, occurring in
both in vitro and in vivo models. Here, we have exploited transgenic
mice that ubiquitously express a ubiquitin fusion degradation
proteasome substrate to directly assess the functionality of
the UPS in R6/2 mice or the slower onset R6/1 mice. Although
expression of N-mutHtt caused a general inhibition of the UPS in
PC12 cells, we did not observe an increase in the levels of proteasome
reporter substrate in the brains of R6/2 and R6/1 mice.
We show that the increase in ubiquitin conjugates in R6/2 mice
can be primarily attributed to an accumulation of large ubiquitin
conjugates that are different from the conjugates observed upon
UPS inhibition. Together our data show that polyubiquitylated
proteins accumulate in R6/2 brain despite a largely operative
UPS, and suggest that neurons are able to avoid or compensate
for the inhibitory effects of N-mutHtt.
A comprehensive framework of E2-RING
E3 interactions of the human ubiquitinproteasome
system
van Wijk, S.J., de Vries, S.J., Kemmeren, P., Huang, A., Boelens, R.,
Bonvin, A.M., Timmers, H.T.M.
Mol Syst Biol (2009) 5:295.
Division of Biomedical Genetics, Department of Physiological
Chemistry, University Medical Center Utrecht, Utrecht, The
Netherlands.
PMID: 19690564
Covalent attachment of ubiquitin to substrates is crucial to
protein degradation, transcription regulation and cell signalling.
Highly specific interactions between ubiquitin-conjugating
enzymes (E2) and ubiquitin protein E3 ligases fulfil essential roles
in this process. We performed a global yeast-two hybrid screen to
study the specificity of interactions between catalytic domains of
the 35 human E2s with 250 RING-type E3s. Our analysis showed
over 300 high-quality interactions, uncovering a large fraction of
new E2-E3 pairs. Both within the E2 and the E3 cohorts, several
members were identified that are more versatile in their interaction
behaviour than others. We also found that the physical
interactions of our screen compare well with reported functional
E2-E3 pairs in in vitro ubiquitination experiments. For validation
we confirmed the interaction of several versatile E2s with E3s
in in vitro protein interaction assays and we used mutagenesis
to alter the E3 interactions of the E2 specific for K63 linkages,
UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B). Our
data provide a detailed, genome-wide overview of binary E2-E3
interactions of the human ubiquitination system.
Other highlighted publications
Muñoz, J., Heck, A.J.
Snapshots of kinase activities
Nat Biotechnol (2009) Oct;27(10):912-3.
PMID: 19816446

abstracts
npc
Albers, H.M.G., Dong, A., van Meeteren, L.A., Egan, D.A.,
Sunkara, M., van Tilburg, E.W., Schuurman, K., van Tellingen,
O., Morris, A.J., Smyth, S.S., Moolenaar, W.H., Ovaa, H.
A boronic acid-based inhibitor of autotaxin reveals rapid
turnover of LPA in the circulation
Proc Natl Acad Sci U S A (2010) Apr 1
Wiederhold, E., Veenhoff, L.M., Poolman, B., Slotboom, D.J.
Proteomics of Saccharomyces cerevisiae organelles
Mol Cell Proteomics (2010) Mar;9(3):431-45.
PMID: 19955081
Mahmoudi, T., Li, V.S., Ng, S.S., Taouatas, N., Vries, R.G.,
Mohammed, S., Heck, A.J., Clevers H.
The kinase TNIK is an essential activator of Wnt target genes
EMBO J (2009) 28; 3329-3340
PMID: 19816403
Witte, M.D., Florea, B.I., Verdoes, M., Adeyanju, O., van der
Marel, G.A., Overkleeft, H.S.
O-GlcNAc peptide epoxyketones are recognized by mammalian
proteasomes
J Am Chem Soc (2009) Sep 2;131(34):12064-5.
PMID: 19658393
Ramadurai, S., Holt, A., Krasnikov, V., van den Bogaart, G.,
Killian, J.A., Poolman, B.
Lateral diffusion of membrane proteins
J Am Chem Soc (2009) Sep 9;131(35):12650-6.
PMID: 19673517
van Wijk S.J., Timmers H.T.M.
The family of ubiquitin-conjugating enzymes (E2s): deciding
between life and death of proteins
FASEB J (2009); Apr;24(4):981-93.
PMID: 19940261
Frederiks, F., Heynen, G.J., van Deventer, S.J., Janssen, H.,
van Leeuwen, F.
Two Dot1 isoforms in Saccharomyces cerevisiae as a result of
leaky scanning by the ribosome
Nucleic Acids Res (2009)37(21):7047-58.
PMID: 19778927
Schaaij-Visser, T.B., Graveland, A.P., Gauci, S., Braakhuis,
B.J., Buijze, M., Heck, A.J., Kuik, D.J., Bloemena, E.,
Leemans, C.R., Slijper, M., Brakenhoff, R.H.
Differential Proteomics Identifies Protein Biomarkers That
Predict Local Relapse of Head and Neck Squamous Cell
Carcinomas
Clin Cancer Res (2009) Dec 15;15(24):7666-7675.
PMID: 19996216
Ng, S.S., Mahmoudi, T., Danenberg, E., Bejaoui, I., de Lau, W.,
Korswagen, H.C., Schutte, M., Clevers, H.
Phosphatidylinositol 3-kinase (PI3K) signaling does not activate
the Wnt cascade
J Biol Chem (2009) 284: 35308-35313
PMID: 19850932
Van Hoof, D., Dormeyer, W., Braam, S.R., Passier, R.,
Monshouwer-Kloots, J., Ward-van Oostwaard, D., Heck, A.J.,
Krijgsveld, J., Mummery, C.L.
Identification of cell surface proteins for antibody-based
selection of human embryonic stem cell-derived cardiomyocytes
J Proteome Res (2010) Mar 5; 9 (3):1610-8
PMID: 20088484
Christin, C., Hoefsloot, H.C., Smilde, A.K., Suits, F., Bischoff,
R., Horvatovich, P.L.
Time Alignment Algorithms Based on Selected Mass Traces for
Complex LC-MS Data
J Proteome Res (2010) Mar 5; 9 (3):1483-95
PMID: 20070124
Mischerikow, N., Spedale, G., Altelaar, M., Timmers, H.T.M.,
Pijnappel, P.W.W.M., Heck, A.J.R.
In-depth profiling of post-translational modifications on the
related transcription factor complexes TFIID and SAGA
J.Proteome Res. (2009)11:5020-5030
PMID: 19731963
Raijmakers, R., Kraiczek, K., de Jong, A., Mohammed, S.,
Heck, A.J.
Exploring the human leukocyte phosphoproteome using a
microfluidic RP-TiO2-RP HPLC Phosphochip coupled to a Q-ToF
mass spectrometer
Anal. Chem. (2010) Feb 1;82(3):824-32
Schaaij-Visser, T.B., Brakenhoff, R.H., Jansen, J.W.,
O’Flaherty, M.C., Smeets, S.J., Heck, A.J., Slijper, M.
Comparative proteome analysis to explore p53 pathway
disruption in head and neck carcinogenesis
J Proteomics. (2009) Jul 21;72(5):803-14.
PMID: 19446051
van Breukelen, B., Georgiou, A., Drugan, M., Taouatas, N.,
Mohammed, S., Heck, A.J.
LysNDeNovo: An algorithm enabling de novo sequencing of Lys-N
generated peptides fragmented by electron transfer dissociation
Proteomics (2010) Mar;10(6):1196-201
PMID: 20077410
Helbig, A.O., de Groot, M.J., van Gestel, R.A., Mohammed, S.,
de Hulster, E.A., Luttik, MA, Daran-Lapujade P, Pronk JT,
Heck AJ, Slijper M.
A three-way proteomics strategy allows differential analysis
of yeast mitochondrial membrane protein complexes under
anaerobic and aerobic conditions
Proteomics (2009) Oct;9(20):4787-98.
PMID: 19750512
Jaworski, J. Kapitein, L.C., Gouveia, S.M., Dortland, B.R.,
Wulf, P.S., Grigoriev, I., Camera, P., Spangler, S.A.,
Di Stefano, P., Demmers, J., Krugers, H., Defilippi, P.,
Akhmanova, A., Hoogenraad, C.C.
Dynamic microtubules regulate dendritic spine morphology
and synaptic plasticity
Neuron (2009) Jan 15;61(1):85-100
PMID: 19146815
| 33

NPC Research Hotels
The NPC offers the newest proteomics technologies and facilities in specialised Research Hotels for joint projects with external
scientists. This overview gives you an impression of our expertise and facilities. If you have any specific questions about our
technologies you can directly contact the respective Hotel Manager. If you have more general questions about our Research
Hotels and the conditions for joint projects, please contact the NPC office at info@npc.genomics.nl.
Cell Sorting Hotel Utrecht
Utrecht University
Many protein complexes differ between cell types. The Cell
Sorting Hotel Utrecht offers equipment and expertise to
isolate specific cell types from plant tissues. The goal is to
extract proteins from sorted cells and subject them to
proteomic analysis. Cell-specific proteomics is a technical
goal in itself and as soon as this procedure has been optimized
for Arabidopsis all plant NPCII groups will have access
to the facility using Arabidopsis, tomato and potato cells.
Hotel Manager
Prof. dr. Ben J.G. Scheres
University Utrecht
Department Biology/Molecular Genetics
Padualaan 8
3584 CH Utrecht
T: +31 30 253 3133
E: b.scheres@uu.nl
34 | NPC Highlights 11 | April 2010
Analytical Hotel Delft
Delft University of Technology
Analytical Hotel Delft provides up to date Peptidomics in all
of its aspects: both qualitative and quantitative analysis of
the subset of the proteome designated as the peptidome;
naturally occurring (native, endogenous) peptides with
important biological (regulatory, signal, (neuro)hormonal)
functions. Focus will be on various peptide chemical
identification strategies, including de novo
sequencing, peptide bioinformatics, the
study of bioactive peptide location, as well
as their (physiological/pharmacological)
activities.
Hotel Managers
Dr. Martijn Pinkse &
Prof. dr. Peter Verhaert
Delft University of Technology
Kluyver Laboratory
Julianalaan 67
2628 BC Delft
T: +31 15 278 2344
E: m.w.h.pinkse@tudelft.nl
Analytical Hotel Utrecht
Utrecht University
The main task of the Utrecht Analytical Hotel is to provide
an expert centre for state of the art proteomics analysis.
In the Hotel, innovative mass spectrometric methods are
developed and implemented for the efficient and detailed
characterization of biomolecules in their relation to their
biological function. The scientific focus ranges from single
protein identifications to the analysis of complex, whole
cell lysate samples and determination of posttranslational
modifications.
Analytical Hotel Rotterdam
Erasmus MC
The Erasmus MC Research Hotel is embedded within the
Erasmus MC Proteomics Center (part of the Department of
Biochemistry) and offers proteomic and mass spectrometric
services for the Dutch research academic community as well
as for the local scientific community. Projects undertaken
will be on a collaborative basis and consultation regarding
study design; data interpretation is provided by people
from the Research Hotel. The Research Hotel develops mass
spectrometry based proteomics methodologies and protein
separation techniques for the qualitative and quantitative
analysis of (sub) proteomes.
Hotel Managers
Dr. Maarten Altelaar &
Prof. dr. Albert Heck
Utrecht University
Biomolecular Mass Spectrometry
and Proteomics Group
Padualaan 8,
3584 CH Utrecht
T: +31 30 253 9554
E: m.altelaar@uu.nl
Hotel Manager
Dr. Jeroen Demmers
Proteomics Center, Erasmus MC
Room Ee679
Dr. Molenwaterplein 50
3015 GE Rotterdam
T: +31 10 703 8124
E: j.demmers@erasmusmc.nl

Analytical Hotel Wageningen
Plant Research International/
Wageningen University
Analytical Hotel Groningen
University of Groningen
hotel managers
The Wageningen Research Hotel is strongly linked to activities
within the Centre for Biosystems Genomics (CBSG),
which aims to decipher the genetic basis of plant specific
processes and to improve plant performance and product
quality. Traits of interest from both scientific and applied
point of view are: disease resistance against pathogens;
metabolic pathways that contribute to flavour production;
consumer-driven qualities of food products (e.g. tomato)
and developmental aspects of plant growth. The NPCII
plant projects are focused on plant development and signal
transduction in plant meristems. Proteomics technologies
will be used in the Hotel to perform protein
profiling for comparative proteomics and to
unravel signalling pathways and the involved
protein complexes.
Hotel Managers
Dr. Twan America &
Prof. dr. Gerco Angenent
Wageningen UR
Plant Research International
Droevendaalsesteeg 1
6708 PB Wageningen
T: +31 317 480 953
E: Angenent@wur.nl
Bioinformatics Hotel Utrecht
Utrecht University
The Bioinformatics Hotel Utrecht mainly focusses on the
development of bioinformatics software and algorithms that
facilitate proteomics research. This can be subdivided in three
research lines.
1. (Raw) data (pre) processing: development and implementation
of algorithms for noise filtering, FDR calculations,
database search software and de novo sequence algorithms.
2. Data storage and dissemination: storage of raw and
processed data into public and local repositories (or LIMS
systems).
3. Knowledge extraction: extract biological information from
pre-processed proteomics data.
Hotel Manager
Dr. Bas van Breukelen
Utrecht University
Biomolecular Mass Spectrometry and
Proteomics Group
Padualaan 8
3584 CH Utrecht
T: +31 30 253 9761
E: b.vanbreukelen@uu.nl
The Analytical Hotel Groningen provides expertise and assistance
in experiments requiring the purification, separation
and high resolution analysis of proteins employing
proteomics tools as well as biochemical and biophysical techniques.
These include membrane and organelle isolation,
ultracentrifugation (analytical and preparative), gel- and
solution (chromatography)-based separation of proteins and
peptides (1- and 2-dimensional) coupled to mass spectrometry.
The Hotel offers collaboration and knowledge transfer
to address questions regarding MS-based identification of
proteins in highly complex mixtures and the biophysical
characterization of individual membrane
proteins, protein complexes and their modifications.
Hotel Managers
Dr. Fabrizia Fusetti &
Prof. dr. Bert Poolman
University of Groningen
Groningen Biomolecular Sciences and
Biotechnology Institute & Centre for
Synthetic Biology
Nijenborgh 4
9747 AH Groningen
T: +31 50 363 4190
E: b.poolman@rug.nl
Cell Culture Hotel Utrecht
UMC Utrecht
The scientific focus of the Cell Culture Hotel Utrecht is to
provide key technologies for targeted proteomic analyses of
embryonic stem cells (ESCs), induced pluripotent stem cells
(iPS) and differentiated cells. This includes selective tagging
of proteins using bacterial artificial chromosomes (BACs) and
viral-mediated gene manipulation. The Hotel offers a variety
of different epitopes for efficient tagging and purification of
target proteins expressed at endogenous levels. This enables
isolation of protein complexes for proteomic
and genomic analyses.
Hotel Managers
Dr. Pim Pijnappel &
Prof. dr. Marc Timmers
University Medical Centre Utrecht
Department of Physiological Chemistry
Division Biomedical Genetics
Universiteitsweg 100
3584 CG Utrecht
T: +31 88 756 8981
E: h.t.m.timmers@umcutrecht.nl
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upcoming events
23-27 May 2010
27 June - 1 July
19-24 September 2010
23-27 October 2010
23 November 2010
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ASMS, Salt Lake City, U.S.A.
Metabolomics 2010, Amsterdam, The Netherlands
9th HUPO World Congress, Sydney, Australia
EuPa, Estoril, Portugal
NGI Life Sciences Momentum, Utrecht, The Netherlands
Combining the best
existing forces
Only innovation on a European level will allow us to keep pace
in the world economy. Are we innovative enough No. Do we
have the basis Yes. We do have excellent research, education
and business centres but knowledge and ideas sometimes still
tend to become clogged up in dead ends. Here is where we
clearly need to improve. Four components to achieve this goal
are people, focus, leadership and entrepreneurship.
Dr. Martin Schuurmans
Chairman - European
Institute of Innovation and
Technology (EIT)
http://eit.europa.eu
People, because they are the ones carrying the knowledge
and drive ideas forward. We need more creative and
entrepreneurial people. Focus and leadership are essential,
because efforts and resources must be effectively
and efficiently concentrated on clear goals. Finally,
entrepreneurship is needed to achieve the goals in terms of
new products, services, business and job creation.
© Photo EIT
With the European Institute of Innovation and Technology
(EIT) we contribute to a structural change in the innovation
ecosystem, based on the full integration of the knowledge
triangle of higher education, research and business/innovation.
To unleash Europe’s innovation potential, we will break up
borders between academia and business, between teaching
and research. At the same time, successful integration requires
excellent partners. We need to continue to strengthen our
capacities and invest more in R&D and higher education.
In particular higher education is crucial because we need
people with the right multi-disciplinary skills and an open,
entrepreneurial mindset driving innovation processes.
The EIT’s Knowledge and Innovation Communities (KICs) are
test beds for this new kind of collaborative, entrepreneurial
approach. While building on strong existing partners and
cooperations, the KICs will have a totally new quality in the way
the three sides of the triangle interact. People in the triangle
will work together face-to-face in integrated teams in a number
of co-location innovation centres in the KIC. They will operate
towards explicit goals and under clear leadership of the KIC,
exploiting every avenue of entrepreneurship. Working together
across co-location centres will also contribute to healthier
brain motion over Europe. For now, the three first KICs focus on
climate change adaptation and mitigation, sustainable energy,
and the future information and communication society. Others
may follow in the future, for example in the field of health.
Maybe proteomics will contribute as well!
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