PCT News Discovery Starts With Sample Preparation - Pressure ...

P C T N e w s
Discovery Starts With Sample Preparation TM
Volume 3, Issue 6 June 2009
Pressure BioSciences, Inc. Launches Its
PCT MicroTube Adapter Kit for Pressure-Enhanced Enzymatic Proteolysis
at the American Society for Mass Spectrometry (ASMS) Annual Meeting
with Supporting Data from Key Investigators
Pressure BioSciences, Inc. (PBIO)
Chief Executive Officer Rang
The NASDAQ Stock Market Opening Bell
What:
Pressure BioSciences visited the NASDAQ MarketSite in
New York City’s Times Square on June 1 st to celebrate the
release of their next generation PCT-based sample
preparation product, the PCT MicroTube Adapter Kit. In
honor of the occasion, Richard T. Schumacher, President
and Chief Executive Officer of Pressure BioSciences, Inc.
[PBIO], rang The NASDAQ Stock Market Opening Bell.
Why:
The PCT MicroTube Adapter Kit was launched at the 57th
Annual Conference of the American Society for Mass
Spectrometry (ASMS) in Philadelphia, on May 31 st through
June 4th, 2009. Pressure Cycling Technology (PCT) uses
cycles of hydrostatic pressure between ambient and ultrahigh
levels to control bio-molecular interactions.
PBI currently focuses its efforts in the development and
sale of PCT-enhanced enzymatic digestion products
designed specifically for the mass spectrometry
marketplace, as well as sample preparation products for
biomarker discovery, soil and plant biology, forensics,
histology, and counter-bioterror applications. The biological
sample preparation market is estimated by the Emmes
Group (2008) to be comprised of up to 80,000 laboratories
and 500,000 researchers worldwide. Since 2007, PBI has
installed over 75 PCT Sample Preparation Systems into
such sites as the NIH, FDA, USDA, Harvard Medical
School’s Harvard Catalyst Laboratory for Innovative
Translational Technologies (“HC LITT”), NYU School of
Medicine, Amgen, Lilly, Novartis, Pacific Northwest
National Labs, and the J. Craig Venter Institute.
C A L E N D A R O F E V E N T S
2009 APS ANNUAL
MEETING
8TH HUPO
WORLD CONGRESS
Portland, Oregon Toronto, Canada
August 1-5, 2009 Sept. 26th-30th, 2009
Pressure-Enhanced Enzymatic Proteolysis
Presentations at the 2009 ASMS
Ultra-Rapid Pressure Digestion and Label-Free Quantitative
Proteomics of Yersiniae Infected Mice Tissues
Kim K. Hixson 1 ; Daniel Lopez Ferrer 1 ; Matthew Bender 2 ; Patricia L.
Worsham 3 ; Karl K. Weitz 1 ; Nate Lawrence 4 ; Amy Rasley 5 ; Therese W.
Clauss 1 ; Ljiljana Pasa-tolic 1 ; Richard D. Smith 1 ; Mary S. Lipton 1
1 Pacific Northwest National Lab, Richland, WA; 2 NBACC, Washington,
DC; 3 USAMRIID, Frederick, MD; 4 Pressure Biosciences, Inc., South
Easton, MA; 5 Lawrence Livermore National Lab, Livermore, CA
Improved Protein Coverage and Throughput in Proteomics Using
On-Line Multiplexed Enzyme Digestions and Targeted MS/MS with
a Modified LTQ-FTICR
Daniel Lopez Ferrer 1 ; Konstantinos Petritis 1 ; Andrei Liyu 1 ; Yufeng
Shen 1 ; Benito Canas 2 ; Kim K. Hixson 1 ; Richard D. Smith 1 ;
Mikhail Belov 1 PNNL / Battelle Northwest, Richland , WA; 2 Universidad
Complutense de Madrid, Madrid, Spain
High-pressure assisted in-gel tryptic digest: qualitative and
quantitative aspects
Michail Alterman 1 ; Melkamu Getie-Kebtie 1 ; Alexander Lazarev 2 ;
Vera S. Gross 2 1 FDA, CBER, Rockville, MD; 2 Pressure Biosciences, Inc,
Woburn, MA;
Proteomics under pressure: Rapid extraction and digestion in a
single tube
Alexander V. Lazarev 1 ; Emily Freeman 2 ; Vera S. Gross 1 ; Greta
Carlson 1 ; Edmund Ting 1 ; Alexander R. Ivanov 2
1Pressure BioSciences, South Easton, MA ; 2Harvard School of Public
Health, Boston, MA
Pressure-Enhanced Enzymatic Proteolysis
J. Behnke, G. Carlson, J. Damasio, C. Dussault, W. Fritzsche,
V. Gross, J. Lanuza, N. Lawrence, A. Lazarev, L. Manning, R.
McCloskey, M. Potter, C. Saravis, R. T. Schumacher,
G. Smejkal, M. Thomson, and E. Ting. Pressure BioSciences, South
Easton, MA
Other PCT Related Presentations
at the
2009 ASMS
Top-Down Indentification of Bacterial Intact Protein Expression
Profile Markers
Melinda A. McFarland; John H. Callahan; Denis Andrzejewski;
Rebecca Bell; Steven M. Musser, FDA/CFSAN, College Park, MD
Discovery of Mitochondrial Protein Biomarkers
of Atrial Fibrillation Using Unique Human Tissue Samples
Maryam Goudarzi1, Mark M. Ross1, Weidong Zhou1, Amy Van Meter1,
Emanuel Petricoin1, Lance Liotta1, Lisa Martin2, Chidima Martin2 and
Niv Ad2 1George Mason University, Manassas, VA; 2Inova Heart &
Vascular Institute, Falls Church, VA
www.pressurebiosciences.com

P C T N e w s
Discovery Starts With Sample Preparation TM
Volume 3, Issue 6 June 2009
Excerpt from PBI Poster
Presented at the 2009 ASMS
Proteomics Under Pressure: rapid
extraction and digestion in a single tube
ALEXANDER V. LAZAREV1; Emily Freeman2; Vera S.
Gross1; Greta Carlson1; Edmund Ting1; Alexander R.
Ivanov2. 1Pressure BioSciences, South Easton, MA ;
2Harvard School of Public Health, Boston, MA
Introduction
Hydrostatic pressure has been previously shown to enhance
enzymatic hydrolysis by trypsin [1, 2], chymotrypsin and
pepsin [3, 4], as well as by Alcalase, Neutrase, Corolase 7089,
Corolase PN-L, and papain [5, 6]. In our preliminary
experiments, we have confirmed the positive effects of
pressure and additional benefits of alternating hydrostatic
pressure (pressure cycling technology, or PCT) for several
enzymes including proteinase K, PNGase F, Lys-C and
lysozyme. However, the mechanisms of pressure-induced
acceleration remain speculative, and the influence of various
chemical agents on enzymatic activity, substrate conformation
and efficiency of the digestion process are not well
understood, which leads to great variability of experimental
results between groups which have attempted to employ PCTenhanced
digestion methods.
The Pressure Cycling Technology Sample Preparation System
(PCT SPS) applies alternating hydrostatic pressure between
ambient and ultra high levels to control molecular interactions
[7]. The PCT SPS has been successfully used in a variety of
applications, including cell and tissue lysis, and the extraction
of proteins, lipids and nucleic acids [8]. Recently, PCT has also
been shown to accelerate enzymatic reactions such as
proteolysis [1]. The PCT SPS (Figure 1) is comprised of a
small, semi-automated bench-top instrument (Barocycler
NEP3229 or NEP2320) and single-use sample processing
containers called PULSE Tubes (Pressure BioSciences, Inc.,
South Easton, MA). Used together, the PULSE Tubes
transmit the pressure generated by the Barocycler to the
sample, resulting in pressure enhanced proteolysis and
accelerated genomic DNA isolation.
PCT MicroTubeTM Adapter Kit*
for
Pressure-Enhanced Enzymatic Proteolysis
See Brochure
with Data and Comments from
Thermo Fisher, FDA, NYU School of Medicine, Mississippi
State University and
Harvard School of Public Health
In this work we explore the stability and catalytic activity of
trypsin and chymotrypsin under the influence of several
chaotropes and organic solvents in combination with
hydrostatic pressure and elevated temperatures. We have
employed chromogenic substrates in order to measure
enzyme activity independent of pressure-induced changes in
substrate protein conformation. Additionally, using high
performance LC-MS analysis, we have tested the effect of the
factors outlined above on efficiency, selectivity, and throughput
of proteolytic digestion. Analysis of the data obtained thus far
leads to a set of guidelines for development of optimized and
highly reproducible pressure-enhanced digestion methods.
Continued on Page 3
www.pressurebiosciences.com

P C T N e w s
Discovery Starts With Sample Preparation TM
Volume 3, Issue 6 June 2009
Excerpt from PBI Poster Presented at the 2009 ASMS (continued)
Proteomics Under Pressure: rapid
extraction and digestion in a single tube
Excerpt from PBI Poster Presented at the 2009 ASMS (continued)
Proteomics Under Pressure: rapid
extraction and digestion in a single tube
A
C
B
Conclusion
Pressure cycling has been shown to significantly improve
proteolysis with a number of enzymes. High hydrostatic
pressure acts synergistically with chaotropes and organic
solvents to boost the effects of these chemicals on protein
denaturation and digestion These effects not only improve
digestion efficiency and save significant time, but also allow
the use of much lower concentrations of denaturants, which
can simplify and improve downstream applications. While such
effects are clearly beneficial, care must be taken in
development of PCT-enhanced digestion methods to avoid
impairment of enzymatic activity due to denaturation of the
enzyme itself. The following points illustrate some of our recent
findings:
Figure 2. PCT MicroTubes. A) Standard PCT MicroTubes. B)
PCT MicroTube cartridge system. C) PCT MicroTubes with gelpicking
caps available in 50 µl, 100 µl and 150 µl sizes.
1. Chymotrypsin activity can be significantly enhanced by
performing digestion using cycled pressure.
2. Under cycled pressure, 0.5 M Guanidine-HCl inhibits trypsin
but not chymotrypsin activities.
3. Under cycled pressure, 4% HFIP enhances trypsin but
inhibits chymotrypsin activities.
Grand Total: 1077
4. Pressure levels above 20,000 psi exhibit negative effect on
trypsin activity.
912
PCT-assisted
243
669
“Conventional”
165
5. Under cycled pressure, trifluoroethanol (TFE) below 15%
enhances trypsin but inhibits chymotrypsin activities (data not
shown).
Enriched with
cytosolic
proteins
834
Enriched with
organelle and
membrane
proteins
6. Under cycled pressure, both methanol and urea can be
included in trypsin digests at low concentration, but become
rapidly inhibitory at higher concentrations.
7. Pressure cycling instrumentation could be used to enhance
cell lysis and protein digestion in the same sample container to
minimize sample handling and potential loss of analytes.
Figure 14. Overlap of detected HepG2 proteomes extracted
by a conventional method and by PCT. The lower plots
demonstrate GO localization terms differentially enriched in the
non-overlapping fraction of detected proteomes.
www.pressurebiosciences.com