production of selected secondary metabolites in transformed ...

Using low-molecular mass pI markers in proteomic staining-free method
for study of posttranslationally modified proteins
Karel Mazanec 1,2 , Karel Šlais 2 and Josef Chmelík 2
1 Institute of Material Chemistry, Faculty of Chemistry, Brno University of Technology,
Purkyňova 118, 612 00 Brno, Czech Republic, e-mail: mazanec@iach.cz
2 Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veveří 97,
602 00 Brno, Czech Republic
INTRODUCTION
At present the knowledge of proteome plays very important role in the study of living
processes. Special attention in our lab is paid to the investigation of the proteome of barely
grains. The aim is to contribute to understanding of malting processes and to selection of
malting barley cultivars. Proteins are the major functional molecules of life made of amino
acids arranged in a linear chain linked together by peptide bonds. The residues in a protein are
often chemically altered in a process known as post-transcriptional modification (PTM):
either before the protein can function in the cell, or as part of control mechanisms. The
identification of the PTM is experimentally difficult.
Due to the fact that many samples, mainly of biological origin, are complex mixtures of
proteins with a wide range of molecular masses, salts and other compounds, proteins should
be separated and purified prior to their identification by mass spectrometry. Traditionally,
most proteomics researches are based upon two-dimensional gel electrophoresis involving
isoelectric focusing (IEF) as one dimension. Separated compounds are focused into very
sharp bends according to their pI values during IEF. They are twice or more concentrated in
this zones. However, ampholytes present in gel and creating pH gradient during IEF
complicate the visualizing the separated proteins by staining and make this method
impracticable for common proteomic utilization.
The aim of this work was to develop and optimize the novel staining-free proteomic
procedure for separation of intact proteins by gel IEF in presence of low-molecular mass pI
markers and subsequent determination of molecular masses of separated compounds in the
gels by MALDI-TOF/TOF MS. The selected set of pI markers includes both colored and
colorless compounds with known low-molecular mass. Thus, the identification of both pI
marker and proteins in the same excised piece of gel by MS technique is expected to give
reliable information about the correct pI values of analyzed proteins even in complex samples.
MATERIALS AND METHODS
pI markers – The substituted phenols (I - Mw 314.08, pI 3.9, yellow color; II - Mw 359.10,
pI 4.3, orange; III - Mw 272.06, pI 5.3, yellow; IV - Mw 252.12, pI 5.7, colorless; V - Mw
285.09, pI 6.4, yellow; VI - Mw 315.10, pI 7.0, yellow; VII - Mw 337.17, pI 7.5, yellow; VIII
– Mw 265.14, pI 7.9, yellow; IX - Mw 363.23, pI 8.4, yellow; X - Mw 267.16, pI 8.9, yellow;
XI - Mw 352.20, pI 9.0, colorless; XII - Mw 333.21, pI 10.1, yellow) used as pI markers were
prepared by the procedure described by Šlais and Friedl 1 at the Institute of Analytical
Chemistry (Brno, Czech Republic). The pI values were determined by potentiometric
titration. The chemical structures of pI markers are shown in Fig. 1.
Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006
Sekce DSP 2006, strana 254