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Us<strong>in</strong>g low-molecular mass pI markers <strong>in</strong> proteomic sta<strong>in</strong><strong>in</strong>g-free method<br />
for study <strong>of</strong> posttranslationally modified prote<strong>in</strong>s<br />
Karel Mazanec 1,2 , Karel Šlais 2 and Josef Chmelík 2<br />
1 Institute <strong>of</strong> Material Chemistry, Faculty <strong>of</strong> Chemistry, Brno University <strong>of</strong> Technology,<br />
Purkyňova 118, 612 00 Brno, Czech Republic, e-mail: mazanec@iach.cz<br />
2 Institute <strong>of</strong> Analytical Chemistry, Academy <strong>of</strong> Sciences <strong>of</strong> the Czech Republic, Veveří 97,<br />
602 00 Brno, Czech Republic<br />
INTRODUCTION<br />
At present the knowledge <strong>of</strong> proteome plays very important role <strong>in</strong> the study <strong>of</strong> liv<strong>in</strong>g<br />
processes. Special attention <strong>in</strong> our lab is paid to the <strong>in</strong>vestigation <strong>of</strong> the proteome <strong>of</strong> barely<br />
gra<strong>in</strong>s. The aim is to contribute to understand<strong>in</strong>g <strong>of</strong> malt<strong>in</strong>g processes and to selection <strong>of</strong><br />
malt<strong>in</strong>g barley cultivars. Prote<strong>in</strong>s are the major functional molecules <strong>of</strong> life made <strong>of</strong> am<strong>in</strong>o<br />
acids arranged <strong>in</strong> a l<strong>in</strong>ear cha<strong>in</strong> l<strong>in</strong>ked together by peptide bonds. The residues <strong>in</strong> a prote<strong>in</strong> are<br />
<strong>of</strong>ten chemically altered <strong>in</strong> a process known as post-transcriptional modification (PTM):<br />
either before the prote<strong>in</strong> can function <strong>in</strong> the cell, or as part <strong>of</strong> control mechanisms. The<br />
identification <strong>of</strong> the PTM is experimentally difficult.<br />
Due to the fact that many samples, ma<strong>in</strong>ly <strong>of</strong> biological orig<strong>in</strong>, are complex mixtures <strong>of</strong><br />
prote<strong>in</strong>s with a wide range <strong>of</strong> molecular masses, salts and other compounds, prote<strong>in</strong>s should<br />
be separated and purified prior to their identification by mass spectrometry. Traditionally,<br />
most proteomics researches are based upon two-dimensional gel electrophoresis <strong>in</strong>volv<strong>in</strong>g<br />
isoelectric focus<strong>in</strong>g (IEF) as one dimension. Separated compounds are focused <strong>in</strong>to very<br />
sharp bends accord<strong>in</strong>g to their pI values dur<strong>in</strong>g IEF. They are twice or more concentrated <strong>in</strong><br />
this zones. However, ampholytes present <strong>in</strong> gel and creat<strong>in</strong>g pH gradient dur<strong>in</strong>g IEF<br />
complicate the visualiz<strong>in</strong>g the separated prote<strong>in</strong>s by sta<strong>in</strong><strong>in</strong>g and make this method<br />
impracticable for common proteomic utilization.<br />
The aim <strong>of</strong> this work was to develop and optimize the novel sta<strong>in</strong><strong>in</strong>g-free proteomic<br />
procedure for separation <strong>of</strong> <strong>in</strong>tact prote<strong>in</strong>s by gel IEF <strong>in</strong> presence <strong>of</strong> low-molecular mass pI<br />
markers and subsequent determ<strong>in</strong>ation <strong>of</strong> molecular masses <strong>of</strong> separated compounds <strong>in</strong> the<br />
gels by MALDI-TOF/TOF MS. The <strong>selected</strong> set <strong>of</strong> pI markers <strong>in</strong>cludes both colored and<br />
colorless compounds with known low-molecular mass. Thus, the identification <strong>of</strong> both pI<br />
marker and prote<strong>in</strong>s <strong>in</strong> the same excised piece <strong>of</strong> gel by MS technique is expected to give<br />
reliable <strong>in</strong>formation about the correct pI values <strong>of</strong> analyzed prote<strong>in</strong>s even <strong>in</strong> complex samples.<br />
MATERIALS AND METHODS<br />
pI markers – The substituted phenols (I - Mw 314.08, pI 3.9, yellow color; II - Mw 359.10,<br />
pI 4.3, orange; III - Mw 272.06, pI 5.3, yellow; IV - Mw 252.12, pI 5.7, colorless; V - Mw<br />
285.09, pI 6.4, yellow; VI - Mw 315.10, pI 7.0, yellow; VII - Mw 337.17, pI 7.5, yellow; VIII<br />
– Mw 265.14, pI 7.9, yellow; IX - Mw 363.23, pI 8.4, yellow; X - Mw 267.16, pI 8.9, yellow;<br />
XI - Mw 352.20, pI 9.0, colorless; XII - Mw 333.21, pI 10.1, yellow) used as pI markers were<br />
prepared by the procedure described by Šlais and Friedl 1 at the Institute <strong>of</strong> Analytical<br />
Chemistry (Brno, Czech Republic). The pI values were determ<strong>in</strong>ed by potentiometric<br />
titration. The chemical structures <strong>of</strong> pI markers are shown <strong>in</strong> Fig. 1.<br />
Sborník soutěže Studentské tvůrčí č<strong>in</strong>nosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006<br />
Sekce DSP 2006, strana 254