production of selected secondary metabolites in transformed ...
production of selected secondary metabolites in transformed ...
production of selected secondary metabolites in transformed ...
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Gel<br />
pos. Name<br />
Prote<strong>in</strong><br />
Number<br />
pI marker<br />
Theor.<br />
pI<br />
No. pI<br />
a Prote<strong>in</strong> Z (Z4) (Major endosperm album<strong>in</strong>) P06293 5.70 II 4.3<br />
b Beta-amylase P16098 5.58 III 5.3<br />
Glucan endo-1,3-beta-glucosidase GV Q02438 6.92<br />
c Prote<strong>in</strong> Z (Z4) (Major endosperm album<strong>in</strong>) P06293 5.70 IV 5.7<br />
d Granule-bound starch synthase 1 P09842 7.06 VI 7.0<br />
Alpha-amylase/subtilis<strong>in</strong> <strong>in</strong>hibitor precursor P07596 7.78<br />
e Glucan endo-1,3-beta-glucosidase GV Q02438 6.92 VII 7.5<br />
f Lichenase II precursor P12257 9.0 IX 8.4<br />
Elongation factor 1-alpha Q40034 9.15<br />
g 26 kDa endochit<strong>in</strong>ase 2 precursor P23951 8.83 XI 9.0<br />
Histone H3 (Fragment) P06353 9.73<br />
h 26 kDa endochit<strong>in</strong>ase 2 precursor P11955 8.54 XII 10.1<br />
Elongation factor 1-alpha Q40034 9.15<br />
Tab.1: The list <strong>of</strong> prote<strong>in</strong>s identified directly <strong>in</strong> the same gel piece as the pI marker. Standard<br />
prote<strong>in</strong>s are bolded.<br />
CONCLUSIONS<br />
The method is based on separation <strong>of</strong> <strong>in</strong>tact prote<strong>in</strong>s by gel IEF along with low-molecular<br />
mass pI markers and subsequent sequential determ<strong>in</strong>ation <strong>of</strong> molecular masses <strong>of</strong> separated<br />
compounds by MALDI-TOF/TOF MS. The identification <strong>of</strong> both pI markers and prote<strong>in</strong>s <strong>in</strong><br />
the same gel piece give reliable <strong>in</strong>formation about the correct pI values <strong>of</strong> particular identified<br />
prote<strong>in</strong>s <strong>in</strong> complex samples. Moreover, it allows omitt<strong>in</strong>g prote<strong>in</strong> sta<strong>in</strong><strong>in</strong>g and desta<strong>in</strong><strong>in</strong>g<br />
procedure, because the Coomassie procedure is <strong>in</strong> the case <strong>of</strong> IEF gels complicated by<br />
presence <strong>of</strong> ampholytes caus<strong>in</strong>g dark background. The suggested procedure shortens roughly<br />
by half the time <strong>of</strong> analysis. The choice <strong>of</strong> appropriate ampholytes allows to modify the pH<br />
gradient and to study only the certa<strong>in</strong> area <strong>of</strong> the pH scale.<br />
There are only twelve ma<strong>in</strong>ly colored pI markers shown <strong>in</strong> this work. Nevertheless, the<br />
number <strong>of</strong> pI markers used may be much higher. The color <strong>of</strong> pI markers is not relevant for<br />
MS determ<strong>in</strong>ation. Focused pI markers may cover whole pH range and from gels narrow<br />
pieces can be cut out, which will allow more precise determ<strong>in</strong>ation <strong>of</strong> identified prote<strong>in</strong>s.<br />
Utilization <strong>of</strong> low-molecular mass pI markers with IEF gels was chosen as a first example <strong>of</strong><br />
their applicability because the gel process can be easily observed visually. There is a good<br />
chance to use this approach also <strong>in</strong> other IEF techniques (e.g. <strong>in</strong> capillaries or on chips).<br />
REFERENCES<br />
[1] Šlais K, Friedl Z. Low-molecular-mass pI markers for isoelectric focus<strong>in</strong>g. Journal <strong>of</strong><br />
Chromatography A 1994; 661: 249.<br />
[2] Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequenc<strong>in</strong>g <strong>of</strong> prote<strong>in</strong>s<br />
from silver sta<strong>in</strong>ed polyacrylamide gels. Analytical Chemistry 1996; 68: 850.<br />
[3] Strohalm M, Santrucek J, Hynek R, Kodicek M. Analysis <strong>of</strong> tryptophan surface<br />
accessibility <strong>in</strong> prote<strong>in</strong>s by MALDI-TOF mass spectrometry; Biochemical and<br />
Biophysical Research Communications 2004; 323: 1134.<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 258