production of selected secondary metabolites in transformed ...

Gel
pos. Name
Protein
Number
pI marker
Theor.
pI
No. pI
a Protein Z (Z4) (Major endosperm albumin) P06293 5.70 II 4.3
b Beta-amylase P16098 5.58 III 5.3
Glucan endo-1,3-beta-glucosidase GV Q02438 6.92
c Protein Z (Z4) (Major endosperm albumin) P06293 5.70 IV 5.7
d Granule-bound starch synthase 1 P09842 7.06 VI 7.0
Alpha-amylase/subtilisin inhibitor precursor P07596 7.78
e Glucan endo-1,3-beta-glucosidase GV Q02438 6.92 VII 7.5
f Lichenase II precursor P12257 9.0 IX 8.4
Elongation factor 1-alpha Q40034 9.15
g 26 kDa endochitinase 2 precursor P23951 8.83 XI 9.0
Histone H3 (Fragment) P06353 9.73
h 26 kDa endochitinase 2 precursor P11955 8.54 XII 10.1
Elongation factor 1-alpha Q40034 9.15
Tab.1: The list of proteins identified directly in the same gel piece as the pI marker. Standard
proteins are bolded.
CONCLUSIONS
The method is based on separation of intact proteins by gel IEF along with low-molecular
mass pI markers and subsequent sequential determination of molecular masses of separated
compounds by MALDI-TOF/TOF MS. The identification of both pI markers and proteins in
the same gel piece give reliable information about the correct pI values of particular identified
proteins in complex samples. Moreover, it allows omitting protein staining and destaining
procedure, because the Coomassie procedure is in the case of IEF gels complicated by
presence of ampholytes causing dark background. The suggested procedure shortens roughly
by half the time of analysis. The choice of appropriate ampholytes allows to modify the pH
gradient and to study only the certain area of the pH scale.
There are only twelve mainly colored pI markers shown in this work. Nevertheless, the
number of pI markers used may be much higher. The color of pI markers is not relevant for
MS determination. Focused pI markers may cover whole pH range and from gels narrow
pieces can be cut out, which will allow more precise determination of identified proteins.
Utilization of low-molecular mass pI markers with IEF gels was chosen as a first example of
their applicability because the gel process can be easily observed visually. There is a good
chance to use this approach also in other IEF techniques (e.g. in capillaries or on chips).
REFERENCES
[1] Šlais K, Friedl Z. Low-molecular-mass pI markers for isoelectric focusing. Journal of
Chromatography A 1994; 661: 249.
[2] Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins
from silver stained polyacrylamide gels. Analytical Chemistry 1996; 68: 850.
[3] Strohalm M, Santrucek J, Hynek R, Kodicek M. Analysis of tryptophan surface
accessibility in proteins by MALDI-TOF mass spectrometry; Biochemical and
Biophysical Research Communications 2004; 323: 1134.
Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006
Sekce DSP 2006, strana 258