production of selected secondary metabolites in transformed ...

RESULTS
More than forty pI markers of different types of structures were studied by MALDI(LDI)-
TOF MS. Twelve selected suitable pI markers are used and more closely characterized in this
work.
At analysis of low-molecular mass pI markers, several features were observed that help to
their identification. In the positive ion mode mass spectra of nitro-substituted pI markers the
characteristic peaks at -16 and -32 Da caused by loss of oxygen atoms during the process of
ionization (also reminded by Strohalm et al. 3 ) can be seen (Fig. 2, spectrum a, c). A
characteristic double-peak pattern is obtained for markers containing a chlorine atom (Fig. 2,
spectrum b, c). These groups of peaks are very helpful for identification of pI markers
especially in complex mixtures where one peak may be hidden with a cluster or by another
well-ionizable substance.
Fig.2: The positive ion mass spectra of the pI markers XII (a); II (b) and I (c) obtained after
extraction from the IEF gel. A characteristic peak patterns are clearly seen.
Isoelectric focusing and gel treatment - The 3/10 carrier ampholytes were used to create pH
gradient in this work. At 50-mm distance of electrodes it means that the slope of the gradient
is 0.14 pH/mm. The color pI markers enable the orientation in gels and supervision of the IEF
process. Each abnormality in pH gradient can be immediately and very easily recognized.
Moreover, the gels can be cut up according to the position of the visible markers. The IEF
gels were immediately after focusing scanned (Fig. 3A) and the bands with focused pI
markers were excised. For comparison, another IEF gel was stained with Coomassie Brilliant
Blue R 250. The focused proteins visualized by Coomassie staining are shown in Fig. 3B,C.
Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006
Sekce DSP 2006, strana 256