production of selected secondary metabolites in transformed ...

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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
Identification of pI markers - First, the pI markers were extracted from each excised gel piece and identified from extracts by MALDI-TOF/TOF MS. The experiments confirmed that pI markers are easily and unambiguously identifiable in extracts from excised gel pieces after IEF. Fig.3: IEF gel of pI markers and proteins scanned immediately after focusing (A); standards (B) and beta-amylase extract (C) after Coomassie staining. The positions of electrodes are marked with E. Letters at right show positions where the proteins were identified (see Tab. 1). Identification of proteins - First experiment was provided with standard proteins (cytochrome c, myoglobin and albumin). After extraction of pI markers from particular gel pieces, the proteins in the same gel pieces were treated by enzyme digestion and identified by MS. These proteins were identified in positions according to their real pI values and validated our approach. Then, the extract from barley malt was used as a model mixture of glycated proteins. The separated protein mixture after the referential Coomassie staining is shown in Fig. 3C. Table 1 shows the proteins identified in the gel pieces from which the pI markers were extracted and identified. Nine proteins were identified in eight excised gel pieces of twelve ones excised according to the locations of the pI markers used. Some proteins were found in two different gel pieces. During malting process the barley proteins are gradually cleaved and modified. This is the reason why some proteins may be found in several gel pieces with different pI values. The differences observed for some other proteins might be caused either by posttranslational modifications or by discrepancies between experimental and theoretically calculated pI values. For more detailed studies, the gels can be excised into a higher number of narrower pieces and the pI values of proteins identified between the locations of pI markers can be interpolated or a higher number of pI markers can be used. The identifications of the proteins were confirmed from the stained gel by the same proteomic protocol. Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006 Sekce DSP 2006, strana 257
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PRODUCTION OF SELECTED SECONDARY ME
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containing ampicillin, IPTG and x-g
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THE SIMPLE METHOD FOR THE RECOGNITI
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Figure 1 Negative-ion mode MALDI-TO
Page 9 and 10: Table 1 Evaluation of negative-ion
Page 11 and 12: Fig. 1: Scheme of the hydride gener
Page 13 and 14: [4] H. Matusiewicz, M. Kopras, J. A
Page 15 and 16: - are hypotriglyceridemic; - exhibi
Page 17 and 18: HYDROPHOBIZED SODIUM HYALURONATE IN
Page 19 and 20: spectrophotometer (AMINCO-Bowman, S
Page 21 and 22: Hyaluronate derivatives showed with
Page 23 and 24: copolymers were additionally functi
Page 25 and 26: Each micelle has a hydrophobic core
Page 27 and 28: STUDY OF THE COPPER(II) IONS NON-ST
Page 29 and 30: CuSO4, Cu(ClO4)2 and Cu2P2O7 of the
Page 31 and 32: total flux [mol.m -2 ] 0.7 0.65 0.6
Page 33 and 34: number of free radicals is very low
Page 35 and 36: Differences can be caused by severa
Page 37 and 38: MEASUREMENT OF THERMOPHYSICAL PROPE
Page 39 and 40: The typical time responses of tempe
Page 41 and 42: Figure 4 illustrates the typical de
Page 43 and 44: sodium sulphate and filtered to a 5
Page 45 and 46: Compare all tested evening primrose
Page 47 and 48: IMPROVED FLUORIMETRIC DETERMINATION
Page 49 and 50: Al F i 80 70 60 50 40 30 20 10 0 0
Page 51 and 52: F i 60 50 40 30 20 10 Data UCL LCL
Page 53 and 54: PHYSICAL-CHEMICAL ASPECTS OF PAINT
Page 55 and 56: Finally, the solubility parameter o
Page 57 and 58: Study of the influence of different
Page 59: Fig.1: Chemical structures of pI ma
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