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Analysis of Proteins

The analysis for proteins present in plasma or a cell extract is a challenging task due to their complexity and the great difference between protein concentrations present in the sample. Simple mixtures of intact proteins can be analyzed by infusion with electrospray ionization and more complex ones by matrix assisted laser desorption ionization. MALDI is more suited for complex mixtures because for each protein an [M+H]+ signal is observed while for ESI multiply charged ions are observed. Surface enhanced laser desorption (SELDI) is a technique for the screening of protein biomarkers based on the mass spectrometric analysis of intact proteins [49]. However in most cases for sensitivity reasons mass spec-

Omeprazole Metabolite

Fig. 1.42 LC-MS profile of omeprazole metabolites spiked in plasma: (A) without mass defect filter, (B) with mass defect filter. Peaks: Ml mono-oxidation metabolite [+16 u, Mass defect (MD) +5 milliunits], M2 reduction and demethylation (-30 u, MD +10 milliunits), M3 mono-oxidation metabolite (+16 u, MD —5 milliunits), M4 reduction (—16 u, +5 milliunits), M5 mono-oxidation metabolite (+16 u, +5 milliunits). Adapted with permission from reference [85].

Fig. 1.42 LC-MS profile of omeprazole metabolites spiked in plasma: (A) without mass defect filter, (B) with mass defect filter. Peaks: Ml mono-oxidation metabolite [+16 u, Mass defect (MD) +5 milliunits], M2 reduction and demethylation (-30 u, MD +10 milliunits), M3 mono-oxidation metabolite (+16 u, MD —5 milliunits), M4 reduction (—16 u, +5 milliunits), M5 mono-oxidation metabolite (+16 u, +5 milliunits). Adapted with permission from reference [85].

trometric analysis is performed at the peptide level after enzymatic digestion. Basically there are two approaches for the identification of complex mixtures of proteins (Fig. 1.43). The first is based on two-dimensional electrophoretic separation of intact proteins followed by trypsin digestion and matrix assisted laser desorption-time of flight (MALDI-TOF) detection. The second approach digests first the protein mixture and the resulting peptides are then separated by a two-dimensional chromatographic procedure using nanoliquid chromatography coupled to nanoelectrospray ionization.

Two-dimensional electrophoresis [86] is a well established technique for the separation of intact proteins. In the first dimension the proteins are separated based on their isolectric point while the second dimension separates them based on their size. The presence on the gel of the proteins is revealed by Coomassie blue or silver staining. Under favorable conditions several thousand spots can be differentiated. The gel is digitized and computer-assisted analysis of the protein spot is performed. The spots of interest are excised either manually or automatically and then digested with trypsin. Trypsin cleaves proteins at the C-terminal side of lysine and arginine. In general one spot represents one protein and the peptides are analyzed by MALDI-TOF to obtain a peptide mass fingerprint. A peptide mass fingerprint involves the determination of the masses of all pep-

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