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Published: Kem. Ind. 57 (5) (2008) 231–243
Paper reference number: KUI-21/2007
Paper type: Review
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Protein Analysis by Mass Spectrometry

N. Galić and M. Cindrić


Soft ionization techniques, electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI) make the analysis of biomolecules by mass spectrometry (MS) possible. MS is used for determination of the molecular weight of peptides and protein, sequence analysis, characterization of protein-ligand interactions etc. The detection limit, resolution and mass accuracy depend on instrument used (Table 1). Impurities (buffers, salts, detergents) can reduce the ion intensities or even totally suppress them, so a separation method (chromatography, 2D-gel electrophoresis) must be used for purification of the sample. Molecular mass of intact protein can be determined by ESI or MALDI MS. Multiply charged ions are produced by ESI MS, while singly charged ions are predominant in MALDI spectra (Fig. 2). Sequence analysis of proteins by MS can be performed using peptide mass fingerprint. In this method, proteins are separated by 2-D gel electrophoresis and digested with specific protease (Table 2) or digested and then separated by two-dimensional chromatography (Fig. 1). The obtained peptide mixtures are analyzed by MS or MALDI-TOF technique. The masses determined by MS are compared with calculated masses from database entries. Different algorithms have been developed for protein identification. Example of posttranslational modifications (N- and O-glycosylation) and protein sequence complex analysis after dual digestion (endoproteinase digestion followed by endoglycosidase digestion) is shown in Fig. 3. It is known that detection of peptides by MS is influenced by intrinsic properties like amino acid composition, the basicity of the C-terminal amino acid, hydrophobicity, etc. Arginine-containing peptides dominate in MS spectra of tryptic digest, so the chemical derivatization of lysine terminal residue by O-methilisourea or 2-methoxy-4,5-1H-imidazole was suggested (Fig. 4). The peptide mass fingerprint method can be improved further by peptide fragmentation using tandem mass spectrometry (collision-induced dissociation, CID or post-source decay, PSD). Different types of fragments and main fragmentation paths are shown in Figs. 6 and 8. Fragmentation pathway of a doubly charged tryptic pentapeptide to b- and y-ions by collision-induced dissociation inside the mass spectrometer is described more in details in Fig. 7. All types of fragment ions are summarized in table 3. Since the any of the peptide bonds can be broken in several ways, the MS/MS spectra are complex, and quite difficult to interpret. Chemical derivatization is used to obtain only or predominantly one type of fragment ions. Sulfonation of N-terminal amino group enhance PSD sequencing, producing mainly y-type fragment ions. The mass difference of two consecutive y-ions corresponds to an amino acid mass, so the peptide sequence can be obtained with minimal or no assistance from genomic data, e. g. de novo protein sequencing is possible. Fig. 9 represents the strategy for the protein identification by mass spectrometry. Various chemical modifications on the peptide fragmentation patterns are shown in Fig. 10.

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mass spectrometry, protein identification, identification of posttranslational modifications, quantitative protein analysis