Peptides mapping

The most commonly utilized chemical cleavage agent is cyanogen bromide (it cleaves the peptide bond on the carboxyl side of methionine residues). V8 protease, produced by certain staphylococci, along with trypsin are two of the more commonly used proteolytic-based fragmentation agents. 

The efficiency of identification greatly depends on the performance of the mass spectrometer. Most notably, the mass accuracy of the instrument, usually determined by studying standards, has a dramatic effect Clearly, the more accurate the measured masses are the narrower is the set of proteins that produce fragments with masses within the tolerance. The number of peptides identified is correlated with the amino acid residue coverage of the original protein. 

Monoisotopic (mi) and average (av) peptide masses from tryptic digestion of human hemoglobin a chain [11] 

In our first example we use a low-performance mass spectrometer. Assuming that five peptides (m/z 729.86,818.95,1072.32,1253.49, and 1530.65) appear in the mass spectrum (e.g., as commonly observed, due to the ion-suppression effect we do not detect all tryptic peptides), the average masses are determined with 2000 ppm mass accuracy, and the search in the SwissProt database is restricted to the proteins of Homo sapiens, the MS-Fit searching tool of Protein Prospector [11] finds 114 entries that are more or less consistent with this data. The relevant section of the mass spectrum is shown in Fig. 2. Note that in this example no impurities complicate the spectrum. 

The top-ranked hit is human hemoglobin a subunit with all five masses matched, but with only 35.5% coverage (in light gray below). 

The other proteins on the list showed fewer number of matching peptides or lower degree of coverage. 

Trypsin cleaves adjacent to arginine and lysine, but has a preference for arginine, particularly at high pH values. In the sequences Arg-X or Lys-X, cleavage is inhibited if there is a proline residue at position X. Cleavage is also inhibited by glutamic acid and aspartic acid, and by the occurrence of additional arginine or lysine residues. 

Chymotrypsin (bovine pancreatic) Tyr-X, Phe-X, Trp-X, Leu-X, Met-X, Ala-X pH 8.0 PMSF, TPCK, aprotinin 

Clostripain (Clostridium histciyticum) Arg-X pH 7.2 Trypsin inhibitor, a2-macroglobulin, leupeptin, antipain 

Endoproteinase Lys-C (Lysobacter enzymogenety Lys-X pH 8.5 TLCK, aprotinin. leupeptin 

Factor Xa (bovine blood) Arg-X in Gly-Arg-X pH 8.3 a2-plasmin, antithrombin III, benzamidine 

and 1337.1. Assuming that these peaks correspond to MH ions, the mass balance can be checked from the molecular weights  

Interestingly, the PA, 0 form of the peptide is also found in normal brain, while longer forms that include additional hydrophobic residues on the carboxy terminus have been implicated in Alzheimer s disease. Thus, further digestion of this same peptide sample with CNBr (which cleaves at methionine residues) resulted in three additional peaks at m/z 431.1, 614.2, and 626.0, corresponding to the peptides  

Tryptic mapping provides a convenient means for comparing recombinant proteins and native proteins. In this technique, recombinant proteins are digested wath trypsin, fractionated on reversed-phase HPLC, and the retention times of their tryptic fragments compared with those from native proteins. Since the amino acid sequence is known and should be the same as that of the native protein, differences in retention 

Glu Rich Mature Toxin Region (Minor Chain) 

MMKFWLAC VP.QEEEYFE AKALPPGSVCDGMESDCKCYGAWHKCRCPWKWH RSEESER SK LFVAAHSFA I AEVPELER I FTGEGPCTCEKGMKETTCITKLHCPHKAEWQLDW I 

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Protein sequencing by creation of overlap peptides (mapping),... 

M. Stromqvist, Peptide mapping using combinations of size-exclusion chromatography, reversed-phase chromatography and capillary electrophoresis , 7. Chromatogr. 667 304-310(1994). 

G. J. Opiteck, J. W. Jorgenson, M. A. Moseley III and R. J. Anderegg, Two-dimensional mia ocolumn HPLC coupled to a single-quadrupole mass spectrometer for the elucidation of sequence tags and peptide mapping , 7. Microcolumn Sep. 10 365-375 (1998). 

The use of the dynamic-FAB probe (see Section 4.4 above) has allowed the successful coupling of HPLC to this ionization technique but there is an upper limit, of around 5000 Da, to the mass of molecules which may be successfully ionized. Problem solving, therefore, often involves the use of chemical methods, such as enzymatic hydrolysis, to produce molecules of a size more appropriate for ionization, before applying techniques such as peptide mapping (see Section 5.3 below). 

Peptide mapping The process of considering the amino acid sequence information from peptides obtained by enzyme digestion in an attempt to derive the (amino acid) sequence of the parent protein. 

Reboud-Ravaux, M. Desvages, G. Chapeville, F. Irreversible inhibition and peptide mapping of urinary plasminogen activator urokinase. FEBS Lett 1982, 140, 58-62. 

Thannhauser, T. W., McWherter, C. A., and Scheraga, H. A., Peptide mapping of bovine pancreatic ribonuclease A by reverse-phase high-performance liquid chromatography. II. A two-dimensional technique for determination of disulfide pairings using a continuous-flow disulfide detection system, Anal. Biochem., 149, 322, 1985. 

Stone, K. L. and Williams, K. R., High-performance liquid chromatographic peptide mapping and amino acid analysis in the subnanomole range, /. Chromatogr., 359, 203, 1986. 

Takahashi, N., Takahashi, Y., Ishioka, N., Blumberg, B., and Putnam, F. W., Application of an automated tandem high-performance liquid chromatographic system to peptide mapping of genetic variants of human serum albumin, J. Chromatogr., 359, 181, 1986. 

Palmieri, R., Peptide mapping by capillary electrophoresis, Appl. Brief DS-774, Beckman Instruments, Fullerton, CA, 1990. 

Bonneil, E., Mercier, M. and Waldron, K.C., Reproducibility of a solid-phase trypsin microreactor for peptide mapping by capillary electrophoresis, Anal. Chim. Acta, 404, 29, 2000. 

English, R. D. Warscheid, B. Fenselau, C. Cotter, R. J. Bacillus spore identification via proteolytic peptide mapping with a miniaturized MALDITOF mass spectrometer Anal. Chem. 2003, 75, 6886-6893. 

This strategy requires a more complex system for sample processing however, it engages the advantages of improved mass accuracy and better sensitivity that derive from working with lower mass ions. Peptide maps from protein mixtures have been demonstrated to provide reliable identifications of microorganisms that are reasonably pure.79-81... 

Wagner, K., Miliotis, T., Marko-Varga, G., Bischoff, R., Unger, K.K. (2002). An automated online multidimensional HPLC system for protein and peptide mapping with integrated sample preparation. Anal. Chem. 74, 809-820. 

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