Digestion of peptides

Figure 13. Tryptic peptides derived from adrenal opioid peptides F and I (see Fig. 7) (from Kimura et a/., 1980). Chromatography was carried out on an Ultrasphere octyl column using a gradient of n-propanol at pH 4.0. (A) Standard a, [Met ]enkephalin-Lys b, [Met ] enkephalin[Arg ] c, [Met ]enkephalin d, [Leu ]enkephalin e, [Met ]enkephalin-Arg -[Phe ]. (B) Tryptic digest of peptide F. (C) Tryptic digest of peptide I. Aliquots of the collected fractions were tested for opioid activity and hydrolyzed for amino acid analysis.

A typical TIC chromatogram from an analysis of peptides resulting from enzymatic digest of myoglobin. The peaks represent individual peptides eluting from an LC column and being mass measured by a spectrometer coupled to it through a dynamic-FAB inlet/ion source. 

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

Figure 9.10 Three-dimensional representation of the data volume of a tryptic digest of ovalbumin. Series of planar slices through the data volume produce stacks of disks in order to show peaks. Reprinted from Analytical Chemistry, 67, A. W. Moore Jr and J. W. Jorgenson, Comprehensive three-dimensional separation of peptides using size exclusion chromatogra-phy/reversed phase liquid chromatography/optically gated capillary zone electrophoresis, pp. 3456-3463, copyright 1995, with permission from the American Chemical Society.

Peptides formed during tryptic digest of Salmonella flagellin were immobilized on the WPG-PG to prepare immunoadsorbents for the isolation of monoreceptor antibodies from rabbit serum against H-antigens of Salmonella spp. [129]. The... 

Besides sensitive methods for the analysis of proteins, bioinformatics is one of the key components of proteome research. This includes software to monitor and quantify the separation of complex samples, e.g., to analyze 2DE images. Web-based database search engines are available to compare experimentally measured peptide masses or sequence ions of protein digests with theoretical values of peptides derived from protein sequences. Websites for database searching with mass spectrometric data may be found at http //www.expasy.ch/tools, http //prospector.ucsf. edu/ and http //www.matrixscience.com. 

The proteolytic digestion of j6-lactoglobulin was carried out with trypsin which, as indicated in Table 5.4 above, is expected to cleave the polypeptide backbone at the carboxy-terminus side of lysine (K) and arginine (R). On this basis, and from the known sequence of the protein, nineteen peptide fragments would be expected, as shown in Table 5.7. Only 13 components were detected after HPLC separation and, of these, ten were chosen for further study, as shown in Table 5.8. 

Trypsin digests of 17 proteins were carried out and the peptides produced then studied by using LC-MS-MS. In summary, these produced between one and six peptides which gave some sequence information by MS-MS. Of these peptides, the number of amino acid residues sequenced ranged from three to thirteen. 

Figure 5.27 Selective detection of lactolated peptides from a tryptic digest of / -lacto-globulins by LC-electrospray-MS-MS, showing (a) the total-ion-cnrrent trace in full-scan mode, and (b) the total-ion-current trace in neutral-loss-scanning mode. Figure from Selective detection of lactolated peptides in hydrolysates by liquid chromatography/ electrospray tandem mass spectrometry , by Molle, D., Morgan, F., BouhaUab, S. and Leonil, J., in Analytical Biochemistry, Volume 259, 152-161, Copyright 1998, Elsevier Science (USA), reproduced with permission from the publisher.

Figure 5.31 LC-electrospray-MS-MS spectrum of the column eluate at around 22 min in the analysis of the peptide mixture from the tryptic digest of glycoprotein TIME-EA4 from silkworm diapause eggs. Reprinted from Bioorg. Med. Chem., 10, Kurahashi, T., Miyazaki, A., Murakami, Y., Suwan, S., Franz, T., Isobe, M., Tani, M. and Kai, H., Determination of a sugar chain and its linkage site on a glycoprotein TIME-EA4 from silkworm diapause eggs by means of LC-ESI-Q-TOF-MS and MS/MS , 1703-1710, Copyright (2002), with permission from Elsevier Science.

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

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