Tryptic

Fig. 12. Tryptic map of it-PA (mol wt = 66,000) showing peptides formed from hydrolysis of reduced, alkylated rt-PA. Separation by reversed-phase octadecyl (C g) column using aqueous acetonitrile with an added acidic agent to the mobile phase. Arrows show the difference between A, normal, and B, mutant rt-PA where the glutamic acid residue, D, has replaced the normal arginine residue, C, at position 275.

CGRP has a wide distribution in the nervous system (19) and was the first peptide to be localized to motoneurons (124). It is also found in primary sensory neurons where it is colocalized with substance P (125). CGRP is derived from a precursor stmcturaHy related to the calcitonin precursor. The latter precursor produces two products, calcitonin itself and katacalcin, while the CGRP precursor produces one copy of CGRP (123). Like other peptides, CGRP is cleaved from its precursor by tryptic breakdown between double basic amino acid residues. 

J. Marknssen, Human Insulin by Tryptic Transpeptidation of Porcine Insulin andBiosynthetic Precursors, MTP Press, Lancaster, U.K., 1987. 

FIGURE 8.13 SEC of casein hydrolyzates. Numbers above the peaks refer to the number of amino acid residues in the typical peptide in the indicated fraction. Column PolyHEA, 200 X 9.4 mm 5 /zm, 200 A. Flow rate 0.5 ml/min. Mobile phase 50 mtA Formic acid. Detection A250. Samples (A) Pancreatin hydrolyzate and (B) tryptic hydrolyzate. (Adapted from Ref. 29 with permission from Silvestre et of. Copyright 1994, American Chemical Society.)... 

Figure 9.6 Surfer-generated chromatoeletropherogram of fluorescamine-labeled tryptic digest of ovalbumin. Reprinted from Analytical Chemistry, 62, M. M. Bushey and J. W. Jorgenson, Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography/capillary zone electrophoresis, pp 978-984, copyright 1990, 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... 

Figure 4.18 Electrospray spectrum from a single chromatographic response in the LC-MS analysis of a tryptic digest. From applications literature published by SCIEX, Concord, Ontario, Canada, and reproduced by permission of MDS SCIEX, a division of MDS Inc.

Experimentation showed that the protein was not glycosylated and that the sequence at the iV-amino acid terminus corresponded to that expected. The C-terminus sequence, however, did not correspond to that predicted and these data were interpreted in terms of the presence of a heterogeneous, truncated, protein. A study of the tryptic digest fragments from this protein with matrix-assisted laser desorption ionization (MALDI) with post-source decay enabled the authors to suggest the positions at which the parent protein had been truncated. 

The LC-FTIR part of the study was concerned with the verification of the presence of particular functional groups within each of the tryptic fragments and will not be considered any further here. 

Table 5.8 Polypeptides detected during the LC-electrospray-MS analysis of the tryptic digest from / -lactoglobulin (/ILG). Reprinted from 7. Chromatogr., A, 763, Tnrnla, V. E., Bishop, R. T., Ricker, R. D. and de Haseth, J. A., Complete structnre elncidation of a globular protein by particle beam hqnid chromatography-Fourier transform infrared spectrometry and electrospray hqnid chromatography-mass spectrometry - Seqnence and conformation of / -lactoglobulin , 91-103, Copyright (1997), with permission from Elsevier Science...

Matrix-associated laser desorption ionization with a time-of-flight mass analyser (MALDl-ToF) was used to examine the crude tryptic peptide mixture from a number of the proteins, without HPLC separation, to provide a mass map, i.e. a survey of the molecular weights of the peptides generated by the digestion process. 

What are the advantages of using MALDl-ToF to examine the crude tryptic peptide mixture ... 

MALDI-ToF is a technique that allows the molecular weights of proteins and peptides to be determined. It is less susceptible to suppression effects than electrospray ionization and thus is able to be used for the direct analysis of mixtures. In the case of a crude tryptic digest, MALDI-ToF will provide a molecular weight profile of the polypeptides present without the analysis time being extended by the need to use some form of chromatographic separation. 

Figure 5.19 MALDI-ToF mass spectrum, providing a molecular-weight profile of the tryptic peptides derived from spot 22 (see Figure 5.18) of the silver-stained two-dimensional gel of the proteins extracted from the yeast S. cerevisiae. From Poutanen, M., Salusjarvi, L., Ruohonen, L., Penttila, M. and KaUddnen, N., Rapid Commun. Mass Spectrom., 15, 1685-1692, copyright 2001. John Wiley Sons Limited. Reproduced with permission.

The electrospray spectrum from the corresponding chromatographic response in the LC-MS analysis of the tryptic digest of the protein after reaction with the inhibitor is shown in Figure 5.24. In addition to the three species found in the digest of the parent protein, two additional polypeptides, with molecular weights of 2439.36 zb 0.07 and 2457.43 zb 0.02 Da, i.e. 70 and 88 Da above... 

Figure 5.23 Electrospray mass spectrum of the tryptic peptide with a retention time of 41.81 min from intact CMY-2 -lactamase. Reprinted from Biochim. Biophys. Acta, 1547, Bonomo, R. A., Liu, J., Chen, Y., Ng, L., Hujer, A. M. and Anderson, V. E., Inactivation of CMY-2 0-lactamase by tazobactam initial mass spectroscopic characterization , 196-205, Copyright (2001), with permission from Elsevier Science.

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.

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