Trypsin digest

Table 5.7 Theoretically predicted polypeptides from the trypsin digestion of S-lacto-globulin (/3LG) . Reprinted from J. Chromatogr., A, 763, Turula, V. E., Bishop, R. T., Ricker, R. D. and de Haseth, J. A., Complete structure elucidation of a globular protein by particle beam liquid chromatography-Fourier transform infrared spectrometry and electrospray liquid chromatography-mass spectrometry - Sequence and conformation of /3-lactoglobulin , 91-103, Copyright (1997), with permission from Elsevier Science...

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 5.22 HC traces obtained from LC-MS analyses of trypsin digests of (a) CMY-2 /3-lactamase, and (b) tazobactam/CMY-2 /S-lactamase. Reprinted from Biochim. Biophys. Acta, 1547, Bonomo, R. A., Liu, 1., Chen, Y., Ng, L., Hujer, A. M. and Anderson, V. E., Inactivation of CMY-2 /S-lactamase by tazobactam initial mass spectroscopic characterization , 196-205, Copyright (2001), with permission from Elsevier Science.

It has been shown [18] that when high cone-voltages were used in conjunction with negative-ion electrospray, phosphopeptides produce diagnostic ions at m/z 63 (P02 ) and m/z 79 (P03 ). LC-MS analysis of a trypsin digest of bovine... 

Nielsen, R. G., Riggin, R. M., and Rickard, E. C., Capillary zone electrophoresis of peptide fragments from trypsin digestion of biosynthetic human growth hormone, /. Chromatogr., 480, 393, 1990. 

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

Release of active pancreatic enzymes directly causes local or distant tissue damage. Trypsin digests cell membranes and leads to the activation of other pancreatic enzymes. Lipase damages fat cells, producing noxious substances that cause further pancreatic and peripancreatic injury. 

Due to the limited peak capacity of the 15 cm analytical column utilized in 2-D nano LC-MS, several elution steps are required to achieve the required separation. The 15 cm analytical column can be replaced with a 100 cm nano LC column to increase the resolution of sample in each step. As shown by Yang,20 a 100 cm column allows the one-step separation of more than 2000 polypeptides from trypsin digest of mouse brain lysate, P2 fraction using XtremeSimple ultrahigh pressure nano LC (Micro-Tech Scientific, Vista, California) and LTQ MS (Thermo Electron, San Jose, California) in 6 hr (Figure 14.16). In addition to the improvement of resolving power with a 100 cm column, it... 

FIGURE 14.17 Ultrahigh resolution nano LC-MS separation of base peak chromatogram of 2351 peptides identified in trypsin digest of mouse brain lysate P2 fraction using Micro-Tech XtremeSimple nano-LC and Thermo Electron LTQ. Column 100 cm x 75 fim C18 column, 3 /mi, 8000 psi column head pressure. Solvent composition time 350 min gradient, 5 to 35% B. Solvent A 2% acetonitrile, 0.1% formic acid. Solvent B 95% acetonitrile, 0.1% formic acid. Data analysis Sequest, PeptideProphet, and Protein Prophet. 

The inverse of the above experiments gave similar results (Whitlock and Stein, 1978). Trypsin-digested histones removed from HeLa core particles can subsequently fold DNA, although DNase I digests the resulting particles more rapidly than the untreated ones. Parallel experiments were performed for chicken erythrocyte core particles (Lilley and Tatchell, 1977). In all cases it could be concluded that it is the trypsin-insensitive carboxy-terminal regions of the histones which are responsible for the folding of the DNA in the nucleosome. 

Huang, S. N., Minassian, H., and Moore, J. D. (1976) Application of immuno-fluorescent staining on paraffin sections improved by trypsin digestion. Lab Invest. 35, 383-390. 

Fig. 1. Core histone modifications. Human histone N-terminal and in some cases C-terminal amino acid sequences are shown. The modifications include methylation (M), acetylation (Ac), phosphorylation (P), ubiquitination (U), and ADP ribosylation (step ladder). The sites of trypsin digestion of histones in nucleosomes are indicated (T).

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