The Tryptic Peptides

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.

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 16. Chromatographic separation of peptides of a tryptic hydrolysate of the a chain of Hb-St. Claude ona O.9 X O cm column of Chromo-bead resin type P at 37°C. The pH gradient is indicated by the broken line. T-1, T-2, etc. refer to the tryptic peptides and the numbers underneath to the positions in the chain. Several peptides are pure and give satisfactory analyses without further purification.

Once the resolution has been optimized as a function of gradient rate, one can continue to fine-tune the separation, raising flow rate and temperature. In a study of temperature and flowrate variation on the separation of the tryptic peptides from rabbit cytochrome c, column performance doubled while analysis time was reduced by almost half using this strategy.97 Commercially available software has been developed to aid in optimization. As a final note, in an industrial laboratory optimization is not completed until a separation has been shown to be rugged. It is a common experience to optimize a separation on one column, only to find that separation fails on a second column of identical type. Reproducibility and rigorous quality control in column manufacture remains a goal to be attained. 

Purify the tryptic peptides by chromatography on a C18 column to remove salts (follow the manufacturer s directions for peptide purification). Dry the eluent and redissolve the peptides in 20 pi 0.1 percent formic acid. 

Figure 4.13 Separation of the tryptic peptides from recombinant HGH using a 120-min linear gradient at 60°C from water + 0.1% TFA to 40 60 water acetonitrile + 0.1% TFA. Column Zorbax SB-C8, 4.6 X 150 mm, 30-nm pore, 5- tm particle size. (Reprinted from W.S. Hancock, R.C. Chloupek, J.J. Kirkland, and L.R. Snyder, J. Chromatogr. A, 686 31 [1994]. With permission from Elsevier Science.)...

Guidotti, G., R. J. Hill, and IV. Konigsberg The structure of human hemoglobin. TI. The sepanition and amino acid composition of the tryptic peptides from the a and p chains. Journ. Biol. Chem. 237, 2184-2195 (1962). 

Konigsberg, W., and R. J. Hill The structure ol human hemoglobins. 111. The sequence of amino acid.s in the tryptic peptides of the a-chain. Journ. Biol. Chem. 237, 2517 -2561 (1962). 

The second approach is the tandem mass spectrometric method (Wilm et ah, 1996 Link et ah, 1999 Yates, 2000). This method relies on fragmentation of individual peptides in the tryptic peptide mixture to gain sequence information. Its main advantage is that sequence information derived from several peptides is much more specific for the identification of a protein than a list of peptide masses. The sequence data can be used to search not only protein sequence databases but also nucleotide databases such as expressed sequence tag (EST) databases and, more recently, even... 

Fig. 9. The chymotryptic peptides of al-CB6 Cl, C2, and C3. The tryptic peptides T 1 -T 5 of C2 are indicated. The numbers of residues per peptide are given in parentheses. The amino acid sequences of Cl, C2, and C3 have been described 67,35,36)...

Der Terrossian, E. Pradel, L.-A. Kassab, R. Desvages, G. Separation of the two non-identical subunits of lombricine kinase from Lumbricus terrestris muscle by chromatography on sepharose-mercurial. Isolation of the tryptic peptide containing its essential thiol group. Eur. J. Biochem., 45, 243-251 (1974)... 

Four tryptic peptides containing intact methionine residue were obtained from the tryptic digest of intact nuclease. Amino acid analysis (32) and NH2-terminus determination by the dinitrofluorobenzene method of the BrCN fragments, the tryptic peptides obtained from the BrCN fragments and the four from native nuclease provided the information required to deduce the linear arrangement of the BrCN fragments (25). 

Kaiser and Krause (57) used HPLC to separate the tryptic peptides in cow s-milk and goat s-milk cheeses and cheeses made from mixtures of these milks. These authors reported that the quantitative detection limit could be as low as 1 % cow s milk in goat s-milk cheese. Mayer et al. (125a) have developed a procedure for the separation of bovine, ovine, and caprine para ic-casein using cation-exchange HPLC. 

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-stain-ing, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method. 

Chang, J. P Kiehl, D. E. Kennington, A. 1997. Separation and characterization of the tryptic peptide mapping of recombinant bovine growth hormone by reversed-phase high-performance liquid chromatography electrospray mass spectrometry. Rapid Commun. Mass Spectrom., 11, 1266-1270. 

The reversed-phase HPLC separation of the tryptic peptides of hemoglobin variants using ammonium acetate buffers, generally 10 mM at pH... 

Fig. 17. High-speed gel permeation separation of the tryptic peptides of human thyro-globulin on dual Waters 1-125 protein columns using a mobile phase comprised of 1 M urea-0.1% HaPO, pH 3.5, at a flow rate of 0.5 ml/min. Reproduced from Hearn era/. (170).

---