Tryptic peptides chromatographic separation

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 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.

Figure 20. Chromatographic separation of thermolytic fragments of peptide T-9 isolated from the tryptic digest of the AE-p-chain of Hl Sheperds Bush. The Dowex 1-X2 column uxis 60 cm long and had an internal diameter of 0.6 cm. The pH gradient is indicated by the broken line. The fragments are identified bu their sequences and the positions they occupy in the p-chain. The sequence of the abnormal T-9 is given at the top of the figure.

Figure 6 High-speed tryptic fingerprint. Horse cytochrome c was digested with trypsin and the peptide chromatographed in acetonitrile water 0.1% TFA at various temperatures and flow rates on a 15 x 0.2-cm PS-DVB column packed with 3-p, 300-A particles, (a) 26°C and 0.5 ml/min. (b) 42°C and 0.7 ml/min. (c) 70°C and 1.1 ml/min. Detection at 220 nm. Note that the resolution rises with the speed of separation.89 (From Swadesh, ]., BioTechniques, 9, 626, 1990. With permission.)...

TABLE 12.1 Chromatographic Modes and Conditions Used for LC-MS Study of Tryptic Peptides Separation Selectivity... 

Hancock, W.S., Chloupek, R.C., Kirkland, J.J., Snyder, L.R. (1994). Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples. I. Optimizing the separation of a growth hormone tryptic digest. J. Chromatogr. A 686, 31 -3. 

Fig. 1.4 Chromatographic separation of tryptic peptides from m-AspAT. A. Sephadex G-50 column chromatography. Fractions I—111 were applied on DEAE-cellulose chromatography (B). B. DE-52 column chromatographies of fractions, I, II and III. Data are from Kagamiyama et al.2S)...

Fig. VIII-1. Column chromatographic separation of tryptic peptides of clupeine 2. The 20-h tryptic hydrolyzate of clupeine 2 (approximately 10 pmoles) was chromatographed on Amberlite CG-50 (1.0 x 30 cm column). Elution was performed with an increasing NaCl concentration in 0.2 M sodium borate buffer stepwise as indicated in the figure at 30 °C and at a flow rate of 2.99 ml/tube/45 min. The color yield was 98 % (from Ando et /., 1962 and...

Table 2. IdenUficaUon of PBPs of E. coU K12 (ATCC 29079) Labeled with F/S labeled p-lactams. The intact log phase cells were treated with the F/S p-lactams for 10 minutes and then disrupted by sonication. After removal of the debris by centrifugation (10,000 rpm, 10 min.), aliquots of the supemate were separated chromatographically as described (39). The F/S labeled peaks were isolated and rechromatographed on a shallower gradient. The purified peaks were divided and subjected to tryptic and chymotiyptic digestion respectively. The digests were analyzed by MALDI-TOF or by SIMS. The resultant mass spectral data were submitted for peptide mass fingerprinting at the UCSF Mass Spectrometry Facility.

---