Tryptic mapping

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.

Ling V., Guzzetta A.W., Canova-Davis E., Stults J.T., Hancock W.S., Covey T.R., and Shushan B.I. (1991), Characterization of the tryptic map of recombinant DNA derived tissue plasminogen activator by high-performance liquid chromatography-electrospray ionization mass spectrometry, Anal. Chem. 63, 2909-2915. 

FIGURE 21 Two HPLC gradient chromatograms (tryptic maps of lysozyme) illustrating the dramatic effect of flow rate (F), gradient time (t ), and void volume (Vg) on analysis time. Figure reprinted with permission from Reference 22. 

Dong, M. W., Tryptic mapping by reversed-phase liquid chromatography. In Brown, P. Ed., Advances in Chromatography, Vol. 32, Marcel Dekker, New York, pp. 21-51, 1992. 

Characterization Carbohydrate map N-terminal sequence Tryptic map (LC/MS) Peptide sequences... 

Figure 2.8 Tryptic map chromatograms of the rt-PA reference standard and a mutant form of rt-PA with a glutamic acid residue in place of the normal arginine residue at position 275. Arrows illustrate the differences in the two chromatograms caused by the substitution. (Reprinted from Ref. 36 with permission.)...

Tryptic Maps of Relaxin and Relaxin B-chain. Digestion of the A-chain of human relaxin with trypsin can theoretically result in the release of five fragments that of the B-chain in the release of six fragments as illustrated in Table II. A typical tryptic map of relaxin B-chain is shown in Figure 2. The peptide was reduced and carboxymethylated with iodoacetic acid before enzymatic digestion. The peptide assignments were made after analysis of the peaks by amino acid hydrolysis for amino acid composition and confirmed by fast atom bombardment mass spectrometry (FAB-MS) as shown in Table IH... 

In contrast to the relative simplicity of relaxin, rt-PA is a large glycosylated protein of approximately 65 kD. Perhaps its greater complexity is best illustrated by a comparison of the tryptic maps of these... 

Figure 2. Tryptic map of human relaxin B-chain. The peptide was reduced with dithiotreitol and alkylated with iodoacetic acid before digestion with trypsin. The chromatography was performed on a Vydac Cis column using TFA-containing mobile phases, and eluted with an acetonitrile linear gradient.

Mass Spectral Analysis of Tryptic Map Peptides of Human Relaxin B-chain... 

Figure 3. Comparison of rt-PA (reduced and carboxymethylated) with relaxin tryptic maps. The chromatography was performed as outlined in Figure 2 legend.

Figure 4. Tryptic map of reduced and carboxymethylated rt-PA heavy and light chains. Chromatography was performed as outlined in Figure 2 legend upper panel heavy chain lower panel light chain. (Reproduced with permission from Ref. 19. Copyright 1989 Elsevier Science Publishers.)...

The authors wish to thank the following people for their work in the development of the analytical procedures utilized in this study Ida Baldonado for the reversed-phase HPLC and tryptic map of relaxin ... 

Figure 3. Tryptic maps of carbonic anhydrase (A), L-asparaginase (B) and myoglobin (C). Column Hy-Tach micropellicular C-18 silica, 105x4.6mm eluent A, 20 mM phosphoric acid adjusted to pH 2.8 with NaOH, eluent B, 60% (v/v) ACN, 20 mM phosphoric acid, pH 2.8 flow rate, 1.0 ml/min. temp., 50°C. Initial column inlet pressure, 278 bars. Protein samples were carboxymethylated and subsequently digested with trypsin following the procedure of Stone et. al.

Fig. 9.3. Tryptic maps of cyctochrome c by (a) nano-LC (250 bar) and (b) pressurized electrochromatography (12 kV, 250 bar (at the inlet vial)). Column 6 cm (20 cm overall length) x 100 pm I.D. packed with ODS (1.5 pm 100 A) eluent 0.1 % v/v trifluoracetic acid in water (pH 2) containing (A) 0 % and (B) 50 % acetonitrile gradient, 0-100 % B in 10 min. Reproduced from [32], with permission.

Determination of the molecular weight difference between the wild- type and the variant TTR obtained by ESI MS limited postulated amino acid substitutions to a relatively small number. Analysis of the tryptic digest of the protein by LC-ESI MS was used to locate more precisely the site of the mutation. Comparison of the tryptic map obtained for the variant with that of a normal sample facilitated detection of the modified tryptic peptide. MS-MS was carried out on the variant tryptic peptide contained in the appropriate chromatographic fractions collected during the LC-ESI MS experiment. 

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