Peptide mobility and mapping

Janini, G. M., Metral, C. J., Issaq, H. J., and Muschik, G. M. (1999). Peptide mobility and peptide mapping in capillary zone electrophoresis — experimental determination and theoretical simulation. J. Chromatogr. A 848, 417-433. 

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.

Mobile phase A—6.9 g KHjPOA pH 2.85 mobile phase B—CHjCN, gradient with 100% A -0-30%B at 0.33%/min-I%/min B until 60% B-I0 min 214 nm USP 23, p. 50, peptide mapping, 20 major peaks purity by electrophoresis and single-chain content by size exclusion [5]... 

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.

FIGURE 6 Tryptic peptide mapping of a recombinant protein. A recombinant protein was labeled with mBBr as in Protocol 15, enzymatically cleaved with trypsin as in Protocol 6, and then chromatographed by RP - HPLC on a Vydac 218TP column. Mobile phases A, 0.1 % TFA in water B, 0.1 % TFA in 80% acetonitrile. Gradient 0-5 min, 0% B 5 - 90 min, 0 - 50% B. The flow rate was 1.0 mL / min. Detection was UV absorbance at 218 nm (top chromatogram) and fluorescence with a 470 nm filter after excitation at 382 nm (bottom chromatogram). 

Fig. 8. The peptide map for the tryptic cleavage of apolipoprotein B which was obtained on a Waters radially compressed analytical system with a mobile phase of 1% triethylam-monium phosphate, pH 3.2, and a gradient of acetonitrile. The flow rate was 1 ml/min. Adapted from Hancock and Sparrow (1981d).

Besides the advantages of short elution times, excellent resolution, and high recoveries, a further benefit of these reversed-phase HPLC methods for peptide mapping is the ease of sample preparation. In many cases the crude digest can be loaded directly. The problems associated with overlapping peptides in an otherwise straightforward application can often be remedied by small changes in the pH of the mobile phase or, alternatively, variation in the polarity and concentration of the counterion. 

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