Tryptic peptides mapping

Methods to measure the structure of biopharmaceuticals include tryptic peptide mapping, HPLC, capillary electrophoresis (CE), mass spectroscopy, and circular dichroism spectra. 

Peptides extracted from casein with N, N-dimethyl formamide have complex electrophoretic patterns identical to those of the fraction first prepared by Long and co-workers and called X-casein (El-Negoumy 1973). These peptides are identical electrophoretically to those released by the action of plasmin, which is present in fresh raw milk, upon asr casein (Aimutis and Eigel 1982). Two of these peptides have tryptic peptide maps and molecular weights identical to those of a pair of the peptides produced by plasmin degradation of asl-casein. These peptides appear to be fragments of a8l-casein which are present in milk as the result of plasmin proteolysis. More definitive information on their primary structure is needed before nomenclature for these fragments can be established. 

Further studies (38) in vivo and in vitro showed that the new enzymes produced by the cross between E. coli and S. marcescens were a dimer containing one monomer of the E. coli type and one monomer of the S. marcescens type. By crossing E. coli with an S. marcescens mutant that produced very little alkaline phosphatase, it was shown that the alkaline phosphatase isozymes produced gave the same tryptic peptide map as the E. coli wild-type enzyme with the exception of two new peptides. 

Tryptic peptide mapping. The technique of generating a chromatographic profile characteristic of the fragments resulting from trypsin enzyme cleavage of the protein. 

Figure 6.47 Somidobove LC/MS tryptic peptide mapping. (Reprinted with permission from Chang et al., 1997. Copyright 1997 John Wiley Sons.)...

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. 

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. 5. Tryptic peptide maps of the maize-branching enzymes showing the similarity between the pair BEIIa/BEIIb and differences between the pair and BEI. Approximately 1.25 nmol of peptide digest was chromatographed on a reverse-phase column. Figure reprinted with permission from Singh and Preiss (1985). 

Tryptic peptide mapping of each lectin revealed half of the expected number of peptides (15-16 peptides of the expected 32-37). The lectins share 15 identical, or closely similar, tryptic peptides441 this suggests that each protein is a molecule consisting of identical halves.441 However, a unique peptide was identified for LcH-A which stained gray with ninhydrin. The amino terminal sequence of the first 25 amino acids of the a- and /1-subunits of the pea and the lentil lectins has been determined. It was found that, of the 25 residues analyzed,442a,b,c the N-terminal cc-chain of the lentil and pea lectins differed only at three positions, and the /1-chains at two positions. 

Immunochemical studies, tryptic peptide mapping, and end-group analysis suggested that the toxin and agglutinin may have one common and one unique type of subunit each.146,147,648,658,660-662 Thus, a comparison of tryptic peptide maps gave146 a ratio of identical to unique peptides of 1 1. Furthermore, antisera to either the toxin or the agglutinin cross-reacted with the other protein.146,147,658,660,662... 

Since these low pi rpST forms accounted for approximately 35 % of the total protein as analyzed in Fig. 1 an understanding of their origin was desirable. In order to determine the chemical identity of these low pi species, preparative IPG was used to isolate sufficient quantities for characterization. Fig. 3 shows an IPG gel of the low pi rpST loaded onto the gel (same sample as lane 4, Fig. 2) and several of the individual bands purified from this sample. Each of these isolated proteins was subjected to tryptic peptide mapping. Peptide maps of normal pi rpST and the rpST from lane 4 in Fig. 3 showed several... 

Figure 16.6 Reversed-phase electrospray LC-MS tryptic peptide map of recombinant tissue plasminogen activator. Reprinted from [60] with permission, 1991, American Chemical Society.

S. Julka, F.E. Regnier, Benzoyl derivatization as a method to improve retention of hydrophilic peptides in tryptic peptide mapping. Anal. Chem., 76 (2004) 5799. 

There is great homology between the Bowman-Birk soybean trypsin inhibitor (89), lima bean trypsin inhibitor IV (75) and the Great Northern trypsin inhibitor II (90) as shown in Table VIII The tryptic peptide maps of Great Northern isoinhibitors I and mb were very similar while isoinhibitors I and II had no peptides in common (15) ... 

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