Peptide mapping fragments

The primary goal of peptide mapping is the verification of the amino acid sequence deduced from the genetic code of the recombinant protein. The protein backbone gets cleaved by typically two or three different endoproteinases like Lys-C, trypsin, and Glu-C to achieve maps with sequence-overlapping peptide fragments. These peptide mixtures can then be separated by LC or CE and analyzed on-line by MS to obtain sequence information. Often simple mass analysis matches the predicted primary sequence of the protein. However, sometimes mutations can lead to isobaric masses of peptides that can be overseen, if no further sequence analysis like N-terminal Edman sequencing and MS/MS is carried out. 

Figure 10.6 Peptide mapping of PIXY321. (A) Cleavage of the primary sequence with Lys-C endoprotease results in 14 theoretical fragments. (B) As detailed in the text, LC-MS analysis of the Lys-C digest shows that a few peptides, notably LI, L7, and L14, elute at several retention times due to heterogeneity of both their glycan structures and amino acid sequence. The insets illustrate the complexity of the mass spectra of glycopeptide LI eluting under peaks 17 (inset C) and 18 (inset D).

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. 

The second part of the proof of colinearity of DNA and protein sequences was the determination of the complete amino acid sequence of tryptophan synthase and peptide mapping (Chapter 3) of fragments of the mutant enzymes. From the peptide maps it was possible to identify altered peptides and to establish the exact nature of the amino acid substitutions present in a variety of different tryptophan auxotrophs. When... 

In peptide mapping, a protein is enzimatically or chemically cleaved into small peptide fragments, and the resultant mixture is separated by HPLC to generate a peptide map, or fingerprint,... 

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

Peptide mapping studies, generated by the cleavage of a protein into peptide fragments, must be highly reproducible and quantitative. Several electropherograms of protein digests have been obtained when chicken ovalbumin was cleaved by trypsin... 

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