Peptide overlap

Protein sequencing by creation of overlap peptides (mapping),... 

Figure 4-7. The overlapping peptide Z is used to deduce that peptides X and Y are present in the original protein in the order X —> Y, not Y X.

The hybrid approach enhances the speed and efficiency of primary structure analysis and the range of proteins that can be sequenced. It also circumvents obstacles such as the presence of an amino-terminal blocking group or the lack of a key overlap peptide. Only a few segments of primary strucmre must be determined by Edman analysis. 

The most frequent application of SPOT-synthesis has been in the preparation of peptide arrays for the identification of linear B-cell epitopes. If the protein antigen is known, a set of overlapping peptides that encompass the entire sequence can be readily synthesized and assayed for binding of antibody (Reineke et al., 1999). The individual residues critical for binding can then be determined by SPOT-synthesis of peptides containing amino acid substitutions. 

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. 

Thus, with this goal In mind, and In order to circumvent the aforementioned problems and render the determination of protein antigenic structures more feasible within a reasonable time, we have devised (5) a novel comprehensive synthetic approach designed to yield the full antigenic profile of the protein under study. This approach consists of the direct synthesis and examination of the immunochemical activities of a series of overlapping peptides having uniform size and overlaps and that encompass the... 

Automated peptide synthesis allowing the construction of overlapping peptide fragments of the macromolecule. 

Martens, W., Greiser-Wilke, I., Harder, T.C., et al. (1995) Spot synthesis of overlapping peptides on paper membrane supports enables the identification of linear monoclonal antibody binding determinants on morbillivirus phosphoproteins. Vet. Microbiol. 44, 289-298. 

Identification of relevant epitopes using overlapping peptides spotted onto membranes... 

Ayuso, R., Reese, G., Lehrer, S.B. 1999b. IgE-binding epitopes of Pen a 1 (shrimp tropomyosin) Localization of antigenic regions using overlapping peptides. Journal of Investigative Medicine 47(2) S66-S66. 

Katragadda M, Alderfer JL, Yeagle PL (2001) Assembly of a Polytopic Membrane Protein Structure from the Solution Structures of Overlapping Peptide Fragments of Bacteriorhodopsin. Biophys J. 81(2) 1029-1036... 

A set of overlapping peptides can be prepared from the above data by making use of the fact that one CNBr peptide must end in methionine, one tryptic peptide must end in either lysine or arginine, and one chymotryptic peptide must end in an aromatic amino acid. The sequence of the peptide is, therefore, Gly-Tyr-Ser-Met-Thr-Lys-Ala-Gly. 

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. 

Maruta, T., Dolimbek, B.Z., Aoki, K.R., Steward, L.E., Atassi, M.Z. (2004). Mapping of the synaptosome-binding regions on the heavy chain of botulinum neurotoxin A by synthetic overlapping peptides encompassing the entire chain. Protein J. 23 539-52. 

Figure 4.25. Overlap Peptides. The peptide obtained by chymotryptic digestion overlaps two tryptic peptides, establishing their order.

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