A labelling method to distinguish isobaric amino acids and amino acid combinations

Abstract

A method for increasing peptide fragmentation by labeling the peptide at the C-terminal end with a guanidinium group or other basic functional group and distinguishing isobaric amino acids and amino acid combinations of asparagine and glycine-glycine; glutamine and glycine-alanine; and / or glutamine and alanine-glycine, during polypeptide sequencing. The method involves: obtaining a peptide of interest and / or digesting a polypeptide of interest with a protease, such as pepsin, chymotrypsin or trypsin, or by chemical cleavage to produce shorter peptides; reacting the obtained and / or generated peptides with a coupling reagent to derivatize the free C-terminal carboxylic acid function of the peptides, thus adding a basic functional group rendering C-terminal peptide fragment ions detectable by mass spectrometry; selecting a charge state of 2+ or more, and fragmenting the derivatized peptides in a mass spectrometer under conditions effective to generate at least w ions; and detecting the w ions by mass spectrometry, and identifying derivatized peptides which incorporate the additional mass of the basic functional group.

Description

technical field [0001] The present invention relates to a polypeptide sequencing method. In particular, the invention relates to distinguishing isobaric amino acids and amino acid combinations during polypeptide sequencing: asparagine and glycine-glycine; glutamine and glycine-alanine; and / or glutamine and alanine-glycine Methods. Background technique [0002] For peptide sequencing, mass spectrometry has been used for decades. Typically, a given peptide is isolated and fragmented. At low collision energies, peptide fragmentation occurs at or near peptide bonds. Based on the exact position of the fragmentation pattern, fragment ions, including the N-terminus of the peptide, were named a, b, or c ions, and their respective complementary fragments from the C-terminus were named x, y, or z, respectively. The y1 ion will represent the first amino acid residue on the C-terminal side and b1 will represent the first amino acid residue on the N-terminal side of the peptide; the ...

AI Technical Summary

Problems solved by technology

However, CPY shows a strong sequence-specific bias in terms of transpeptidation and has been mostly studied using simple peptide mixtures (Breddam et al., 1980) or proteins (Xu et al. 2011)

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