Discrimination of Isoleucine and Leucine by Dimethylation-Assisted MS3

Sheila Maibom-Thomsen, Søren Heissel, Ejvind Mørtz, Peter Højrup, Jakob Bunkenborg

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Protein sequencing by mass spectrometry has transformed the field of biopharmaceutical analysis, but a missing part in the analytical toolkit is the ability to distinguish between the isomeric residues isoleucine and leucine because it is a requisite for efficient analysis of the primary structure of proteins. To address this need, we have developed a novel mass spectrometric method that combines reductive dimethylation and MS3 fragmentation with LCMS peptide mapping. The dimethylation of peptide N-termini leads to intense a1-ions upon collision-induced fragmentation, and further fragmentation of the isoleucine/leucine a1-ion leads to informative spectra with fragments that can discriminate between the two isomers. The methodology of a1-directed MS3 was applied to two antibodies in combination with the proteases trypsin, thermolysin, chymotrypsin, and pepsin to generate peptides exposing N-terminal I/L residues.

TidsskriftAnalytical Chemistry
Udgave nummer15
Sider (fra-til)9055–9059
StatusUdgivet - 2018


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