Selective 351 nm photodissociation of cysteine-containing peptides for discrimination of antigen-binding regions of IgG fragments in bottom-Up liquid chromatography-tandem mass spectrometry workflows

Victoria C. Cotham, Yariv Wine, Jennifer S. Brodbelt

Research output: Contribution to journalArticlepeer-review

Abstract

Despite tremendous inroads in the development of more sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies for mass spectrometry-based proteomics, there remains a significant need for enhancing the selectivity of MS/MS-based workflows for streamlined analysis of complex biological mixtures. Here, a novel LC-MS/MS platform based on 351 nm ultraviolet photodissociation (UVPD) is presented for the selective analysis of cysteine-peptide subsets in complex protein digests. Cysteine-selective UVPD is mediated through the site-specific conjugation of reduced cysteine residues with a 351 nm active chromogenic Alexa Fluor 350 (AF350) maleimide tag. Only peptides containing the AF350 chromophore undergo photodissociation into extensive arrays of b- and y-type fragment ions, thus providing a facile means for differentiating cysteine-peptide targets from convoluting peptide backgrounds. With the use of this approach in addition to strategic proteolysis, the selective analysis of diagnostic heavy-chain complementarity determining regions (CDRs) of single-chain antibody (scAb) fragments is demonstrated.

Original languageEnglish
Pages (from-to)5577-5585
Number of pages9
JournalAnalytical Chemistry
Volume85
Issue number11
DOIs
StatePublished - 4 Jun 2013
Externally publishedYes

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