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Anal Chem. 2013 Oct 15;85(20):9664-70. doi: 10.1021/ac401978w. Epub 2013 Sep 23.

Covalent labeling with isotopically encoded reagents for faster structural analysis of proteins by mass spectrometry.

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  • 1Department of Chemistry, University of Massachusetts , Amherst, Massachusetts 01003, United States.

Abstract

Covalent labeling and mass spectrometry (MS) are increasingly being used to obtain higher-order structure of proteins and protein complexes. Because most covalent labels are relatively large, steps must be taken to ensure the structural integrity of the modified protein during the labeling reactions so that correct structural information can be obtained. Measuring labeling kinetics is a reliable way to ensure that a given labeling reagent does not perturb a protein's structure, but obtaining such kinetic information is time and sample intensive because it requires multiple liquid chromatography (LC)-MS experiments. Here we present a new strategy that uses isotopically encoded labeling reagents to measure labeling kinetics in a single LC-MS experiment. We illustrate this new strategy by labeling solvent-exposed lysine residues with commercially available tandem mass tags. After tandem MS experiments, these tags allow the simultaneous identification of modified sites and determination of the reaction rates at each site in a way that is just as reliable as experiments that involve multiple LC-MS measurements.

PMID:
24010814
[PubMed - indexed for MEDLINE]
PMCID:
PMC3819941
Free PMC Article
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