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J Am Soc Mass Spectrom. 2012 Dec;23(12):2132-9. doi: 10.1007/s13361-012-0476-x. Epub 2012 Sep 11.

Minimizing back exchange in the hydrogen exchange-mass spectrometry experiment.

Author information

1
Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 1006 Stellar-Chance Labs, 422 Curie Boulevard, Philadelphia, PA 19104, USA. benth@mail.med.upenn.edu

Abstract

The addition of mass spectrometry (MS) analysis to the hydrogen exchange (HX) proteolytic fragmentation experiment extends powerful HX methodology to the study of large biologically important proteins. A persistent problem is the degradation of HX information due to back exchange of deuterium label during the fragmentation-separation process needed to prepare samples for MS measurement. This paper reports a systematic analysis of the factors that influence back exchange (solution pH, ionic strength, desolvation temperature, LC column interaction, flow rates, system volume). The many peptides exhibit a range of back exchange due to intrinsic amino acid HX rate differences. Accordingly, large back exchange leads to large variability in D-recovery from one residue to another as well as one peptide to another that cannot be corrected for by reference to any single peptide-level measurement. The usual effort to limit back exchange by limiting LC time provides little gain. Shortening the LC elution gradient by 3-fold only reduced back exchange by ~2%, while sacrificing S/N and peptide count. An unexpected dependence of back exchange on ionic strength as well as pH suggests a strategy in which solution conditions are changed during sample preparation. Higher salt should be used in the first stage of sample preparation (proteolysis and trapping) and lower salt (<20 mM) and pH in the second stage before electrospray injection. Adjustment of these and other factors together with recent advances in peptide fragment detection yields hundreds of peptide fragments with D-label recovery of 90% ± 5%.

PMID:
22965280
PMCID:
PMC3515739
DOI:
10.1007/s13361-012-0476-x
[Indexed for MEDLINE]
Free PMC Article

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