期刊
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
卷 23, 期 12, 页码 2132-2139出版社
SPRINGER
DOI: 10.1007/s13361-012-0476-x
关键词
Amide hydrogen exchange; Hydrogen/deuterium exchange; Back exchange; HXMS; HDX MS; Deuterium recovery; HX MS
资金
- NIH [RO1 GM031847, GM08275]
- NSF [MCB1020649]
- Mathers Charitable Foundation
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 similar to 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 %.
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