Advantages of isotopic depletion of proteins for hydrogen/deuterium exchange experiments monitored by mass spectrometry

George M. Bou-Assaf, Jean E. Chamoun, Mark R. Emmett, Piotr G. Fajer, Alan G. Marshall

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

Solution-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein?protein interactions and conformational changes in biological systems, especially when traditional methods such as X-ray crystallography or nuclear magnetic resonance are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein molecular weight(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addition, different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of 13C and/or 15N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species ( 12Cc1Hh14N n16Oo32Ss), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data reduction.

Original languageEnglish (US)
Pages (from-to)3293-3299
Number of pages7
JournalAnalytical Chemistry
Volume82
Issue number8
DOIs
StatePublished - Apr 15 2010
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry

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