title: Minimizing back exchange in the hydrogen exchange-mass spectrometry experiment.
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%.
authors.affiliation: 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
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Walters
authors.forename: Alec
authors.initials: A
authors.lastname: Ricciuti
authors.forename: Leland
authors.initials: L
authors.lastname: Mayne
authors.forename: S Walter
authors.initials: SW
authors.lastname: Englander
mesh_terms: D000577:Amides / Q000737:chemistry; D041961:Deuterium Exchange Measurement / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry*; D033902:Periplasmic Binding Proteins / Q000737:chemistry; D059629:Signal-To-Noise Ratio; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013696:Temperature
chemical_list: D000577:Amides; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D010455:Peptides; D033902:Periplasmic Binding Proteins