Minimizing Back Exchange in the Hydrogen Exchange-Mass Spectrometry Experiment

Benjamin T. Walters 0 1 Alec Ricciuti 0 1 Leland Mayne 1 S. Walter Englander 0 1 0 Graduate Group in Biochemistry and Molecular Biophysics, University of Pennsylvania , Philadelphia, PA 19104, USA 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 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 (G20 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 %. - T hydrogens with the hydrogens in water depends on he naturally occurring exchange of protein amide and therefore can provide detailed information about protein structure, biophysical properties, and functional behavior, in Benjamin T. Walters and Alec Ricciuti contributed equally. principle resolved to the amino acid level. This powerful capability has been very widely exploited in HX NMR studies but routine NMR analysis is limited to relatively small, highly soluble proteins that are available in quantity and labeled with stable isotopes. Hydrogen exchange investigations of larger and biologically more interesting protein systems can be achieved by a proteolytic fragmentation method [1] followed by mass spectrometry analysis [25]. In this method, protein samples taken from an H-D exchange experiment are proteolytically fragmented and separated in preparation for MS analysis to determine the quantity and position of carried D-label at a fragment-resolved level. The comparison of high quality data for many overlapping fragments promises to provide HX information at the amino acid-resolved level [68]. This is the capability that has made the HX NMR method so valuable. A problem is that some D-label is variably lost during sample preparation due to back exchange in the H2O solutions used. The different residues in any given peptide fragment unavoidably lose Dlabel at different rates [9], and this residue-level variability cannot be reconstructed and corrected when one has only fragment-level data. The problem can only be minimized by reducing the level of back exchange. A related HX MS method uses electron transfer or capture dissociation to produce a series of protein fragments that differ by only one terminal residue [3, 1020]. Comparison of results for overlapping fragments can then resolve the position and quantity of carried D-label at the individual residue level by simple subtraction. This method can use direct whole molecule sample injection and thus minimizes the sample preparation steps that allow back exchange. However, it seems likely that analysis of large proteins will still rely on submolecular protein fragments prepared as just described. In this case, back exchange will continue to be a problem. Because back exchange quickly degrades HX MS analysis, it continues to receive a great deal of attention [8, 12, 2129]. The typical level of D-label recovery reported in the fragment separation literature is about 70 % (30 % back exchange). Higher reported values generally depend on results for only one or a few peptides. However, we find that different peptide fragments experience a wide range of back exchange values. Among other implications, any computational correction for back exchange using reference peptides will be flawed. This is true even for direct measurement of back exchange in the peptide of interest since different amide sites will be labeled in experimental and reference situations. We systematically (...truncated)