Simultaneous Reduction and Digestion of Proteins with Disulfide Bonds for Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

Abstract

Proteolyzed peptides provide the basis for mass-analyzed hydrogen/deuterium exchange (HDX) for mapping solvent access to various segments of solution-phase proteins. Aspergillus saitoi protease type XIII and porcine pepsin can generate peptides of overlapping sequences and high sequence coverage. However, if disulfide bonds are present, proteolysis can be severely limited, particularly in the vicinity of the disulfide linkage(s). Disulfide bonds cannot be reduced before or during the H/ D exchange reaction without affecting the protein higher-order structure. Here, we demonstrate simultaneous quench/digestion/reduction following H/D exchange, for subsequent mass analysis. Proteolysis is conducted in the presence of TCEP·HCl and urea, and all other steps of the H/D exchange and analysis are maintained. This method yields dramatically increased sequence coverage and localization of solvent exposed segments for mass-analyzed solution-phase H/D exchange of proteins containing disulfide bonds.

The Protein Targeting Factor Get3 Functions as ATP-Independent Chaperone under Oxidative Stress Conditions

“…Since pepsin digestion of TCEP-reduced Get3 ox was indistinguishable from the digestion pattern of Get3 red that was never oxidized, we conducted all subsequent pH quench and pepsin digests in the presence of 100 mM TCEP. By using this method, which has been previously established to avoid differences in pepsin coverage when comparing oxidized and reduced proteins (Zhang et al, 2010), we were able to directly compare the mass differences in the individual Get3 peptides over time (Table S1). As shown in Figure S3C and Figure 4, analysis of the H/D exchange rates in Get3 ox versus Get3 red peptides revealed astounding differences.…”

Hydrogen/deuterium exchange mass spectrometry and optical spectroscopy as complementary tools for studying the structure and dynamics of a membrane protein

“…Nonetheless, results obtained for a number of other systems [26,27,29] suggest that spatially-resolved HDX/ESI-MS studies on membrane proteins will soon cease to be considered a fringe area. We are confident that that this development will be spurred by ongoing improvements of the HDX and digestion workflow [85][86][87], possibly in combination with the application of electron-based dissociation techniques [88,89].…”

Chemical inhibition of prometastatic lysyl-tRNA synthetase–laminin receptor interaction

“…Briefly, 5 μl of a sample (20 μM) was mixed with 45 μl of 20 mM HEPES, pH 7.5, and 150 mM NaCl in D 2 O to initiate each HDX reaction. HDX incubation periods were 0.5, 1, 2, 4, 8, 15, 30, 60, 120, and 240 min, each followed by simultaneous quench and proteolysis 50 . HDX was quenched by 1:1 (v/v) addition of protease type XIII solution in 1.0% formic acid to reduce the pH to ~2.3 and to initiate the 2-min proteolysis.…”