Platform Dependencies in Bottom-up Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract

Hydrogen-deuterium exchange mass spectrometry is an important method for protein structure-function analysis. The bottom-up approach uses protein digestion to localize deuteration to higher resolution, and the essential measurement involves centroid mass determinations on a very large set of peptides. In the course of evaluating systems for various projects, we established two (HDX-MS) platforms that consisted of a FT-MS and a highresolution QTOF mass spectrometer, each with matched front-end fluidic systems. Digests of proteins spanning a 20 -110 kDa range were deuterated to equilibrium, and figures-of-merit for a typical bottom-up (HDX-MS) experiment were compared for each platform. The Orbitrap Velos identified 64% more peptides than the 5600 QTOF, with a 42% overlap between the two systems, independent of protein size. Precision in deuterium measurements using the Orbitrap marginally exceeded that of the QTOF, depending on the Orbitrap resolution setting. However, the unique nature of FT-MS data generates situations where deuteration measurements can be inaccurate, because of destructive interference arising from mismatches in elemental mass defects. This is shown through the analysis of the peptides common to both platforms, where deuteration values can be as low as 35% of the expected values, depending on FT-MS resolution, peptide length and charge state. Hydrogen-deuterium exchange mass spectrometry (HDX-MS)1 provides a powerful means to study the link between protein structure and function (1). The method involves a chemical process in which labile hydrogens within a protein are exchanged with hydrogen from bulk water. When D 2 O is used in place of H 2 O, a mass shift results at every point of exchange, but it is the backbone amide hydrogens that offer exchange rates on a measurable timescale (2, 3). Measuring an amide hydrogen exchange rate can provide access to conformational dynamics, stability, and the interaction characteristics in that location of structure (4, 5). H/D exchange rates have be used to explore mechanisms of protein folding (6), determine the allosteric impact of post-translational modifications and ligand binding (7, 8), define truncation points for enhancing crystallization success (9), and they have also found a role in mapping interactions between proteins (10). Applications have stepped outside of primary research to include the characterization of protein drugs for stability and similarity testing (11-13). The capacity to provide such information has attracted increased attention from regulatory bodies and is generating a push for standardizing HDX methods. Mass spectrometers are very effective tools for measuring exchange rates, from whole proteins down to the individual amide levels. Classical methods of rate measurement have used NMR (3), but mass spectrometry offers all the advantages of speed, sensitivity and scale that have made the tool so useful in proteomics. Measurements at the peptide level provide an important intermediate resolution. As with bottom-up proteomics, r...

Isotope Labeling of Biomolecules

“…More recent FT-based instruments offer even higher resolution, but suffer from potential destructive interference effects that can result in inaccurate deuteration measurements (Burns, Rey, Baker, & Schriemer, 2013). Another consideration with the mass spectrometer is that the heating during ESI will also result in some back-exchange (Katta & Chait, 1993).…”

Mass Spec Studio for Integrative Structural Biology

“…The samples were quenched and digested at 10°C for 2.5 min using nepenthesin (Rey et al, 2013, pH 2.4. The sample was injected into an automated quadrupole time-of-flight-based HX-MS system previously described, employing an AB Sciex 5600 TripleTOF in MS mode (Burns et al, 2013) for mass analysis (positive ion mode, m/z 300–1,250). Samples were run in quadruplicate for each nucleotide state and time-point.…”

Lysine residues in the N-terminal huntingtin amphipathicα-helix play a key role in peptide aggregation

“…Mass spectrometry (MS), however, is capable of site‐specific analyses of intact proteins through the use of both ‘top‐down’ and ‘bottom‐up’ techniques coupled to isotopic labeling strategies, such as HDX . ‘Bottom‐up’ HDX techniques are the most prevalent in the literature . For these methods, protein labeling occurs in a deuterium‐rich solvent, such as D 2 O.…”

“…[29][30][31] 'Bottom-up' HDX techniques are the most prevalent in the literature. [32][33][34][35] For these methods, protein labeling occurs in a deuterium-rich solvent, such as D 2 O. The exchange is quenched by rapid addition of a highly protic solvent, such as formic or acetic acid, to pH 3 and the temperature is decreased to 0°C.…”