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 high-resolution 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. These findings are supported by simulations of Orbitrap transients, and highlight that caution should be exercised in deriving centroid mass values from FT transients that do not support baseline separation of the full isotopic composition.
Highlights
From the ‡Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada, §Cultivated Code, Inc
We demonstrate that the Orbitrap system returns greater sequencing depth and marginally better precision than the 5600, the measurement accuracy is strongly influenced by destructive interference arising from unequal mass defects between 13C and 2H
System and Samples—A comparison of data from different MS platforms requires the analysis of identical samples, frontend fluidic systems and data processing routines to minimize any bias in the analysis (Fig. 1)
Summary
Platform Dependencies in Bottom-up Hydrogen/Deuterium Exchange Mass Spectrometry*□S. Hydrogen-deuterium exchange mass spectrometry is an important method for protein structure-function analysis. Most users of the HDX-MS method are migrating from low resolution to high resolution systems, operated in a single-stage MS mode This includes FT-MS and higher-resolution QTOF platforms, in this study, we explore how an LTQ Orbitrap Velos (Thermo) and a 5600 TripleTOF (AB Sciex) influence the measurement of deuteration data for proteins of increasing size. We demonstrate that the Orbitrap system returns greater sequencing depth and marginally better precision than the 5600, the measurement accuracy is strongly influenced by destructive interference arising from unequal mass defects between 13C and 2H This has implications for any application that involves centroid mass determinations, beyond HDX-MS
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