Article pubs.acs.org/ac
Resolving Isotopic Fine Structure to Detect and Quantify Natural Abundance- and Hydrogen/Deuterium Exchange-Derived Isotopomers Qian Liu,† Michael L. Easterling,‡ and Jeffrey N. Agar*,§ †
Department of Chemistry, Brandeis University, Waltham, Massachusetts 02453, United States Bruker Daltonics Inc., Billerica, Massachusetts 01821, United States § Department of Chemistry, Northeastern University, Boston, Massachusetts 02115, United States ‡
S Supporting Information *
ABSTRACT: Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is used for analyzing protein dynamics, protein folding/unfolding, and molecular interactions. Until this study, HDX MS experiments employed mass spectral resolving powers that afforded only one peak per nominal mass in a given peptide’s isotope distribution, and HDX MS data analysis methods were developed accordingly. A level of complexity that is inherent to HDX MS remained unaddressed, namely, various combinations of natural abundance heavy isotopes and exchanged deuterium shared the same nominal mass and overlapped at previous resolving powers. For example, an A + 2 peak is comprised of (among other isotopomers) a two-2H-exchanged/zero-13C isotopomer, a one-2H-exchanged/one-13C isotopomer, and a zero-2H-exchanged/two-13C isotopomer. Notably, such isotopomers differ slightly in mass as a result of the ∼3 mDa mass defect between 2H and 13C atoms. Previous HDX MS methods did not resolve these isotopomers, requiring a natural-abundance-only (before HDX or “time zero”) spectrum and data processing to remove its contribution. It is demonstrated here that high-resolution mass spectrometry can be used to detect isotopic fine structure, such as in the A + 2 profile example above, deconvolving the isotopomer species resulting from deuterium incorporation. Resolving isotopic fine structure during HDX MS therefore permits direct monitoring of HDX, which can be calculated as the sum of the fractional peak magnitudes of the deuterium-exchanged isotopomers. This obviates both the need for a time zero spectrum as well as data processing to account for natural abundance heavy isotopes, saving instrument and analysis time.
H
not readily exchange such that the extent of deuteration correctly reflects protection characteristics of the intact system. These exchanged peptides are spatially separated via liquid chromatography (LC) and introduced to the mass spectrometer where measured peptide masses are compared to similar data sets of nonexchanged systems to determine the extent of deuteration for mapping structural protection features. Improving the realized efficacy of this approach requires independent optimization of all component processes including
ydrogen/deuterium exchange mass spectrometry (HDX MS) is used to characterize protein structure, dynamics, and the interactions between proteins and other molecules. Popular applications include folding and unfolding rates; how solvent conditions, mutation, post-translational modification, and interaction with other molecules affect protein structures; and the quality of biologics. Reviews of the mechanisms and many applications of HDX MS are available.1−3 While standardized approaches to HDX MS have not yet been realized, the workflow is somewhat conserved in the literature. Typically, proteolytic (classically nonspecific) degradation of proteins or protein complexes is carried out in solution under slow exchange reaction conditions. Under these “quench” conditions, nonprotected peptide fragments should © 2013 American Chemical Society
Received: October 16, 2013 Accepted: December 13, 2013 Published: December 13, 2013 820
dx.doi.org/10.1021/ac403365g | Anal. Chem. 2014, 86, 820−825
Analytical Chemistry
Article
proteolytic digestion conditions,4 LC separation methods,5−7 and tandem mass spectrometry (MS/MS) fragmentation techniques.8−12 Automation of these steps in combination with MS/MS of exchange peptides lead to high-throughput platforms (DXMS),13,14 while further gains in robustness coupled with automated data interpretation15−22 recently culminated in an automated commercial HDX MS platform.7 Various combinations of natural abundance isotopomers and the isotopomers resulting from HDX shared the same nominal mass and their peaks therefore overlapped at previous MS resolving powers.23 This complexity, specifically various combinations of natural abundance isotopomers and deuterated isotopomers, is recalcitrant to chromatographic separation. Whereas a Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) at a resolving power >375 000 was able to mass resolve small molecule keto and enol isomers after deuterium labeling,24 higher resolving power is required for (normally larger) peptides. Although such fine structure was not previously resolved, it was shown to influence the peak shape of peptides analyzed by an FTICR MS instruments at resolving power ∼100 00023 and should therefore also affect peak shape for Orbitrap instruments and potentially low mass peptides on modern quadrupole-time-of-flights (Q-TOFs). Recent advances in FTICR MS technology, for example, the compensated dynamically harmonized ICR cell25 and the higher field Orbitrap with optimized geometry, should enable the routine detection of isotopic fine structure and even make its detection difficult to avoid. Ultrahigh resolving power FTICR MS was employed to analyze the isotopic fine structure of a model peptide following HDX, which permitted detection of, as well as characterization of the relative abundance of, peptide isotopomers with precise number of deuterium incorporation separable from isotopic species with the same nominal mass due to the distribution of natural abundance isotopes. Such data could be collected on an LC-compatible time scale (∼2 s) with commercially available technology. Specific data analysis methods were found to be required for high resolving power data and were therefore developed in order to account for resolving HDX-related isotopomers from isotopomers present from the distribution of natural abundance isotopes. It was further demonstrated that the isotopic fine structure affected peak shape in any FTICR MS instrument (commercial ICR or Orbitrap), and failure to account for this decreased the accuracy of HDX MS data analysis.
was generally less than 0.1 ppm over m/z 430 to 1110. Samples were directly infused with a built-in syringe pump at 150 μL/h flow rate. Nebulizer gas was 1 bar and drying gas was 2.2 L/min at 180 °C. Control parameters during acquisition included the following: capillary voltage = −4500 V, source declustering potential = 34 V, source accumulation time = 0.001 s, ion accumulation time = 0.15 s, time-of-flight = 0.001 s, and sidekick extraction voltages = −1.5 V. Data was acquired using either 4 M or 64k data acquisition points with a quadrupole isolation window of m/z 20 or 30 (samples were pure and isolation was used to separate a given charge state). Unless otherwise noted, spectra were generated by averaging 16 scans. HDX Data Processing. Both high-resolution (4 M acquisition points, 4.61 s transient length) and low-resolution (64k acquisition points, 0.07 s transient length) spectra were calibrated internally using the theoretical substance P isotopic mass (
