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 a… Show more
“…We have included a detailed tutorial for this experiment type, in addition to a functional description (Supplemental Experimental Procedures). The HX package supports high-resolution MS. Deuteration data and deuterium distribution values can be extracted from any expanded isotopic envelope, and the framework supports the incorporation of alternative algorithms for deuteration analysis (Liu et al, 2014). …”
SUMMARY
The integration of biophysical data from multiple sources is critical for developing accurate structural models of large multiprotein systems and their regulators. Mass spectrometry (MS) can be used to measure the insertion location for a wide range of topographically sensitive chemical probes, and such insertion data provide a rich, but disparate set of modeling restraints. We have developed a software platform that integrates the analysis of label-based MS data with protein modeling activities (Mass Spec Studio). Analysis packages can mine any labeling data from any mass spectrometer in a proteomics-grade manner, and link labeling methods with data-directed protein interaction modeling using HADDOCK. Support is provided for hydrogen/ deuterium exchange (HX) and covalent labeling chemistries, including novel acquisition strategies such as targeted HX-tandem MS (MS2) and data-independent HX-MS2. The latter permits the modeling of highly complex systems, which we demonstrate by the analysis of microtubule interactions.
“…We have included a detailed tutorial for this experiment type, in addition to a functional description (Supplemental Experimental Procedures). The HX package supports high-resolution MS. Deuteration data and deuterium distribution values can be extracted from any expanded isotopic envelope, and the framework supports the incorporation of alternative algorithms for deuteration analysis (Liu et al, 2014). …”
SUMMARY
The integration of biophysical data from multiple sources is critical for developing accurate structural models of large multiprotein systems and their regulators. Mass spectrometry (MS) can be used to measure the insertion location for a wide range of topographically sensitive chemical probes, and such insertion data provide a rich, but disparate set of modeling restraints. We have developed a software platform that integrates the analysis of label-based MS data with protein modeling activities (Mass Spec Studio). Analysis packages can mine any labeling data from any mass spectrometer in a proteomics-grade manner, and link labeling methods with data-directed protein interaction modeling using HADDOCK. Support is provided for hydrogen/ deuterium exchange (HX) and covalent labeling chemistries, including novel acquisition strategies such as targeted HX-tandem MS (MS2) and data-independent HX-MS2. The latter permits the modeling of highly complex systems, which we demonstrate by the analysis of microtubule interactions.
“…To better define the chemical composition of PTMs and to enable quantitative metabolic labeling experiments in future studies, we developed ultrahigh‐resolution MS methods. In this series of manuscripts, we presented fast and accurate algorithms and programs for modeling spectra with resolved isotopic fine structure . Isotopic fine structure can be used, among other things, to determine the elemental composition of PTMs and to discern between isobaric modifications, including the S 1 and O 2 modifications we observe on the Cys111 residue of SOD1.…”
Conformational change and modification of proteins are involved in many cellular functions. However, they can also have adverse effects that are implicated in numerous diseases. How structural change promotes disease is generally not well understood. This perspective illustrates how mass spectrometry (MS), followed by toxicological and epidemiological validation, can discover disease-relevant structural changes and therapeutic strategies. We (with our collaborators) set out to characterize the structural and toxic consequences of disease-associated mutations and post-translational modifications (PTMs) of the cytosolic antioxidant protein Cu/Zn-Superoxide dismutase (SOD1). Previous genetic studies discovered > 180 different mutations in the SOD1 gene that caused familial (inherited) amyotrophic lateral sclerosis (fALS). Using HDX-MS, we determined that diverse disease-associated SOD1 mutations cause a common structural defect – perturbation of the SOD1 electrostatic loop. X-ray crystallographic studies had demonstrated that this leads to protein aggregation through a specific interaction between the electrostatic loop and an exposed beta-barrel edge strand. Using epidemiology methods, we then determined that decreased SOD1 stability and increased protein aggregation are powerful risk factors for fALS progression, with a combined hazard ratio > 300 (for comparison, a lifetime of smoking is associated with a hazard ratio of ∼15 for lung cancer). The resulting structural model of fALS etiology supported the hypothesis that some sporadic ALS (sALS, ∼80% of ALS is not associated with a gene defect) could be caused by post-translational protein modification of wild-type SOD1. We developed immunocapture antibodies and high sensitivity top-down MS methods, and characterized PTMs of wild-type SOD1 using human tissue samples. Using global-HDX, X-ray crystallography, and neurotoxicology we then characterized toxic and protective subsets of SOD1 PTMs. To cap this perspective, we present proof-of-concept that post-translational modification can cause disease. We show that numerous mutations (N→D; Q→E), which result in the same chemical structure as the PTM deamidation, cause multiple diseases.
“…For a given charge state of an ion, the distance between deuterium exchange-associated peaks in an isotopically resolved distribution is constant (the mass difference between 2 H and 1 H divided by charge). Thus, starting from the monoisotopic m/z , the m/z ’s associated with deuterium-incorporated forms of the natural abundance monoisotopic molecule, the “pseudomonoisotopic peaks” [ 43 ], can be calculated. For example, in the case of human substance P (C-terminally amidated RPKPQQFFGLM), the monoisotopic [M + 2H] 2+ is found at m/z 674.3713 and the pseudomonoisotopic m/z’s corresponding to incorporation of 1, 2, or 3 deuterium are m/z 674.8745, m/z 675.3776, and m/z 675.8808, respectively (Figure 2 A).…”
Section: Methodsmentioning
confidence: 99%
“…However, as resolving power increases, the mass defect associated with different atomic nuclei binding energies leads to the detection to the isotopic fine structure. Indeed, at ultrahigh resolving powers (Figure 1 A) deuterium exchange-associated peaks become distinct from natural abundance heavy isotopic species, enabling a more direct detection of the distribution of deuterium incorporation [ 43 ]. Figure 1 Resolving power and acquisition time required to observe deuterium-associated isotopic fine structure is charge state dependent.…”
BackgroundHydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is confounded by deuterium exchange-associated peaks overlapping with peaks of heavy, natural abundance isotopes, such as carbon-13. Recent studies demonstrated that high-performance mass spectrometers could resolve isotopic fine structure and eliminate this peak overlap, allowing direct detection and quantification of deuterium incorporation.ResultsHere, we present a graphical tool that allows for a rapid and automated estimation of deuterium incorporation from a spectrum with isotopic fine structure. Given a peptide sequence (or elemental formula) and charge state, the mass-to-charge ratios of deuterium-associated peaks of the specified ion is determined. Intensities of peaks in an experimental mass spectrum within bins corresponding to these values are used to determine the distribution of deuterium incorporated. A theoretical spectrum can then be calculated based on the estimated distribution of deuterium exchange to confirm interpretation of the spectrum. Deuterium incorporation can also be detected for ion signals without a priori specification of an elemental formula, permitting detection of exchange in complex samples of unidentified material such as natural organic matter. A tool is also incorporated into QUDeX-MS to help in assigning ion signals from peptides arising from enzymatic digestion of proteins. MATLAB-deployable and standalone versions are available for academic use at qudex-ms.sourceforge.net and agarlabs.com.ConclusionIsotopic fine structure HDX-MS offers the potential to increase sequence coverage of proteins being analyzed through mass accuracy and deconvolution of overlapping ion signals. As previously demonstrated, however, the data analysis workflow for HDX-MS data with resolved isotopic fine structure is distinct. QUDeX-MS we hope will aid in the adoption of isotopic fine structure HDX-MS by providing an intuitive workflow and interface for data analysis.
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