Improved Sequence Resolution by Global Analysis of Overlapped Peptides in Hydrogen/Deuterium Exchange Mass Spectrometry

Fajer, Piotr G.; Bou-Assaf, George M.; Marshall, Alan G.

doi:10.1007/s13361-012-0373-3

Cited by 33 publications

(43 citation statements)

References 23 publications

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“…Peptides may be detected with more sensitivity than proteins, and samples of considerably higher complexity can be interrogated (11). Analytically, this shifts the focus toward optimizing protein digestion, to cover 100% of the sequence and generate a high degree of overlapping fragments to increase opportunities for localizing the deuterium label at high resolution (12)(13)(14).…”

mentioning

confidence: 99%

Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry

Rey

Yang

Burns

et al. 2013

Molecular & Cellular Proteomics

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Studies of protein dynamics, structure and interactions using hydrogen/deuterium exchange mass spectrometry (HDX-MS) have sharply increased over the past 5-10 years. The predominant technology requires fast digestion at pH 2-3 to retain deuterium label. Pepsin is used almost exclusively, but it provides relatively low efficiency under the constraints of the experiment, and a selectivity profile that renders poor coverage of intrinsically disordered regions. In this study we present nepenthesin-containing secretions of the pitcher plant Nepenthes, commonly called monkey cups, for use in HDX-MS. We show that nepenthesin is at least 1400-fold more efficient than pepsin under HDX-competent conditions, with a selectivity profile that mimics pepsin in part, but also includes efficient cleavage C-terminal to "forbidden" residues K, R, H, and P. High efficiency permits a solution-based analysis with no detectable autolysis, avoiding the complication of immobilized enzyme reactors. Relaxed selectivity promotes high coverage of disordered regions and the ability to "tune" the mass map for regions of interest. Nepenthesin-enriched secretions were applied to an analysis of protein complexes in the nonhomologous endjoining DNA repair pathway. The analysis of XRCC4 binding to the BRCT domains of Ligase IV points to secondary interactions between the disordered C-terminal tail of XRCC4 and remote regions of the BRCT domains, which could only be identified with a nepenthesin-based workflow. HDX data suggest that stalk-binding to XRCC4 primes a BRCT conformation in these remote regions to support tail interaction, an event which may be phosphoregulated. We conclude that nepenthesin is an effective alternative to pepsin for all HDX-MS applications, and especially for the analysis of structural transitions among intrinsically disordered proteins and their binding partners. Molecular & Cellular

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confidence: 99%

Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry

Rey

Yang

Burns

et al. 2013

Molecular & Cellular Proteomics

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“…We find that the effort to achieve residue resolution is not limited by a difficult calculational barrier (17,18,20) but by experimental difficulties. To reach this goal, it is necessary to obtain many sequentially overlapping peptide fragments that cover the experimental protein several times over.…”

Section: Hdx-ms | Isotope Pattern | Protein Biophysicsmentioning

confidence: 92%

“…More penetrating conclusions could be drawn if it were possible to extend structural resolution to the individual amino acid level. Recently reported efforts have moved in this direction (15,(17)(18)(19)(20)(21)(22)(23)(24).…”

Section: Hdx-ms | Isotope Pattern | Protein Biophysicsmentioning

confidence: 99%

“…However, in the absence of a common terminus, the D occupancy of the overhanging residues cannot be obtained by simple subtraction of the D-dependent mass centroids of neighboring peptides, as for the ECD/ETD dataset. Site-resolution analysis must consider simultaneously the multiple distributed segments and overhangs in the entire peptide collection (15,(17)(18)(19)(20). We find that this kind of analysis can be greatly improved by the use of an aspect of the MS data that has been ignored before, namely the multiple peaks in the isotopic envelope of each peptide fragment rather than just the single parameter mass centroid.…”

Section: Fig 1 Diagrams Two Sets Of Peptides With Different Overlap mentioning

confidence: 99%

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Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis

Kan

Walters

Mayne

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

132

162

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Hydrogen exchange technology provides a uniquely powerful instrument for measuring protein structural and biophysical properties, quantitatively and in a nonperturbing way, and determining how these properties are implemented to produce protein function. A developing hydrogen exchange-mass spectrometry method (HX MS) is able to analyze large biologically important protein systems while requiring only minuscule amounts of experimental material. The major remaining deficiency of the HX MS method is the inability to deconvolve HX results to individual amino acid residue resolution. To pursue this goal we used an iterative optimization program (HDsite) that integrates recent progress in multiple peptide acquisition together with previously unexamined isotopic envelope-shape information and a site-resolved backexchange correction. To test this approach, residue-resolved HX rates computed from HX MS data were compared with extensive HX NMR measurements, and analogous comparisons were made in simulation trials. These tests found excellent agreement and revealed the important computational determinants.HDX-MS | isotope pattern | protein biophysics U nlike any other method, hydrogen exchange (HX) behavior encodes detailed quantitative information at amino acid resolution on the biophysical factors that produce protein function-structure, structure change, interactions, dynamics, and energetics. HX behavior is very sensitive to these properties, and the chemistry (1, 2) and structural physics (3, 4) of HX processes in these terms are now well understood. The ability of HX to measure protein biophysical properties and their implementation in protein function has been demonstrated over the last 30 y by many NMR studies. However, routine NMR analysis is limited to small highly soluble proteins that can be obtained in quantity (multi mgs), isotopically labeled ( 15 N, 13 C), and studied at millimolar concentration. A developing technology, HX measured and analyzed by mass spectrometry (HX MS) (5-16) can extend this proven capability to the much larger and more complex protein systems that make biology work. The method requires only picomoles of protein at submicromolar concentrations; it can be used to study the properties and functioning of proteins at any condition one chooses, and the experimental protein need not even be very pure.For HX MS analysis, a protein sample taken from any H-D exchange experiment is quenched into slow HX conditions, proteolytically fragmented, and the peptide fragments are separated and analyzed by HPLC and mass spectrometry. The number of D atoms carried on each peptide fragment is given by its measurable increase in mass, usually calculated as the increment in the peptide mass centroid. These results provide structural information resolved to the level of individual fragments and are broadly able to indicate where in the protein important behavior occurs (5-16). More penetrating conclusions could be drawn if it were possible to extend structural resolution to the individual amino acid level. Re...

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“…16 This approach was later improved by constructing a two-stage sampling method to produce quantitative rate constants. 17 Finally, the program HDSite introduces further improvements, such as adding information from the shape of the MS envelope. 18 …”

Section: Introductionmentioning

confidence: 99%

A Residue-Resolved Bayesian Approach to Quantitative Interpretation of Hydrogen–Deuterium Exchange from Mass Spectrometry: Application to Characterizing Protein–Ligand Interactions

et al. 2016

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Characterization of interactions between proteins and other molecules is crucial for understanding the mechanisms of action of biological systems and, thus, drug discovery. An increasingly useful approach to mapping these interactions is measurement of hydrogen/deuterium exchange (HDX) using mass spectrometry (HDX-MS), which measures the time-resolved deuterium incorporation of peptides obtained by enzymatic digestion of the protein. Comparison of exchange rates between apo- and ligand-bound conditions results in a mapping of the differential HDX (ΔHDX) of the ligand. Residue-level analysis of these data, however, must account for experimental error, sparseness and ambiguity due to overlapping peptides. Here, we propose a Bayesian method consisting of a forward model, noise model, prior probabilities, and a Monte Carlo sampling scheme. This method exploits a residue-resolved exponential rate model of HDX-MS data obtained from all peptides simultaneously, and explicitly models experimental error. The result is the best possible estimate of ΔHDX magnitude and significance for each residue given the data. We demonstrate the method by revealing richer structural interpretation of ΔHDX data on two nuclear receptors: vitamin D-receptor (VDR) and retinoic acid receptor gamma (RORγ). The method is implemented in HDX Workbench and as a standalone module of the open source Integrative Modeling Platform.

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Improved Sequence Resolution by Global Analysis of Overlapped Peptides in Hydrogen/Deuterium Exchange Mass Spectrometry

Cited by 33 publications

References 23 publications

Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry

Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry

Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis

A Residue-Resolved Bayesian Approach to Quantitative Interpretation of Hydrogen–Deuterium Exchange from Mass Spectrometry: Application to Characterizing Protein–Ligand Interactions

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