Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis |
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Authors: | Zhong-Yuan Kan Benjamin T. Walters Leland Mayne S. Walter Englander |
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Affiliation: | aJohnson Research Foundation, Department of Biochemistry and Biophysics, and;bGraduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104 |
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Abstract: | 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 back-exchange 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.Unlike 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 (15N, 13C), 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. Recently reported efforts have moved in this direction (15, 17–24).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. It is also necessary to solve the back-exchange problem, the loss of D label, and the information it carries, which unavoidably occurs during the sample preparation process before sample injection into the mass spectrometer. We have described experimental and computational methods that can produce and rigorously identify and characterize hundreds of peptide fragments (25, 26) and that minimize back exchange (27). This paper demonstrates a straightforward approach that uses these capabilities together with previously unexamined envelope-shape information (isotopic peak amplitude ratios) and a site-resolved back-exchange correction to analyze HX MS results to amino acid resolution. |
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Keywords: | HDX-MS isotope pattern protein biophysics |
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