Many Overlapping Peptides for Protein Hydrogen Exchange Experiments by the Fragment Separation-Mass Spectrometry Method |
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Authors: | Leland Mayne Zhong-Yuan Kan Palaniappan Sevugan Chetty Alec Ricciuti Benjamin T Walters S Walter Englander |
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Institution: | (1) Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 1006 Stellar-Chance Labs, 422 Curie Boulevard, Philadelphia, PA 19104, USA;(2) Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;(3) Department of GI, Nutrition, and Hepatology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA |
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Abstract: | Measurement of the naturally occurring hydrogen exchange (HX) behavior of proteins can in principle provide highly resolved
thermodynamic and kinetic information on protein structure, dynamics, and interactions. The HX fragment separation-mass spectrometry
method (HX-MS) is able to measure hydrogen exchange in biologically important protein systems that are not accessible to NMR
methods. In order to achieve high structural resolution in HX-MS experiments, it will be necessary to obtain many sequentially
overlapping peptide fragments and be able to identify and analyze them efficiently and accurately by mass spectrometry. This
paper describes operations which, when applied to four different proteins ranging in size from 140 to 908 residues, routinely
provides hundreds of useful unique peptides, covering the entire protein length many times over. Coverage in terms of the
average number of peptide fragments that span each amino acid exceeds 10. The ability to achieve these results required the
integrated application of experimental methods that are described here and a computer analysis program, called ExMS, described
in a following paper. |
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