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

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 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...

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome

“…3A, red), indicating the binding of antibody to the corresponding protein segments. We used the HDsite program (20) to more closely define the amino acid positions of HX slowing. HDsite exploits measured D content and the shape of isotopic peptide envelopes, corrects for D-to-H back exchange that occurs during sample analysis, and compares sequentially overlapping peptides.…”

“…H-to-D exchange was measured for MDTCS both in free solution and when bound to an immobilized column-bound scFv, by the HX MS methods previously described (20)(21)(22)(23).…”

“…We used previously described HX MS methods (22,23) together with the ExMS analysis program (21) to identify and characterize many peptide fragments of ADAMTS13. Given an extensive overlapping peptide dataset, the HDsite analysis program (20) is then able to reduce HX MS data to near-single-residue resolution (Fig. 3B, red).…”

“…Nevertheless, the HDsite analysis can discriminate and quantify the D-occupancy of different sites because it exploits more insightful information contained in the isotopic shape of each peptide envelope. The HDsite analysis is able to compute the different residue D-occupancies within each peptide, but it is not possible to assign the different occupancies to specific residues using only single-peptide information (20,45). HDsite identifies the occupied sites by comparing envelope shape information from overlapping peptides that contain the same amino acids.…”

“…However, HX MS has so far been limited to fairly low structural resolution not sufficient for pinpointing binding sites. We exploited recent advances in HX MS methodology and data analysis to determine antibody-binding sites at near-amino acid resolution (20)(21)(22)(23).…”