Abstract
BackgroundProtein-amide proton hydrogen-deuterium exchange (HDX) is used to investigate protein conformation, conformational changes and surface binding sites for other molecules. To our knowledge, software tools to automate data processing and analysis from sample fractionating (LC-MALDI) mass-spectrometry-based HDX workflows are not publicly available.ResultsAn integrated data pipeline (Solvent Explorer/TOF2H) has been developed for the processing of LC-MALDI-derived HDX data. Based on an experiment-wide template, and taking an ab initio approach to chromatographic and spectral peak finding, initial data processing is based on accurate mass-matching to fully deisotoped peaklists accommodating, in MS/MS-confirmed peptide library searches, ambiguous mass-hits to non-target proteins. Isotope-shift re-interrogation of library search results allows quick assessment of the extent of deuteration from peaklist data alone. During raw spectrum editing, each spectral segment is validated in real time, consistent with the manageable spectral numbers resulting from LC-MALDI experiments. A semi-automated spectral-segment editor includes a semi-automated or automated assessment of the quality of all spectral segments as they are pooled across an XIC peak for summing, centroid mass determination, building of rates plots on-the-fly, and automated back exchange correction. The resulting deuterium uptake rates plots from various experiments can be averaged, subtracted, re-scaled, error-barred, and/or scatter-plotted from individual spectral segment centroids, compared to solvent exposure and hydrogen bonding predictions and receive a color suggestion for 3D visualization. This software lends itself to a "divorced" HDX approach in which MS/MS-confirmed peptide libraries are built via nano or standard ESI without source modification, and HDX is performed via LC-MALDI using a standard MALDI-TOF. The complete TOF2H package includes additional (eg LC analysis) modules.Conclusion"TOF2H" provides a comprehensive HDX data analysis package that has accelerated the processing of LC-MALDI-based HDX data in the authors' lab from weeks to hours. It runs in a standard MS Windows (XP or Vista) environment, and can be downloaded or obtained from the authors at no cost.
Highlights
Protein-amide proton hydrogen-deuterium exchange (HDX) is used to investigate protein conformation, conformational changes and surface binding sites for other molecules
The standard "Opti-Time of flight (TOF)" plate used by the authors for HDX experiments accommodates up to 192 samples deposited within pre-etched spots denoted according to rows (A – H) and columns (1 – 24) with each having a unique alphanumeric "Spot_label"
The functions described form a core package of interconnected programs ("Solvent "Explorer/TOF2H") for the semi-automated processing and analysis of HDX data generated via an Liquid Chromatography (LC)-Matrix-Assisted Laser Desorption (MALDI) workflow, from raw spectra and instrument-generated peaklists through back exchange (BE)-corrected, combined deuterium uptake rate plots
Summary
Protein-amide proton hydrogen-deuterium exchange (HDX) is used to investigate protein conformation, conformational changes and surface binding sites for other molecules. Software tools to automate data processing and analysis from sample fractionating (LC-MALDI) massspectrometry-based HDX workflows are not publicly available. Polypeptide backbone amide proton-deuterium exchange (HDX) analysis provides a powerful approach for understanding protein backbone solvent accessibility and conformational change. Our current path around these challenges has been a combination of the above, namely mass-accurate MALDI-TOF analysis in combination with the rapid reversed phase LC nano-fractionation of HDX experimental samples. This is preceded by a series of preliminary LCMS/MS analyses in order to build, saturate, a nonredundant peptide library (done via an LC-MALDI-TOF/ TOF approach). In place of simple LC retention time analysis we correlate "Z" number (critical organic modifier concentration) [1] between HDX and MS/MS library experiments, for each peptide irrespective of LC instrument or solvent mixture
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