Two tools for applying chromatographic retention data to the mass-based identification of peptides during hydrogen/deuterium exchange experiments by nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry

Two tools are described for integrating LC elution position with mass-based data in hydrogen-deuterium exchange (HDX) experiments by nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry (nanoLC/MALDI-MS, a novel approach to HDX-MS). The first of these, 'TOF2H-Z Comparator', highlights peptides in HDX experiments that are potentially misidentified on the basis of mass alone. The program first calculates normalized values for the organic solvent concentration responsible for the elution of ions in nanoLC/MALDI HDX experiments. It then allows the solvent gradients for the multiple experiments contributing to an MS/MS-confirmed peptic peptide library to be brought into mutual alignment by iteratively re-modeling variables among LC parameters such as gradient shape, solvent species, fraction duration and LC dead time. Finally, using the program, high-probability chromatographic outliers can be flagged within HDX experimental data. The role of the second tool, 'TOF2H-XIC Comparator', is to normalize the LC chromatograms corresponding to all deuteration timepoints of all HDX experiments of a project, to a common reference. Accurate normalization facilitates the verification of chromatographic consistency between all ions whose spectral segments contribute to particular deuterium uptake plots. Gradient normalization in this manner revealed chromatographic inconsistencies between ions whose masses were either indistinguishable or separated by precise isotopic increments.