Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology
František Filandr, Vladimir Sarpe, Shaunak Raval, D. Alex Crowder, Morgan F. Khan, Pauline Douglas, Stephen J. Coales, Rosa Viner, Aleem Syed, John A. Tainer, Susan P. Lees‐Miller, David C. Schriemer
Abstract
Abstract We present a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS 2 ) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-curation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS 1 -based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS 2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-curation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.