Frantisek Filandr

HX-MS2 : Automating Data Validation and Expanding the Scope of Deuteration Analysis

Frantisek Filandr1, Vladimir Sarpe1, Alex Crowder1, Morgan Khan1, David Schriemer1

1. University of Calgary, Calgary, AB, Canada

Introduction: Hydrogen/deuterium exchange mass spectrometry (HX-MS) has been performed using MS1-based measurements since the 1990’s, which restricts it to the detection of high quality and well-isolated isotopic envelopes. There are two problems with this conventional mode of operation. First, the conventional mode restricts applications to protein systems of limited complexity as peptide attrition rates scale with protein size. Second, manual validation of peptide selections is very time consuming and prone to operator error. It is possible to obtain deuteration values from fully scrambled and mass[1]resolved CID fragment ions collected in MS2 mode. However, a robust implementation of this finding is still not available. Here, we show that data independent acquisition (DIA) modes can create an automated “2D” HX-MS method.

Methods: A microflow based HX system and an HDX-PAL autosampler were connected to Sciex Triple TOF 6600 and Thermo Scientific Q Exactive Plus to analyze deuterated phosphorylase B in DIA mode, using standard digestion and quench methods. To analyze the deuteration data resulting from DIA-based acquisitions, a new module was developed for the Mass Spec Studio 2.0. Undeuterated samples were first analyzed in Data Dependent Acquisition (DDA) mode to generate a library of peptide IDs and retention times and used in the analysis of deuterated samples analyzed in DIA mode. Here, we collected kinetics data on the native protein and replicate differential HX data to compare the native and perturbed states. Optimized workflows were developed and applied to several other protein systems.

Preliminary data: We completed a build of AutoHX, a new software package that measures deuterium incorporation from MS1 and MS2 domain data, collected using DIA-based data acquisition systems. The software is built as a plug-in to the Mass Spec Studio and requires the input of peptide features. AutoHX aligns chromatograms for peptide and fragment ions, selects CID fragments for deuteration calculation based on computer vision algorithms, and chooses between MS1 or MS2 derived deuteration values informed by the quality of the underlying identification. We collected HX-DIA data on phosphorylase b, a ~100kDa protein, for the two platforms. Both generated many CID fragments for the majority of peptides detected in the digest. The deuteration precision derived from the fragment data was found to be a function of the number of fragments used in the calculation. We obtain excellent sequence coverage, redundancy, and low deuteration error even with the selection of as few as three fragments. Deuterium uptake curves from a kinetics experiment show that deuteration values from “clean” MS1 signals are virtually identical to MS2-derived values. When differences exist between MS1 and MS2 analyses (due to spectral overlap for example) we show that autovalidated MS2 fragment data can be used to override MS1 data and boost sequence coverage. We also show that deuteration differences between different protein states can be faithfully represented by MS2 domain data. The impact on automation is considerable. We show that full multifile projects can be processed with little to no user intervention, at the click of a button. Any intervention is based solely on selecting filters for the underlying data, creating a verifiable link between data quality metrics and the final deuteration report. DIA-based HX-MS2 is the routine data collection mode in our lab now, and other examples of projects will be shown for illustration.

Novel aspect: A fully automated “2D” HX-MS method for complex mixture analysis based on DIA technology.

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