Computational method allowing Hydrogen-Deuterium Exchange Mass Spectrometry at single amide Resolution

Chris Gessner,John J Skinner,Dionysios P Pantazatos,Thomas J Walsh,Gert Bange,Virgil L Woods,Sabrina Bédard,Wieland Steinchen

doi:10.1038/s41598-017-03922-3

Abstract

Hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (HDXMS) is a rapid and effective method for localizing and determining protein stability and dynamics. Localization is routinely limited to a peptide resolution of 5 to 20 amino acid residues. HDXMS data can contain information beyond that needed for defining protein stability at single amide resolution. Here we present a method for extracting this information from an HDX dataset to generate a HDXMS protein stability fingerprint. High resolution (HR)-HDXMS was applied to the analysis of a model protein of a spectrin tandem repeat that exemplified an intuitive stability profile based on the linkage of two triple helical repeats connected by a helical linker. The fingerprint recapitulated expected stability maximums and minimums with interesting structural features that corroborate proposed mechanisms of spectrin flexibility and elasticity. HR-HDXMS provides the unprecedented ability to accurately assess protein stability at the resolution of a single amino acid. The determination of HDX stability fingerprints may be broadly applicable in many applications for understanding protein structure and function as well as protein ligand interactions.

Highlights

The ultimate advantages of HDXMS in many areas of life sciences[10,11,12,13,14,15] has led to a rapid expansion of this technology[1] and fostered the development of software packages for high-throughput analysis[16, 17]
In order to generate a sufficient amount of overlapping peptides covering the complete amino acid sequence of spectrin, we employed a combination of the proteases pepsin and protease type XIII from Aspergillus saitoi (FPXIII) during LCMS analysis
W21 and W94 have already been shown to significantly confer stability of the triple helical core[27, 28], consolidating the validity of our High resolution (HR)-HDXMS approach to determine a stability fingerprint. These results clearly demonstrate that our HR-HDXMS approach allows unraveling the contribution of single amino acids to the overall dynamics of protein structures exemplified by spectrin

Summary

Introduction

The ultimate advantages of HDXMS in many areas of life sciences[10,11,12,13,14,15] has led to a rapid expansion of this technology[1] and fostered the development of software packages for high-throughput analysis[16, 17]. Analysis of HDXMS data at single amino acid resolution would lead to a much greater insight and understanding of protein structure, dynamics and function. We present a software tool enabling HDXMS analysis at highest resolution (HR-HDXMS) by determining single amide HDX rates from acquired HDXMS centroid data. HR-HDXMS utilizes D-incorporation from multiple overlapping peptide fragments and multiple D-labeling time points to determine single amide HDX rates in proteins. These can be collectively presented as the HDX stability fingerprint of a given protein. Our application demonstrates the utility of HR-HDXMS and HDX stability profiles for evaluating and understanding protein structure and function

Objectives

Methods

Results

Conclusion