Hekate: Software Suite for the Mass Spectrometric Analysis and Three-Dimensional Visualization of Cross-Linked Protein Samples

Andrew N Holding,Meindert H Lamers,Elaine Stephens,J Mark Skehel

doi:10.1021/pr4003867

Andrew N Holding, Meindert H Lamers + Show 2 more

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https://doi.org/10.1021/pr4003867

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Abstract

Chemical cross-linking of proteins combined with mass spectrometry provides an attractive and novel method for the analysis of native protein structures and protein complexes. Analysis of the data however is complex. Only a small number of cross-linked peptides are produced during sample preparation and must be identified against a background of more abundant native peptides. To facilitate the search and identification of cross-linked peptides, we have developed a novel software suite, named Hekate. Hekate is a suite of tools that address the challenges involved in analyzing protein cross-linking experiments when combined with mass spectrometry. The software is an integrated pipeline for the automation of the data analysis workflow and provides a novel scoring system based on principles of linear peptide analysis. In addition, it provides a tool for the visualization of identified cross-links using three-dimensional models, which is particularly useful when combining chemical cross-linking with other structural techniques. Hekate was validated by the comparative analysis of cytochrome c (bovine heart) against previously reported data.1 Further validation was carried out on known structural elements of DNA polymerase III, the catalytic α-subunit of the Escherichia coli DNA replisome along with new insight into the previously uncharacterized C-terminal domain of the protein.

Highlights

With the field of structural biology moving toward the analysis of larger macromolecular complexes, there is an increasing need for alternative or combinatorial methods for characterization of these complexes
Chemical cross-linking has for nearly 40 years been used to investigate the structure of protein complexes with one of the earliest examples being the use of dimethyl suberimidate to locate neighboring proteins within the ribosomes of Escherichia coli.[11]
The input file is formatted as either Mascot Generic Format (MGF) or mzXML, along with details of the isotopic label used and mass accuracy tolerances

Summary

Introduction

With the field of structural biology moving toward the analysis of larger macromolecular complexes, there is an increasing need for alternative or combinatorial methods for characterization of these complexes. The analysis of protein−protein interactions, native protein structures, and the structure of protein complexes by mass spectrometry is a rapidly advancing field that can be used alone or in combination with structural approaches such as protein crystallography,[2,3] single particle electron microscopy,[4,5] and small-angle X-ray scattering.[6,7] Mass spectrometric techniques such as hydrogen−deuterium exchange (HDX)[8] and the analysis of macromolecular complexes by native mass spectrometry[9,10] can provide useful low-resolution information on the interaction between proteins in a complex. Chemical cross-linking has for nearly 40 years been used to investigate the structure of protein complexes with one of the earliest examples being the use of dimethyl suberimidate to locate neighboring proteins within the ribosomes of Escherichia coli.[11] More recently, these methods have been combined with mass spectrometry to provide spatial information.[12−15]. The effects experimental conditions have on the structures determined by different techniques such as NMR and X-ray crystallography have previously been discussed.[19]

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