On the Reproducibility of Label-Free Quantitative Cross-Linking/Mass Spectrometry

Fränze Müller,Juri Rappsilber,Tania Auchynnikava,Zhuo Angel Chen,Lutz Fischer

doi:10.1007/s13361-017-1837-2

Abstract

Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuccinimidyl] suberate (BS3)-cross-linked human serum albumin (HSA) and evaluated how reproducible cross-linked peptides can be identified and quantified by LC-MS analysis. To make QCLMS accessible to a broader research community, we developed a workflow that integrates the established software tools MaxQuant for spectra preprocessing, Xi for cross-linked peptide identification, and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 124 were subsequently quantified across 10 analyses with coefficient of variation (CV) values of 14% (injection replica) and 32% (reaction replica). Thus our results demonstrate that the reproducibility of QCLMS is in line with the reproducibility of general quantitative proteomics and we establish a robust workflow for MS1-based quantitation of cross-linked peptides.Graphical ᅟ

Highlights

Cross-linking/mass spectrometry (CLMS) has become a powerful tool aiding the structural analysis of proteins and their complexes [1,2,3,4,5] since its onset almost two decades ago [6, 7]
We demonstrate that cross-linked residue pairs are identified with reproducibility and saturation characteristics that resembles random sampling in standard shotgun proteomics [37]
Additional injections improve the number of identifications and increase variability between runs caused by random sampling

Summary

Introduction

Cross-linking/mass spectrometry (CLMS) has become a powerful tool aiding the structural analysis of proteins and their complexes [1,2,3,4,5] since its onset almost two decades ago [6, 7]. Previous studies have used CLMS to investigate the structures of single proteins [8], multi-protein complexes [9], and protein–protein interaction networks [10, 11]. The proteins in these studies are often undergoing dynamic conformational changes, which are difficult to determine and visualize by

Methods

Results

Conclusion