On the reproducibility of label-free quantitative cross-linking/mass spectrometry

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 pre-processing, Xi for cross-linked peptide identification and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 146 (66% of those identified) could be imported into Skyline and 124 (84% of those imported) were subsequently quantified 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Fränze Müller  

LAB HEAD: Juri Rappsilber

PROVIDER: PXD007250 | Pride | 2018-01-02

REPOSITORIES: Pride