Quantitative Cross-linking/Mass Spectrometry Using Isotope-labeled Cross-linkers and MaxQuant.
The conceptually simple step from cross-linking/mass spectrometry (CLMS) to quantitative cross-linking/mass spectrometry (QCLMS) is compounded by technical challenges. Currently, quantitative proteomics software is tightly integrated with the protein identification workflow. This prevents automatically quantifying other m/z features in a targeted manner including those associated with cross-linked peptides. Here we present a new release of MaxQuant that permits starting the quantification process from an m/z feature list. Comparing the automated quantification to a carefully manually curated test set of cross-linked peptides obtained by cross-linking C3 and C3b with BS(3) and isotope-labeled BS(3)-d4 revealed a number of observations: (1) Fully automated process using MaxQuant can quantify cross-links in our reference data set with 68% recall rate and 88% accuracy. (2) Hidden quantification errors can be converted into exposed failures by label-swap replica, which makes label-swap replica an essential part of QCLMS. (3) Cross-links that failed during automated quantification can be recovered by semi-automated re-quantification. The integrated workflow of MaxQuant and semi-automated assessment provides the maximum of quantified cross-links. In contrast, work on larger data sets or by less experienced users will benefit from full automation in MaxQuant.
From the ‡Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK;
Zhuo A
0000-0003-0811-8348
ZA
Chen
From the ‡Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK;
Lutz
0000-0003-4978-0864
L
Fischer
§Computational Systems Biochemistry, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
Jürgen
J
Cox
From the ‡Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK; ¶Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany juri.rappsilber@ed.ac.uk.
Juri
0000-0001-5999-1310
J
Rappsilber
D003432:Cross-Linking Reagents / Q000737:chemistry*; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods; D040901:Proteomics / Q000379:methods*; D012984:Software
D003432:Cross-Linking Reagents