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Quantitative cross-linking/mass spectrometry using isotope-labeled cross-linkers and MaxQuant

By Zhuo A Chen, Lutz Fischer, J├╝ergen Cox, Juri Rappsilber

Posted 30 May 2016
bioRxiv DOI: 10.1101/055970 (published DOI: 10.1074/mcp.M115.056481)

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 BS3 and isotope-labeled BS3-d4 revealed a number of observations: 1) Fully automated process using MaxQuant can quantify cross-links in our reference dataset 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.

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