Quantitative cross-linking/mass spectrometry using isotope-labelled cross-linkers.
UNLABELLED
Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue-resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. We found two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible.


BIOLOGICAL SIGNIFICANCE
Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue-resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so. We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Our work lays the foundations of studying the molecular details of biological processes at greater ease than this could be done so far.
['Fischer|Lutz|L|', 'Chen|Zhuo Angel|ZA|', 'Rappsilber|Juri|J|']
D002247:Carbon Isotopes D003432:Cross-Linking Reagents D006801:Humans D007553:Isotope Labeling D013058:Mass Spectrometry D040901:Proteomics D012709:Serum Albumin D012984:Software
Quantitation Cross-linking Mass spectrometry Proteomics Protein dynamics Structural biology