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Quantitative photo-crosslinking mass spectrometry reveals protein structure response to environmental changes

ABSTRACT: Conformational changes of protein structures depend on their chemical and physical environment. Studying conformational changes depending on environmental parameters is notoriously difficult as many methods of structural biology are affected by parameters like temperature or pH. To make such conformational changes accessible to quantitative crosslinking mass spectrometry (QCLMS) approaches, crosslinking chemistry must be invariant to different conditions. We propose this can be achieved by photo-inducible crosslinkers, which are not influenced by changes in environmental parameters. Here, we introduce a workflow combining photo-crosslinking using 4,4’-azipentanoate (sulfo-SDA) with our recently developed data-independent acquisition (DIA)-QCLMS. In this study, we use this novel photo-DIA-QCLMS approach to quantify pH-dependent conformational changes in human serum albumin and cytochrome C. Both proteins show pH dependent conformational changes resulting in acidic and alkaline transitions. 93% and 95% unique residue pairs (URP) were quantifiable across triplicates for HSA and cytochrome C, respectively. Abundance changes of URPs and hence conformational changes of both proteins, were visualized using hierarchical clustering. For HSA we distinguished the N-F and the N-B form from the native conformation. Additionally, we observed for cytochrome C acidic and basic conformations. In conclusion, photo-DIA-QCLMS distinguished pH-dependent conformers of both proteins.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

SUBMITTER: Fränze Müller

LAB HEAD: Juri Rappsilber

PROVIDER: PXD012939 | Pride | 2019-07-01

REPOSITORIES: Pride

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