Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology

Adam Belsom, Michael Schneider, Lutz Fischer, Oliver Brock, Juri Rappsilber

Research output: Contribution to journalArticlepeer-review


Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low resolution of cross-linking has so far precluded its use in determining structures de novo. Cross-linking resolution has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high-resolution photo-cross-linking with conformational space search to investigate the structure of human serum albumin (HSA) domains, from purified samples, and in it's native environment, human blood serum. Our approach is able to determine HSA domain structures with good accuracy: RMSD to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8 Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on HSA demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.

Original languageEnglish
Pages (from-to)1105-1116
JournalMolecular & Cellular Proteomics (MCP)
Issue number3
Publication statusPublished - 18 Sep 2015


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