Enrichment of Cross-Linked Peptides Using Charge-Based Fractional Diagonal Chromatography (ChaFRADIC)
- 5 January 2017
- journal article
- Published by American Chemical Society (ACS) in Journal of Proteome Research
- Vol. 16 (2), 459-469
- https://doi.org/10.1021/acs.jproteome.6b00587
Abstract
Chemical cross-linking of proteins is an emerging field with huge potential for the structural investigation of proteins and protein complexes. Owing to the often relatively low yield of cross-linking products, their identification in complex samples benefits from enrichment procedures prior to mass spectrometry analysis. So far, this is mainly accomplished by using biotin moieties in specific cross-linkers or by applying strong cation exchange chromatography (SCX) for a relatively crude enrichment. We present a novel workflow to enrich cross-linked peptides by utilizing charge-based fractional diagonal chromatography (ChaFRADIC). On the basis of two-dimensional diagonal SCX separation, we could increase the number of identified cross-linked peptides for samples of different complexity: pure cross-linked BSA, cross-linked BSA spiked into a simple protein mixture, and cross-linked BSA spiked into a HeLa lysate. We also compared XL-ChaFRADIC with size exclusion chromatography-based enrichment of cross-linked peptides. The XL-ChaFRADIC approach is straightforward, reproducible, and independent of the cross-linking chemistry and cross-linker properties.Keywords
Funding Information
- Bundesministerium für Bildung und Forschung
- Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen
- Senatsverwaltung für Wirtschaft, Technologie und Forschung des Landes Berlin
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