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An optimized enrichment strategy for improved mass spec analysis of chemically crosslinked peptides

Reputable Mentor II
Reputable Mentor II
Leigh Foster1, Ryan Bomgarden1, Erum Raja1, Chris Etienne1, Rosa Viner2, John Rogers1
ASMS 2018
Purpose: To optimize the sample preparation and enrichment workflow using an azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (aaDSBSO) crosslinker for studying protein-protein interactions. Methods: Amine-reactive, MS-cleavable crosslinkers including DSSO and aaDSBSO were used to crosslink standard proteins and cell lysates. Crosslinked samples were reduced, alkylated and digested with trypsin/LysC before enrichment and LC-MS analysis. For azido-containing crosslinkers, samples were labeled with different biotinylation probes before reduction, alkylation, and digestion, and enriched using different biotin capture resins. Enriched/fractionated BSA samples were separated using a 15cm Thermo Scientific™ EASY-Spray™ column and Thermo Scientific™ UltiMate™ 3000 UHPLC system in 75min gradient, followed by the detection on the Thermo Scientific™  Orbitrap™ Fusion™ Tribrid™ mass spectrometer.  Data were analyzed using Thermo Scientific™ Proteome Discoverer™ 2.2 software and XlinkX node. Results: Our aaDSBSO sample preparation workflow optimized protein crosslinking, biotinylation, enrichment, elution, acid-cleavage and clean up resulting in more crosslinked peptide identifications for BSA and HeLa cell proteins while decreasing the total sample preparation time from 3.5 days to under 2 days.

1Thermo Fisher Scientific, Rockford, IL; 2Thermo Fisher Scientific, San Jose, CA
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Last update:
‎10-15-2021 11:57 AM
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