on 06-12-201801:58 PM - edited on 10-15-202111:57 AM by Closed Account
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