Ryan Bomgarden1, Erum Raja1, Chris Etienne1; Fan Liu4, Albert Heck4, Mathias Mueller2, Rosa Viner3
Purpose: To improve identification of intra- and inter-protein interactions though analysis of chemically crosslinked peptides.
Methods: Different amine-reactive, homobifuctional crosslinkers including disuccinimidyl suberate (DSS), bis-sulfosuccinimidyl suberate (BS3), disuccinimidyl sulfoxide (DSSO)1 and disuccinimidyl dibutyric urea (i.e. NHS-BuUrBu-NHS)2 (Figure 1) were compared for protein crosslinking labeling efficiency and crosslinked peptide identification using MS2 and MS3 fragmentation methods. A Thermo Scientific™ Orbitrap™ Fusion Lumos™ Tribrid™ mass spectrometer was used for crosslinked peptides analysis. Data analysis was performed by Thermo Scientific™ Proteome Discoverer™ using a XlinkX3 software node.
Results: For both DSSO and BuUrBu, we identified over 40 BSA inter-crosslinked peptides using MS2-MS3 approach compared to less than 20 using MS2 CID for DSSO. We also compared these crosslinkers using an E. coli whole cell lysate. Our results show an increase number of identified peptides after crosslinking using the MS2-MS3 in combination with EThcD method compared to CID/EThcD MS2 method.
1Thermo Fisher Scientific, Rockford, IL
2Thermo Fisher Scientific, Bremen, Germany
3Thermo Fisher Scientific, San Jose, CA
4Utrecht University, the Netherlands
United States
