on 06-19-201708:28 AM - edited on 10-15-202105:11 AM by Closed Account
1Rosa Viner, 2Ryan Bomgarden, 2Kratika Singhal, 2Sergei Snovida, 3Craig Gutierrez, 3Lan Huang ASMS 2017 Poster Purpose: To evaluate multiple, widely used enrichment/fractionation techniques and benchmark newly developed strong cation exchange (SCX) spin columns for cross-linked peptide analysis using a Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer.
Methods: Different amine-reactive, homobifuctional crosslinkers were used to crosslink BSA (monomer) and yeast enolase (homodimer) proteins. Crosslinked samples were reduced, alkylated and digested with trypsin before MS analysis. Cross-linked peptides were pre-fractionated on SCX stage tips, SCX spin columns or a peptide size exclusion chromatography (SEC) column. An Orbitrap Fusion Lumos mass spectrometer was used for crosslinked peptide analysis. Data analysis was performed with Thermo Scientific™ Proteome Discoverer™ 2.2 software using a XlinkX1 software node.
Results: Newly designed spin columns containing a polymer-based strong cation exchange resin were used for selective enrichment of cross-linked peptides. Our simplified 2 fractions salt step gradient enabled identification a similar number of cross-linked peptides as SEC based fractionation.
1 Thermo Fisher Scientific, San Jose, CA
2 Thermo Fisher Scientific, Rockford, IL
3 University of California, Irvine, Irvine, CA