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Enrichment Strategies for Improving Mass Spec Analysis of Chemically Crosslinked Peptides

Reputable Mentor II
Reputable Mentor II
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
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Last update:
‎10-15-2021 05:11 AM
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