Rosa Viner1, Kai Fritzemeier2 , Frank Berg2 , Torsten Ueckert2 , Leigh Foster3 , Ryan Bomgarden3 , Richard A. Scheltema 4 , Albert J.R. Heck 4 , Clinton Yu5 , Lan Huang5 ;
Purpose: Development of complete analytical workflow in Thermo Scientific™ Proteome Discoverer™ 2.3 software for identification and multiplex quantitation of cross-linked peptides and proteins.
Methods: Cytochrome C and rabbit 20S proteasome complex in PBS buffer (pH 7.4) or HEPES buffer (pH 8.0) were reacted with DSSO in a molar ratio of 1:5 or 1:100 (protein:cross-linker) for 1 h at room temperature. Cross-linked proteins were then digested and TMT labeled according to manufacturer’s instructions. Cross-linked peptides were pre-fractionated on SCX spin columns or a peptide size exclusion chromatography (SEC) column. A Thermo Scientific™ Orbitrap™ Fusion Lumos™ Tribrid™ mass spectrometer was used for crosslinked peptide analysis. Data analysis was performed with Thermo Scientific™ Proteome Discoverer™ 2.3 software using a XlinkX 2.0 software node.
Results: Quantitation of Multiplexed, Isobaric-labeled cross (X)-linked peptides or QMIX1 is a multiplexed QXL-MS strategy that combines MS-cleavable cross-linkers with isobaric labeling reagents and novel hybrid mass spec acquisition methods. We successfully applied this workflow to measure changes in the structural dynamics of rabbit 20s proteasome complex in the presence of different concentrations of SDS.
1 Thermo Fisher Scientific, San Jose, CA
2 Thermo Fisher Scientific, Bremen, Germany
3 Thermo Fisher Scientific, Rockford, IL
4 Utrecht University, Utrecht, The Netherlands
5 University of California, Irvine, Irvine, CA
United States
