Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Evaluation of FAIMS technology for mass spec analysis of chemical cross-linked peptides. (#900)

Rosa Viner 1 , Leigh Foster 2 , Ryan Bomgarden 2 , Michael W Belford 1 , Romain Huguet 1 , Eloy R Wouters 1
  1. Thermo Fisher Scientific, San Jose, CA, United States
  2. Thermo Fisher Scientific, Rockford, IL, USA


Chemical cross-linking in combination with mass spectrometry is a powerful method to determine protein-protein or nucleic acid-protein interactions. This method has been applied to recombinant and native protein complexes and, more recently, to whole cell lysates or intact unicellular organisms in efforts to identify protein-protein interactions on a global scale. However, this method suffers from low identification rates as the typical yield of cross-linked peptides is less than 1 % of total identified peptides. In this study, we compared widely used enrichment/fractionation techniques and the newly developed High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) device for cross-linked peptides.


Amine-reactive, homo-bifunctional crosslinkers, including DSS, DSSO, and DSBU were used to crosslink protein and protein complex standards. Cross-linked samples were reduced, alkylated and digested with trypsin for MS analysis. Cross-linked peptides were pre-fractionated using SCX. Samples were analyzed by LC-MS/MS on the Orbitrap mass spectrometers with/out the Thermo Scientific™ FAIMS Pro™ Interface. Data were analyzed using Thermo Scientific™ Proteome Discoverer™ 2.3 and XlinkX node 2.0.


The identification of cross-linked peptides by LC-MS/MS presents significant analytical challenges due to their low abundance and higher charge state distribution compared to tryptic peptides. Selective enrichment/fractionation of cross-linked peptides by SCX fractionation using an offline LC approach is widely used for improved interaction sites identification; however, identification rates only increase by a maximum of 2-3 folds. To further enhance the detection of cross-linked peptides, we evaluated the use of a FAIMS device for gas-phase fractionation. Different compensation voltages (CV) between -40 and -90V were tested with 5V resolution. Analysis of the identical samples with a FAIMS device in place, using optimized methods, produced the same number of identified cross-linked peptides as after enrichment/fractionation.


FAIMS alone or in combination with enrichment/pre-fractionation significantly improved identification rates of cross-linked peptides in all samples.