Session: Parallel session 2 - Interactomics
FAIMS-based Gas Phase Fractionation (FAIMS-GPF) boosts in-vitro and proteome-wide cross-linking mass spectrometry (XL-MS) workflows.
Cross-linking mass spectrometry (XL-MS) is central in structural mass spectrometry. If simple workflows are often sufficient for purified complexes, developments are still needed to overcome challenges at proteome-wide levels. While extensive off-line fractionation (SEC, CEX, etc.) is reported in pilot studies (Klykov et al., 2018), it requires high amounts of starting material, extensive sample preparation and long instrument times. Here we provide and apply an empowered XL-MS workflow that compromises sensitivity and MS instrument time using only FAIMS-based gas phase fractionation (GPF), on CAK complex (137 kDa) and HeLa cell lysates.
CAK and native HeLa lysate were reacted with DSSO (200:1 and 2mM respectively) and further denatured, reduced, alkylated and digested with Trypsin/LysC. After peptides cleanup, LC-MS/MS was done on an Ultimate 3000 (90min) coupled to an Eclipse Orbitrap Tribrid (Thermo), in DDA. For GPF, FAIMS Pro interface was used with 1 Compensation Voltage (CV) value per LC run. XLinkX in Proteome Discoverer 2.5.0 was used for XLs-ID.
Analysis of unfractionated CAK complex led to the identification of 33 XLs, and analysis of unfractionated Hela lysate led to 161 XLs. To evaluate the link between CV value applied in FAIMS and the charge states filtered, we screened 11 different values for both samples. Increased proportion of >3+ charges in the -55V run logically led to the highest number of XL identified. We next evaluated the overlap between the different CV values and demonstrate that using only 4 different CVs runs (4CV-GPF) for CAK allowed to identify 87 XLs (+260%), highlighting previously unknown interactions. For Hela lysate, using only a 6-CV GPF allowed to reach 800 XLs identified (+500%) with an increased coverage of proteins interactions and high structural relevance.
Our FAIMS-GPF method unprecedentedly boost XL-MS results for both purified complexes and proteome-wide studies, in only 3 days and using <3µg of sample (with triplicates).
CAK and native HeLa lysate were reacted with DSSO (200:1 and 2mM respectively) and further denatured, reduced, alkylated and digested with Trypsin/LysC. After peptides cleanup, LC-MS/MS was done on an Ultimate 3000 (90min) coupled to an Eclipse Orbitrap Tribrid (Thermo), in DDA. For GPF, FAIMS Pro interface was used with 1 Compensation Voltage (CV) value per LC run. XLinkX in Proteome Discoverer 2.5.0 was used for XLs-ID.
Analysis of unfractionated CAK complex led to the identification of 33 XLs, and analysis of unfractionated Hela lysate led to 161 XLs. To evaluate the link between CV value applied in FAIMS and the charge states filtered, we screened 11 different values for both samples. Increased proportion of >3+ charges in the -55V run logically led to the highest number of XL identified. We next evaluated the overlap between the different CV values and demonstrate that using only 4 different CVs runs (4CV-GPF) for CAK allowed to identify 87 XLs (+260%), highlighting previously unknown interactions. For Hela lysate, using only a 6-CV GPF allowed to reach 800 XLs identified (+500%) with an increased coverage of proteins interactions and high structural relevance.
Our FAIMS-GPF method unprecedentedly boost XL-MS results for both purified complexes and proteome-wide studies, in only 3 days and using <3µg of sample (with triplicates).