Ltq orbitrap xl system

After the cell-surface associated protein fraction was separated by SDS-PAGE, protein bands were stained with Coomassie Brilliant Blue and excised. Peptides were prepared for LC-MS/MS analysis by in-gel digestion with trypsin. LC-MS/MS analysis was performed using a LTQ-orbitrap XL system (Thermo Scientific), as described by Tanaka et al. [68 (link)]. The MS/MS spectra of the identified peptides were searched against our own database, which contains SpaA, flagellin, and CmoA sequences, and against non-redundant protein sequences in the National Center for Biotechnology Information (NCBI) database, using a MASCOT server (Matrix Science).

All the IMAC and TiO₂ fractions were analyzed separately by LC-MSⁿ using an LTQ linear ion trap or an LTQ-Orbitrap XL system equipped with a microESI ion source (ThermoFisher, San Jose, CA). For qualitative studies, a full MS scan followed by eight MS/MS scans on the most abundant precursor signals were acquired. For quantitative purposes, the three more abundant precursors from each full MS were submitted to four MS/MS analyses (three PQD and one CID scan) in the linear ion trap. Eight precursors per full scan were selected in the case of the LTQ-Orbitrap each submitted to two different MS/MS analyses (one CID and one HCD). In all cases, a subsequent MS³ scan was performed when a neutral loss of −49, −32.7 or −24.5 (loss of H₃PO₄ for the +2, +3 and +4 charged ions, respectively) was detected among the 10 most intense ions in the CID MS/MS spectra. MS³ scans allow identification of peptides with poor MS² sequence data in qualitative analyses. In quantitative analyses, MS³ scans also allow to assign peptides with insufficient CID MS² data but with valid TMT or iTRAQ reporter ion data from the corresponding PQD or HCD scans.

PEX19-F125^Bpa and PEX3ΔN-WT at the final concentration of 10 μM were incubated in the buffer (20 mM K-HEPES (pH 7.5), 150 mM CH₃COOK, 10% glycerol) at room temperature for 5 min. The reaction was UV-irradiated as described in the Bpa crosslinking assay. The PEX19-F125xPEX3ΔN-WT crosslinked band was resolved by SDS-PAGE and then stained with SimplyBlue SafeStain (Invitrogen). The excised gel piece was further digested with trypsin (Sigma) and AspN (Roche). The digested peptides were extracted using the extraction buffer (75% acetonitrile and 0.1% TFA),⁵³ (link) and analyzed by LTQ Orbitrap XL system (Thermo Fisher Scientific) from KBSI (Korea Basic Science Institute) MS analysis service.
MS raw data were searched using pLink2 software (version 2.3.9),⁴² (link) with three missed cleavages, carbamidomethylation of cysteines as a fixed modification, and oxidation of methionines as a variable modification. Peptide mass was set to 400 to 10,000 Da, and peptide length of amino acid was set to 4 to 40 residues. The precursor tolerance and the fragment tolerance were 10 ppm and 20 ppm, respectively. The results were filtered by a false discovery rate of 5% and a precursor mass accuracy of ±10 ppm.