Ltq linear ion trap

A commercial DESI source from Prosolia, Inc. was used for the N-alkylation experiments and MS/MS analysis. A homebuilt DESI ion source similar to the commercial source available from Prosolia, Inc. was used for the Suzuki experiments. A Thermo LTQ linear ion trap was used to acquire data. DESI-MS experiments were performed in both positive and negative ion mode over the range m/z 50–500. The ion source parameters are detailed in the ESI.† Membrane substrates were scanned horizontally at a rate between 5000 and 10 000 μm s^–1 using vertical steps of 500 μm. The MS injection time was set so as to produce square pixels of 500 by 500 μm. Data were first converted using a in-house software into a format compatible with Biomap. Biomap (freeware, ; https://ms-imaging.org/wp/biomap/) was used to generate the selected ion images by choosing appropriate ion thresholds.

The pink extracellular pigment produced by C. albicans from both L-and D-tryptophan and the pink extracellular pigment produced by C. neoformans var. grubii, C. neoformans var. neoformans, and C. gattii were eluted from a TLC plate and dissolved in 50% methanol. Mass spectrometric measurements of the pink pigment were performed on a LTQ linear ion trap mass spectrometer (LTQ, Thermo, San Jose, CA) using electrospray positive ionization. The samples were diluted into 50% methanol: water (1∶10 v/v) and infused into the mass spectrometer at a flow rate of 3 µL/min. Tandem mass spectrometry (MS/MS) was performed using an isolation width of 1.5 m/z and a normalized collision energy of 25–35%. Several additional pigments and fluorescent compounds were eluted from a TLC plate, dissolved in 80% methanol and the molecular mass of each compound was determined.

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.

Photolabeling of Rap1 protein using azi-isoflurane and azi-sevoflurane was performed as previously described [16 (link)]. Azi-isoflurane and azi-sevoflurane are photoaffinity probes, developed by incorporating a diazrinyl moiety (CHN₂, 40 Da) into isoflurane and sevoflurane, respectively [17 (link)]. Azi-isoflurane or azi-sevoflurane (final concentration, 100 μM) was equilibrated with Rap1 protein (1 mg/mL) and 500 μM isoflurane or sevoflurane in a reaction volume of 300 μL for 10 min and then exposed to 300-nm light under a Rayonet RPR-3000 Lamp (Southern New England Ultraviolet Co.; Branford, CT, USA) in a 1-mm path-length quartz cuvette for 25 min. The protein was separated on an SDS-polyacrylamide gel and stained with Coomassie G-250. The protein gel band was excised for liquid chromatography (LC)-mass spectrometry (MS)/MS. After trypsin digestion, samples were injected into a nano-LC column with online electrospray into a LTQ linear ion trap (Thermo Fisher Scientific). Raw data were acquired with XCalibur (Thermo Fischer Scientific), and Sequest software (Scripps Research Institute, La Jolla, CA, USA) was used to search for b and y ions against the sequence of Rap1 for adducts of the appropriate mass (196 Da).