Ltq orbitrap elite system

High quality chemicals and reagents were purchased from standard sources such as Sigma-Aldrich (St. Louis, MO, USA) and VWR International (Radnor, PA, USA). Deionized water (18.2 Ω) used for making solutions was obtained from Milli-Q Direct Ultrapure Water System from Millipore (Billerica, MA, USA). All intermediates were characterized by proton nuclear magnetic resonance (¹H NMR) and mass spectrometry (MS) analysis, and the purity of compounds was analyzed by high-performance liquid chromatography (HPLC). ¹H NMR data were collected on a Bruker 400 MHz spectrometer (Bruker, Billerica, MA) using standard parameters, while chemical shifts are reported in ppm (δ) in reference to residual non-deuterated solvent. Electrospray ionization (ESI) MS analysis was performed on new compounds with an LTQ Orbitrap Elite system (ThermoFisher Scientific, Waltham, MA, USA) at the Mass Spectrometry and Biomarker Discovery Core facility of the Cedars-Sinai Medical Center, Los Angeles, CA.

For in-solution digests, 6 μg of protein was reduced with 5 mM dithiothreitol (DTT) (AppliChem) for 20 min at RT and subsequently alkylated with iodoacetamide (Sigma-Aldrich) for 20 min at 37°C. Trypsin (Thermo Scientific) was added in a 1:50 enzyme–protein ratio and digested overnight at 37°C. Samples separated via SDS–PAGE were stained by Coomassie. Each gel lane was cut into 20 pieces and prepared for LC/MS analysis as described (Hecht et al., 2019 (link)). LC/MS and bioinformatical analysis were carried out as described above, with the exception of shortening the LC gradient to 65 min in total. Employing an LTQ Orbitrap Elite system (Thermo Fisher Scientific) online coupled to an U3000 RSLCnano (Thermo Fisher Scientific), samples were analyzed as described (Mohr et al., 2015 (link)), with modifications (Hecht et al., 2019 (link)) and picking the 20 most intense ions from the survey scan for CID fragmentation. Singly charged ions were rejected and m/z of fragmented ions were excluded from fragmentation for 60 s. MS2 spectra were acquired employing the LIT at rapid scan speeds.

Protein samples were prepared for mass spectrometry measurements as previously described (62 (link)). Briefly, the indicated protein complex was subjected to SDS–PAGE. When the electrophoresis was carried out, the sample became a compact slit-like band after loading, the protein band was excised from a Coomassie-stained SDS–PAGE gel and fragmented into small pieces. Thereafter, the proteins were reduced using 10 mM dithiothreitol for 30 min at 55 °C, followed by alkylation with 55 mM iodoacetamide for 30 min at room temperature in the dark. Overnight digestion at 37 °C was performed with 0.6 μg of trypsin (Proteomics-grade, Promega) in 25 mM NH₄HCO₃. The resulting peptides were acidified using 0.1% formic acid (FA) to stop trypsin digestion (63 (link)). After the desalting procedure, the peptides were analyzed by the LTQ Orbitrap Elite system and Easy-nLC 1000 system (Thermo Fisher Scientific).