Similar protocols

Protocol publication

[…] Insect cell-expressed 3 m hsPRC2 samples (5 µM) were prepared with and without the addition of RNA (40 µM) in stock buffer (50 mM Tris, pH 7.5, 100 mM KCl, 1 mM TCEP, 5% glycerol) for differential HDX-MS analysis. Deuterium exchange was conducted on a temperature-controlled HDX2 autosampler (Leap Technologies). Aliquots of sample (4 µL) were mixed (1:4) with deuterium exchange buffer (D2O 25 mM Tris, 150 mM NaCl) at 4°C. Exchange was conducted across five time points (10 s, 60 s, 360 s, 3600 s, 28,800 s) and run in replicates (triplicate/quadruplicate). After a defined exchange time, the sample was transferred by a chilled syringe, and quenched/denatured at 1°C with addition of 20 µL of chilled quench buffer (3.2 M guanidine hydrochloride, 0.8% formic acid). Quenched samples were injected into the chiller box, which housed the sample loop, protease column and trap/analytical columns at 3°C. Blank injections were inserted between every sample to minimize potential carryover.A three pump scheme was employed for peptide detection using Vanquish UPLC pumps (Dionex). A loading pump running 0.1% formic acid at 200 µl/min carried sample sequentially across the pepsin/protease XIII immobilized column (NovaBio Assay) and 2.1 × 5 mm CSH C18 trap column(Waters) for 2 min. Peptides were desalted for an additional 30 s, while the protease column was back-flushed with 0.05% TFA. Peptides were subsequently eluted/separated across a Kinetex C8 2.1 × 50 mm analytical column via a 5.7-min gradient of mobile phase B (8%–37% ACN, 0.1% formic) employing a binary pump gradient with mobile phase A (0.1% formic acid) and mobile phase B (acetonitrile 0.1% formic acid) at 150 µL/min.Data acquisition and processing involved multiple software platforms. Mass spectra were acquired on a Thermo Fusion-Lumos mass spectrometer running XCalibur 2.1 across a scan range of 375–1300 m/z. Orbitrap resolution was set at 60,000 for charge state selection of +2 to + 5 peptides. Peptide fragmentation for peptide identification employed both HCD and EThcd tandem MS2 acquisition. Electrospray source settings were set at high-gas flow (27 sheath, nine aux) and temperature (150°C) to handle the 150 µl/min LC flow rate, while minimizing potential deuterium back-exchange. Peptide pools were generated in Thermo Proteome Discoverer 2.1 with a directed search against the protein sequence, using Sequest HT search and a fixed value PSM validator (0.05 Delta Cn). Tolerances were set at five ppm mass accuracy with 0.6 Da fragment. Deuterium exchange was determined with HDExaminer software. For peptide deuterium uptake, the first two residues and prolines were excluded from calculations. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet, HDExaminer
Application MS-based untargeted proteomics
Organisms Homo sapiens, Chaetomium thermophilum
Diseases Neoplasms
Chemicals Amino Acids, Hydrogen