DOI: 10.25345/c50r9mh8j
title: MassIVE MSV000100329 - TMTpro_16plex_labeling of RAW264_7 cells
title: TMTpro-16plex-labeling of RAW264.7 cells High-pH reversed-phase fractionation of TMTpro-16plex-labeled RAW264.7 peptides After TMTpro sample pooling, 100 ug of the multiplexed peptides were subjected to a combined cleanup and high-pH reversed-phase fractionation workflow using solid-phase extraction (SPE) cartridges (1 cc, 50 mg C18 sorbent; Sep-Pak, Waters) operated on a vacuum manifold. Fractionation buffers consisted of 10 mM ammonium formate, adjusted to pH 10, and containing varying concentrations of acetonitrile (ACN). Equilibration and washing steps were performed with 1 mL of solution per step, while fractions were collected in 600 uL volumes. Equilibration, sample loading, and washing. Each cartridge was first equilibrated with 100% ACN, followed by two washes with 0.2% formic acid (FA). The 100 ug peptide mixture was then loaded onto the cartridge, followed by one additional wash with 0.2% FA and one wash using a 10% ACN fractionation buffer. High-pH fractionation. Peptides were eluted in a stepwise manner using 10 mM ammonium formate (pH 10) containing 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, and 80% ACN, yielding a total of 10 fractions. The eluates were dried in a vacuum concentrator and resuspended in 0.2% FA prior to LC-MS analysis. LC-MS analysis of TMTpro-16plex-peptides For LC-MS analysis, a Vanquish Neo UHPLC system (Thermo Fisher Scientific) was coupled to an Orbitrap Ascend mass spectrometer (Thermo Fisher Scientific) equipped with a Nanospray Flex ion source. Peptide separation was performed on a 40 cm in-house-packed fused-silica column (C18, 1.7 um, 130 A) maintained at 50 C using a custom-built column heater. The spray voltage was set to 2 kV, and the ion transfer tube temperature was 275 C. TMTpro-labeled peptide fractions (500 ng total per injection) were separated at a flow rate of 300 nL/min using a 109-min active gradient from 14% to 41% solvent B (solvent A: 0.2% formic acid in water; solvent B: 80% acetonitrile with 0.2% formic acid). MS analysis was performed using an MS3-SPS-RTS (MultiNotch Synchronous Precursor Selection Real-Time Search) strategy on the Orbitrap Ascend mass spectrometer. MS1 acquisition and MS2 precursor filtering. Full MS1 scans were acquired in the Orbitrap at a resolution of 60,000 over a scan range of 400-1600 m/z. The maximum injection time was 50 ms, with an automatic gain control (AGC) target of 4 x 10e5 (normalized AGC target = 100%). The RF lens was set to 30%. Monoisotopic peak determination (MIPS) was set to peptide, and the isolation window center was defined as the most abundant peak. Precursor charge states of 2-5 were included. Dynamic exclusion was enabled for 20 s with a +-5 ppm mass tolerance. MS2 acquisition and MS3 precursor filtering. MS2 spectra were acquired in the ion trap using a 0.8 m/z isolation window and collision-induced dissociation (CID) at a normalized collision energy (NCE) of 34%, with an activation time of 10 ms and an activation Q of 0.25. Multistage activation was disabled. The ion trap scan rate was set to Turbo over a scan range of 400-1600 m/z, with a normalized AGC target of 100% and a maximum injection time of 23 ms. Real-time searching (RTS) was enabled against the UniProt Mouse Reference Proteome (UP000000589, including common contaminants; downloaded June 2024). Search parameters were as follows: enzyme = Trypsin/P; static modifications = carbamidomethyl (C, +57.0215 Da) and TMTpro16plex (Kn, +304.2071 Da); variable modifications = oxidation (M, +15.9949 Da); maximum of 1 missed cleavage and up to 2 variable modifications per peptide. Under Peak Selection and Threshold Settings, TMT SPS MS3 mode was enabled and maximum search time was set to 80 ms. Scoring thresholds were set to: Xcorr = 1.4, dCn = 0.1, precursor mass tolerance +- 20 ppm, and charge state = 2. In the compound filter field, entries containing the keyword contam_ were rejected. Additional precursor selection filters were as follows: precursor selection range = 400-1600 m/z; precursor ion exclusion tolerance = +-10 ppm; and isobaric tag loss exclusion = TMTpro. MS3 acquisition. MS3 spectra were acquired in the Orbitrap with synchronous precursor selection (SPS) enabled, selecting 10 SPS precursors using a 0.8 m/z isolation window. MS3 scans were acquired at a resolution of 45,000 (at m/z 200) over a scan range of 110-1000 m/z, with a normalized AGC target of 250% and a maximum injection time of 105 ms. High-energy collision dissociation (HCD) was applied at 55% NCE. The total cycle time (MS1 through MS3) was 3 s.