All samples were subjected to SP3 sample preparation (78). Briefly, proteins were denatured, reduced and alkylated, and subsequently digested with Trypsin and Lys-C proteases. Tandem mass tag (TMT) 11plex (Pierce) labeling was used for peptide multiplexing and quantification. Samples were mixed, desalted by solid-phase extraction (Seppak 1 cc/50 mg, Waters), and fractionated by basic reversed-phase fractionation on a quaternary Agilent 1290 Infinity II Ultra-Performance Liquid Chromatography (UPLC) system equipped with a Kinetex Evo-C18 column (150 mm by 2.1 mm, 2.6 μm, 100 Å, Phenomenex). Fractions were concatenated into eight final samples, dried down, and resuspended in 2% acetonitrile, 0.1% trifluoroacetic acid (TFA) before MS analysis. All samples were analyzed on an Orbitrap Fusion Lumos (Thermo Fisher Scientific) that was coupled to a 3000 RSLCnano UPLC (Thermo Fisher Scientific). Samples were loaded on a PepMap trap cartridge {300 μm [inner diameter (I.D.)] by 5 mm, C18, Thermo Fisher Scientific} with 2% acetonitrile and 0.1% TFA at a flow rate of 20 μl/min. Peptides were separated over a 50-cm analytical column [PicoFrit, 360 μm (outer diameter), 75 μm (I.D.), 10-μm tip opening, noncoated, New Objective] that was packed in-house with Poroshell 120 EC-C18, 2.7 μm (Agilent). Solvent A consisted of 0.1% formic acid in water. Elution was performed at a constant flow rate of 250 nl/min using a 180-min method: 8 to 33% solvent B (0.1% formic acid in 80% acetonitrile) within 120 min, 33 to 48% solvent B within 25 min, 48 to 98% buffer B within 1 min, followed by column washing and equilibration. The Fusion Lumos mass spectrometer was equipped with a FAIMS Pro device, which was operated at standard resolution using two alternating CVs (compensation voltages) of −50 and −70 V (cycle time for each was set to 1.5 s). Data acquisition was performed with an MS3-based, data-dependent method in positive ion mode. Advanced peak determination was deactivated. MS survey scans were acquired from 375 to 1500 mass/charge ratios (m/z) in profile mode at a resolution of 120,000. The AGC (automatic gain control) target was set to 4 × 105 charges, enabling a maximum injection time of 50 ms. Peptides with charge states 2 to 6 were subjected to collision-induced dissociation fragmentation [fixed collision energy (CE) = 35% and AGC = 1 × 104] and analyzed in the linear ion trap at a resolution of 125,000 Da/s. The isolation windows for peptides with charge states 2, 3, and ≥ 4 were set to 1.2, 0.7, and 0.5 m/z, respectively. For MS3 acquisition, synchronous precursor selection was enabled to select 5 (2+ peptides) or 10 (3+ to 6+ peptides) fragment ions for higher-energy collisional dissociation (HCD) fragmentation [normalized collision energy (NCE) = 65%, AGC = 1 × 105, and maximum injection time = 105 ms]. MS3 scans were acquired in the Orbitrap at a resolution of 30,000. Precursors were dynamically excluded for 45 s. Raw files were processed with Proteome Discoverer 2.3 (Thermo Fisher Scientific) using SEQUEST HT for peptide identification. Peptide-spectrum matches were filtered to a 1% false discovery rate (FDR) level using Percolator using a target/decoy approach. The protein FDR was set to 1%. Further data processing was performed in R and Perseus (v. 1.6.2.3). Only those proteins that were identified by at least two peptides were included in the analysis. All contaminant proteins were filtered out. A three-step normalization procedure was applied. First, the total intensity of each TMT channel was normalized to correct for mixing errors. Next, the common channel in both TMT sets was used for internal reference scaling (79) to correct for batch effects. Afterward, the data were normalized, applying trimmed mean of M values using the edgeR package. Statistical analysis was performed using two-sample t tests [fudge factor s0 was set to 0.1 (80)]. Resulting P values were corrected for multiple testing using a permutation-based FDR approach.

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