CDK6 271123 6Plex TMT.csv

Dryad dataset for <b>Genome wide association analysis of neutrophil granularity identifies CDK6 as a regulator of primary granules</b>. iScienceDataset DOI: 10.5061/dryad.t1g1jwtfgDescription of the data and file structure4 CDK6 KO (knockout) mouse marrow derived neutrophils are compared with 4 WT (wild type) mouse marrow derived neutrophils.The raw data files were processed and quantified using Proteome Discoverer software v2.4 (Thermo Scientific) and searched against the UniProt Mouse database (downloaded July 2021: 35859 entries) using the SEQUEST HT algorithm. Peptide precursor mass tolerance was set at 10ppm, and MS/MS tolerance was set at 0.6Da. Search criteria included oxidation of methionine (+15.995Da), acetylation of the protein N-terminus (+42.011Da) and Methionine loss plus acetylation of the protein N-terminus (-89.03Da) as variable modifications and carbamidomethylation of cysteine (+57.0214) and the addition of the TMTpro mass tag (+304.207) to peptide N-termini and lysine as fixed modifications.Code/softwareAfter analysis in Proteome Discoverer 2.1, the data were processed and further analysed in the R statistical computing environment. The protein groups were reassessed by an in-house script which selects a master protein firstly by ID and quantitation metrics, then by the annotation quality of uniprot accessions.Access informationOther publicly accessible locations of the data:NAData was derived from the following sources:NA: As Rice et al⁶⁴. Protein lysates were obtained from frozen cell pellets of isolated neutrophils. 1 × 10⁶ cells were washed in PBS and resuspended in RIPA buffer supplemented with 1:100 halt protease and phosphatase inhibitor (Thermo Fisher Scientific) and 10 mM EDTA. Samples were sonicated immediately before further processing. Protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and 100 µg of protein used for Tandem Mass Tag (TMT) mass spectrometry.50 µg of each sample was digested with trypsin (1.25 µg; 37°C, overnight), labelled with TMT 11 plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific), and the labelled samples were pooled. A 100 µg aliquot of the pooled sample was evaporated to dryness, resuspended in 5% formic acid, and then desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters). Eluate from the SepPak cartridge was again evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) before fractionation by high pH reversed-phase chromatography using an UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 µm, 2.1 × 150 mm; Waters) in buffer A, and peptides were eluted with an increasing gradient of buffer B (20 mM ammonium hydroxide in acetonitrile, pH 10) from 0–95% over 60 min. The resulting fractions (15 in total) were evaporated to dryness and resuspended in 1% formic acid before analysis by nano-LC MSMS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific).High pH RP fractions were further fractionated using an Ultimate 3000 nano-LC system in line with an Orbitrap Fusion Tribrid mass spectrometer. In brief, peptides in 1% (vol/vol) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Thermo Fisher Scientific). After washing with 0.5% (vol/vol) acetonitrile, 0.1% (vol/vol) formic acid peptides were resolved on a 250 mm × 75 µm Acclaim PepMap C18 reverse phase analytical column (Thermo Fisher Scientific) over a 150-min organic gradient, using seven gradient segments (1–6% solvent B over 1 min, 6–15% B over 58 min, 15–32% B over 58 min, 32–40% B over 5 min, 40–90% B over 1 min, held at 90% B for 6 min, and then reduced to 1% B over 1 min) with a flow rate of 300 nl min⁻¹. Solvent A was 0.1% formic acid and solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were ionized by nano-electrospray ionization at 2.0 kV using a stainless-steel emitter with an internal diameter of 30 µm (Thermo Fisher Scientific) and a capillary temperature of 275°C.All spectra were acquired using an Orbitrap Fusion Tribrid mass spectrometer controlled by Xcalibur 2.1 software (Thermo Fisher Scientific) and operated in data-dependent acquisition mode using a synchronous precursor selection-MS3 workflow. FTMS1 spectra were collected at a resolution of 120,000, with an automatic gain control (AGC) target of 200,000 and a max injection time of 50 ms. Precursors were filtered with an intensity threshold of 5,000, according to charge state (to include charge states 2–7), and with monoisotopic peak determination set to peptide. Previously interrogated precursors were excluded using a dynamic window (60 s ± 10 ppm). The MS2 precursors were isolated with a quadrupole isolation window of 1.2 m/z. ITMS2 spectra were collected with an AGC target of 10,000, max injection time of 70 ms, and CID collision energy of 35%.For FTMS3 analysis, the Orbitrap was operated at 50,000 resolution with an AGC target of 50,000 and a max injection time of 105 ms. Precursors were fragmented by high-energy collision dissociation at a normalised collision energy of 60% to ensure maximal TMT reporter ion yield. Synchronous precursor selection was enabled to include up to 10 MS2 fragment ions in the FTMS3 scan.