DOI: 10.25345/c5xn26
title: MassIVE MSV000086051 - TNK1 BaF3 phosphoproteomics Andersen
title: Quantitative LC MS/MS was performed on 4 uL of each sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 3 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed in a data dependent acquisition (DDA) mode of acquisition with a r=120,000 (@ m/z 200) full MS scan from m/z 375 to 1500 with a target AGC value of 2e5 ions. MS/MS scans were acquired at Rapid scan rate (Ion Trap) with an AGC target of 5e3 ions and a max injection time of 200 ms. The total cycle time for MS and MS/MS scans was 2 sec. A 20 sec dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each sample injection was approximately 2 hours. A total of 16 UPLC MS/MS analyses (excluding conditioning runs, but including 4 replicate SPQC injections) were performed; the SPQC pool containing an equal mixture of each sample was analyzed after every 4 samples throughout the entire sample set (4 times total). Quantitative LCMS/MS Analysis. Next, data was imported into Proteome Discoverer 2.3 (Thermo Scientific Inc.) and all LC MS/MS runs were aligned based on the accurate mass and retention time of detected ions (features) which contained MS/MS spectra using Minora Feature Detector algorithm in Proteome Discoverer. Relative peptide abundance was calculated based on area under the curve (AUC) of the selected ion chromatograms of the aligned features across all runs. Peptides were annotated at a maximum 1% peptide spectral match (PSM) false discovery rate. Missing values were imputed after sample loading and trim mean (top and bottom 10%) normalization in the following manner. If less than half of the values are missing in a treatment group, values are imputed with an intensity derived from a normal distribution defined by measured values within the same intensity range (20 bins). If greater than half values are missing for a peptide in a group and a peptide intensity is &gt; 5e6, then it was concluded that peptide was misaligned and its measured intensity is set to 0. All remaining missing values are imputed with the lowest 5% of all detected values. The following analyses are based on these normalized values. The MS/MS data was searched against the SwissProt M. musculus database (downloaded in Apr 2018) and an equal number of reversed-sequence decoys for false discovery rate determination. Mascot Distiller and Mascot Server (v 2.5, Matrix Sciences) were utilized to produce fragment ion spectra and to perform the database searches. Database search parameters included fixed modification on Cys (carbamidomethyl) and variable modifications on Meth (oxidation); Asn/Gln (deamidation); Ser/Thr/Tyr (phosphorylation).