Computational protocol: Quantitative Profiling of DNA Damage and Apoptotic Pathways in UV Damaged Cells Using PTMScan Direct

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Protocol publication

[…] PTMScan Direct is adapted from the PhosphoScan method developed at Cell Signaling Technology []. The method and reagent validation strategy are previously described []. [...] Immunoprecipitated peptides were resuspended in 0.125% formic acid and separated on a reversed-phase C18 column (75 mm ID × 10 cm) packed into a PicoTip emitter (~8 mm ID) with Magic C18 AQ (100 Å × 5 mm). Peptides were eluted using a 72 min linear gradient of acetonitrile in 0.125% formic acid delivered at 280 nL/min. Tandem mass spectra were collected in a data-dependent manner with an LTQ-Orbitrap Velos or LTQ-Orbitrap Elite mass spectrometer running XCalibur 2.0.7 SP1 using a top-twenty MS/MS method, a dynamic repeat count of one and a repeat duration of 30 s. Real time recalibration of mass error was performed using lock mass with a singly charged polysiloxane ion m/z = 371.101237 []. MS/MS spectra were evaluated using SEQUEST 3G and the SORCERER 2 platform (v4.0, Sage-N Research, Milpitas, CA, USA) []. Human samples were searched against the NCBI homo sapiens FASTA database, updated on 27 June 2011. Results were filtered using a mass accuracy of +/− 50 ppm for precursor ions and 1 Da for product ions. Enzyme specificity was limited to trypsin, with at least one K or R terminus required per peptide and up to four mis-cleavages allowed. Cysteine carboxamidomethylation, specified as a static modification, oxidation of methionine residues, was allowed, and phosphorylation was allowed on serine, threonine and tyrosine residues. Reverse decoy databases were included for all searches to estimate false positive rates, and data was filtered using a 5% false discovery rate in the Peptide Prophet module of SORCERER 2. Search results were further filtered by mass accuracy based on clustering of forward database assignments (−/+ 5 ppm). Results were also filtered using criteria specific to each PTMScan Direct Reagent, including whether a particular peptide was targeted by an antibody in the reagent or homologous to a target, a minimum MS1 intensity filter of 20,000 in at least one sample run, and a higher abundance of the peptide in the PTMScan Direct Reagent IAP than in a control IAP using empty Protein G beads.Label-free quantification was performed using proprietary software as previously described [,,]. MS1 peak intensities across all samples were retrieved from the ion chromatogram files using a mass precision of −/+ 5 ppm and a retention time window of 5 min. Retention time warping (or chromatographic alignment) was performed across binary comparisons to allow retrieval of the correct peak intensity. Peak intensities for all peptides that changed in abundance between treatments were manually reviewed in the ion chromatogram files to ensure accuracy and where necessary peak height measurements were replaced with peak areas. The pervanadate treated cell line mixture (“CST Cell Line” in ) was run as a separate sample with each study as a rich source of modified peptides. This allowed the label-free quantification software to search for all validated peptides across the experimental samples for a more complete quantitative analysis. […]

Pipeline specifications

Software tools PhosphoScan, Comet
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Multiple Sclerosis