Computational protocol: Phosphoproteomics Profiling of Human Skin Fibroblast Cells Reveals Pathways and Proteins Affected by Low Doses of Ionizing Radiation

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Protocol publication

[…] Phosphopeptides were identified from MS/MS spectra using SEQUEST (Sequest Cluster version 27 revision 12 from Bioworks 3.2, ThermoElectron Corp., Waltham, MA) by searching against the Human IPI database (version 3.20, 61,225 protein sequences, www.ebi.ac.uk/IPI, European Bioinformatics Institute, Cambridge, UK). The search parameters were: (1) Fully tryptic peptide termini (allowing ≤2 missed cleavages) (amino- and carboxy-termini were considered tryptic termini). (2) Dynamic modifications: an addition of 79.9663 Da to serine, threonine, and tyrosine residues (phosphorylation). (3) Static modifications: an addition of 14.0157 Da to aspartic acid, glutamic acid, and the carboxy-terminus (methyl esterification). (4) Precursor ion mass tolerance: ±0.05 Da. (5) Fragment ion mass tolerance: ±0.5 Da (m/z). (6) Maximum number of the same amino acid that can be dynamically modified in a phosphopeptide: 3. The false discovery rate (FDR) of phosphopeptide identification was controlled at ≤ 0.5% using our in-house developed software. This program also measured the probability of correct phosphorylation site localization in each identified peptide by calculating the AScore for each phosphorylation site using Gygi's approach, which is shown in . [...] We use both spectral count and peak area methods to evaluate the changes of phosphopeptides. The total spectral counts for each identified phosphopeptide in each radiation condition and their ratios were used to semi-quantitatively estimate their abundance in each condition. The G-test was used to assess significance of spectral count data at the 95% confidence level. We also used the peak areas of phosphopeptides identified in each of four MS technical replicates to evaluate the phosphopeptide abundance changes. Cross-correlation analyses of each replicate (supplementary ) were performed and generated scores ranging from 0.79 to 0.93, where 100% correlation between replicates would yield a score of 1. Briefly, the peak area for each phosphopeptide was extracted from the MS data applying the MASIC program developed in-house and used to assess phosphopeptide abundance. To obtain significant changed phosphopeptides under control and each radiation condition, the T-test was performed for the phosphopeptides identified from each condition across four MS technical replicates, and p<0.05 was used to filter the non-significantly changed proteins. All significantly changed phosphopeptides were combined for the final list using either spectral count or peak area methods. […]

Pipeline specifications

Software tools Comet, Ascore, MASIC
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Neoplasms