Computational protocol: Histone H3 lysine 27 acetylation is altered in colon cancer

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Protocol publication

[…] The acquired MS/MS raw data files were preprocessed with Mascot Distiller (version 2.2.1, Matrix Science), and the resulting peak lists were searched against the Homo sapiens entries of the SwissProt database (version 05.10.2012, 20306 sequences) using Mascot (version 2.2.03, Matrix Science). The search parameters were as follows: enzyme specificity: semitrypsin; maximum number of missed cleavages: 2; protein mass: unrestricted; parent ions mass error tolerance: 5 ppm; fragment ions mass error tolerance: 0.02 Da; fixed modifications: Carbamidomethylation (C); and variable modifications: Acetyl (K) (42.010565 Da), Methyl (K) (14.015650 Da), Dimethyl (K) (28.031300 Da), Trimethyl (K) (42.046950 Da), Methyl (R) (14.015650 Da), Dimethyl (R) (28.031300 Da), Deamidated (R) (0.984016 Da), Phospho (ST) (79.966331 Da) and Oxidation (M) (15.994915 Da).The statistical significance of peptide identifications was determined using a target/decoy database search approach and a previously described procedure that provided q-value estimates for each peptide spectrum match (PSMs) in the data set [,]. Only PSMs with q-values ≤ 0.01 were regarded as confidently identified.Additional acceptance criteria were used for assessing confidence of modified peptides. In the first step, the exact position of the modifications in the sequence was established by an adopted version of the phosphoRS algorithm []. Next, the MS/MS spectra were inspected manually for accurate fragment ions assignment. Finally, selected types of sites were rejected as potential experimental artifacts. Those included: lysine methylations on the C-terminus of the sequence or detected in peptides with acidic residues (possible artifacts of methyl esterification of the carboxylic group) and peptides with deamidation on the C-terminal arginine (tryptic cleavage after a deamidated residue have been recently shown as a highly unlikely event []).Proteins represented by less than two peptides, or identified by a subset of peptides from another protein, were excluded from further analysis. Proteins matching the same set of peptides were grouped together into clusters. All the steps involved in Mascot results processing were performed using MScan, a proprietary Java application available at http://proteom.ibb.waw.pl/mscan. Multiple alignment of protein sequences and visualization of the detected post-translational modification sites were performed using CLC Sequence Viewer (CLC Bio). […]

Pipeline specifications

Software tools Mascot Distiller, ptmRS, MSCAN, CLC Sequence Viewer
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Colonic Neoplasms, Neoplasms, Colorectal Neoplasms