Computational protocol: Proteomic Analysis of Serum from Patients with Major Depressive Disorder to Compare Their Depressive and Remission Statuses

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Protocol publication

[…] We identified proteins from the peptide masses analyzed. In brief, tandem mass data from the Q-TOF instrument was converted to mzXML by Trapper 4.3.1 (Trans-Proteomic Pipeline, ISB, Seattle, WA, USA) for database searching in SEQUEST (Sorcerer 3.5; Sage-N Research, Lajolla, CA, USA). A UniProKB proteomes database (released in November 2012, HUMAN) was used to search each triplicate run with the following parameters: precursor mass tolerance, 20 ppm; product ion mass tolerance, 50 ppm; 2 missed cleavages allowed; fully tryptic peptides only; fixed modification of carbamidomethyl cysteine; variable modifications of oxidized methionine; and N-terminal carbamylation. A false positive rate of 5% was used for this result. In the case of a protein, we evaluate the quality of protein identification with an error rate of less than 1% from ProteinProphet (Trans-Proteomic Pipeline, ISB, Seattle, WA, USA). [...] Each patient's protein expression was quantified for the depression and remission statuses using IDEAL-Q software (http://ms.iis.sinica.edu.tw/IDEAL-Q). IDEAL-Q software enables label-free quantification, which determines the relative amount of proteins in biological samples. Unlike other protein quantification, label-free quantification does not have to bind to an isotope. In brief, IDEAL-Q was used to continuously process all peptides in each LC-MS/MS run, both identified and unidentified, to quantify as many peptides as possible. Then, the predicted elution time was used to detect peak clusters of the assigned peptide. Detected peptide peaks were processed by statistical and computational methods and further validated by a signal-to-noise ratio >3, correct charge state, and isotopic distribution criteria to filter out noisy data. Based on the peptide ratio distribution, the peptide normalization step could be processed. The protein abundance ratio was determined by the weighted average of non-degenerate peptides. [...] Bioinformatics analysis was carried out to confirm whether biomarker candidate proteins could differentiate mood status (depression vs. remission). The functional annotation network for Gene Ontology terms was visualized and statistically evaluated using ClueGO (v 2.0.6) plug-ins integrated in Cytoscape v 3.0.1 (ISB, Seattle, WA, USA). The protein expression profile of biomarker candidates was imported to the DanteR software v 1.0.0.10 (http://omics.pnl.gov/software) for proteomic analysis. Statistical calculations, such as hierarchical clustering, correlation studies, and principal component analyses were carried out using the DanteR software. The correlation coefficient between samples was calculated for all paired samples in a correlation matrix. GeneGo MetaCore v 6.15 (GeneGo, St. Joseph, MI, USA) was used to assign functional pathways. The significant pathway maps based on GeneGo analysis were mapp-ed and analyzed. […]

Pipeline specifications

Software tools Comet, ProteinProphet, IDEAL-Q, ClueGO, DanteR, MetaCore
Application MS-based untargeted proteomics
Organisms Homo sapiens