Computational protocol: Identification of Phosphorylated Cyclin-Dependent Kinase 1 Associated with Colorectal Cancer Survival Using Label-Free Quantitative Analyses

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Protocol publication

[…] The raw data generated by an LTQ-Orbitrap MS were converted to an open file representation mzXML format using an open-source program, msInspect [], which was developed by the Computational Proteomics Laboratory (CPL); operational analysis was then conducted ( to acquire peak information after peak detection, monoisotopic peak determination, and original mass calculation. Then, a TSV format file was produced for further iPhos software analysis. To determine the potential phosphopeptide signals, the iPhos program was used; a mass shift of -79.966 Da (or multiples of -79.966) due to dephosphorylation reactions was found in the detection by comparing the phosphopeptide signals and the corresponding dephosphorylated peptide signals. The iPhos program is an open source application written in Python 2.6 that operates in Windows ( [, ]. The iPhos program is designed to generate an inclusion list of potential phosphopeptide candidates for future targeted LC-MS/MS analysis. [...] Data from the 12 LC-MS runs were aligned by using Progenesis QI software (Nonlinear Dynamics, Newcastle upon Tyne, UK). One run was chosen as the alignment reference, and the other runs were automatically aligned to the reference according to their retention times. Each run was illustrated by a proprietary algorithm as a two-dimensional feature map as m/z value versus retention time. Features with fewer than three isotopes and only one charge were masked at this point and excluded from further analyses. The control and treated runs were then divided into two appropriate groups, and the raw abundances (areas under the peaks) of all the features were normalized. The features (features with charges of < +2 were masked and excluded from further analyses) were used to calculate a normalization factor to correct the experimental variation between the runs. The iPhos program added an extra function, which utilized the parameters m/z value, RT adjustment, and charge state linked up with qualitative identification and quantitative proteomics analysis in phosphoproteomics research. […]

Pipeline specifications

Software tools msInspect, Progenesis QI
Application MS-based untargeted proteomics
Organisms Homo sapiens
Chemicals Tyrosine