Computational protocol: Evaluating the use of HILIC in large-scale, multi dimensional proteomics: Horses for courses?

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Protocol publication

[…] The raw MS data from the Q-Exactive Orbitrap (Thermo Scientific) were processed with the MaxQuant software package (version Proteins and peptides were identified against the UniProt reference proteome database (August 2013) using the Andromeda search engine , . The following search parameters were used: mass deviation of 6 ppm on the precursor and 0.5 Da on the fragment ions; Tryp/P for enzyme specificity; two missed cleavages. Carbamidomethylation on cysteine was set as a fixed modification. Oxidation on methionine; phosphorylation on serine, threonine, and tyrosine; hydroxylation on proline and acetylation at the protein N-terminus were set as variable modifications. Thresholds for the identification of phosphopeptides were Delta Score = 6 and Andromeda score = 40. The false discovery rate was set to 5% for positive identification of proteins, peptides, and phosphorylation sites.Most of the subsequent data analysis was done in R version 3.1.3 using Rstudio 0.98.1091 and the package ggplot2 ; the sequence coverage analysis was done using Perseus and the GO-terms enrichment analysis using the Cytoscape , app BiNGO. […]

Pipeline specifications

Software tools MaxQuant, Andromeda, Ggplot2, Perseus, BiNGO
Applications Miscellaneous, MS-based untargeted proteomics
Organisms Equus caballus