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Pipeline publication

[…] 4.1.14, Thermo Fisher Scientific). A precursor mass tolerance of 10 ppm and a product ion mass tolerance of 0.02 Da were applied allowing not more than one missed cleavage for trypsin. Fixed modifications included carbamidomethylation of Cys and TMT9-plex labeling for Lys and N-terminal. Variable modifications contained phosphorylation on Ser/Thr/Tyr and deamidation of Asn. The TMT datasets were quantified using the centroid peak intensity with the “reporter ions quantifier” node. To ensure a high-confident identification of peptides, we used the Mascot percolator algorithm (q value filter set to 0.01), Mascot and Sequest HT peptide rank 1 and a cut-off value of Mascot score ≥ 22 as well as Sequest HT ΔCn of 0.1. Moreover, a cut-off value of Xcorr score for charge states of +1, +2, +3 and +4 higher than 1.5, 2, 2.25 and 2.5, respectively, were considered for further analysis. Subsequently, these peptides were filtered against a Decoy database resulting into a false discovery rate (FDR) of lower than 0.01 (FDR < 0.01). PhosphoRS was used to localise phosphorylation sites with a confidence filter of 99%. Six biological replicates without missing values were considered for the statistical analysis. Quantification was performed on the log2-values of the measured peptide intensities and the data were normalised based on the median. Modified peptides were merged with the R Rollup function (http://omics.pnl.gov/software/danter) allowing for one-hit-wonders and using the mean of the normalised intensities for each peptide. Quantification of proteins was obtained by merging the unmodified peptides with the R Rollup function considering at least 2 unique peptides not allowing for one-hit-wonders and using the mean of the intensities. Subsequently, the mean over the experimental conditions for each peptide in each replicate was subtracted in order to merge the data from different replicates. Proteins, phosphopeptides and formerly sialylated N-linked glycopeptides with a consensus motif for N-linked glycosylation (NXS/T/C; where X # P) were considered to be significantly deregulated if they were identified by at […]

Pipeline specifications

Software tools Comet, ptmRS, DanteR