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Pipeline publication

[…] was set to 500 ms. Orbitrap online calibration using internal lock mass calibration on m/z 371.10123 from polydimethylcyclosiloxane was used. Multistage activation/pseudoMS3 was enabled with a neutral loss mass list of 24.49, 32.66, 48.999, 97.97, 195.94, and 293.91 Da for neutral loss of phosphoric acid [H3PO4] at charge states +1, +2, +3 and +4, respectively, 195.94, 97.97 and 48.999 Da for neutral loss of two phosphoric acids [2H3PO4] at charge states +1, +2 and +4, respectively, and 293.91 Da for neutral loss of three phosphoric acids [3H3PO4] at charge state +1. The AGC for MS/MS acquisition in the LTQ was set to 1 × 104, the max IT was set to 100 ms., ProteomeDiscoverer 1.3 (Thermo), SEQUEST search algorithm and PhosphoRS were used for peptide identification phosphorylation site mapping of the first experiment. Precursor mass tolerance was set to 5 ppm and fragment ion tolerance to 0.8 Da and maximum 2 missed cleavages were allowed. Phosphorylation of serine, threonine and tyrosine (+79.966 Da) and oxidation of methionine (+15.995 Da) were set as dynamic modifications. Search was done against Arabidopsis TAIR10 protein database where common contaminants were added. Search results were filtered and only high confidence peptides with XCorr 2.0 or more were used for further analysis. Relative quantification was done with ProtMAX 2012 tool version 2.14 and DanteR software. For that, the Thermo RAW files were converted to mzXML format with MassMatrix MM File Conversion Tool and quantification was done in ProtMAX by summing parent ion intensities. The quantified phosphopeptides were Log10 transformed, EigenMS normalized and missing values were imputed using k-nearest neighbour (KNN) method., The data from experiment 2 were analysed with MaxQuant 1.4 and Andromeda search algorithm. Mass tolerance for precursor was set to 5 ppm and for fragment masses to 0.8 Da. The maximum FDR was set to 1%. Three missed cleavages were allowed because the phosphorylation near to a tryptic site can hinder digestion. Dynamic modifications allowed were phosphorylation (STY), methionine oxidation and protein N-terminal acetylation. Further data analysis, normalization and transformation were done with Perseus 1.5 and Microsoft Excel. Phosphopeptides that are discussed in detail were quantified by using MS1 fil […]

Pipeline specifications

Software tools Comet, ptmRS, DanteR, EigenMS