Computational protocol: The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis

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Protocol publication

[…] Spectra were searched against a concatenated database of Human (IPI, version 3.66) and Toxoplasma (toxoDB, release 6.1) proteins using SEQUEST , with 15 ppm precursor mass tolerance, trypsin specificity with up to two missed cleavages, static modification of cysteine (carbamidomethylation, +57.0215) and variable modification of serine, threonine, and tyrosine (phosphorylation, +79.9663), methionine (oxidation, +15.9949) lysine (SILAC 13C(6)15N(2), +8.0142), and arginine (SILAC 13C(6), +6.0201). Phosphorylation site localization was assessed using the Ascore algorithm . All datasets were filtered using the target-decoy method , to a false discovery rate (FDR) of <1% on the peptide and <3% on the protein level. Phosphopeptides were combined into phosphosites based on their localization probabilities, and phosphosites were further filtered to an FDR of <1% for each phosphorylatable residue (S,T,Y) using the peptide score provided by Ascore. All peptides matching to the human proteome were removed to exclude peptides from the parasite that are identical with the host and where quantification is thus not reliable. SILAC quantification was achieved by analysis of the MS1-intensity peaks using the VISTA algorithm . Quantifications were scored using: closeness of log2 H/L to 1∶1, signal to noise of each isotopic partner, and a VISTA confidence metric that accounts for chromatography quality. Weighted averages were calculated using these scores for sites and proteins for which more than one identification was made. These weighted averages are intentionally conservative, in an attempt to eliminate false-positives from the tails of the distribution. Unweighted average and standard deviation calculations have been included as well. Phosphopeptides were categorized as either mono-, bis-, or tris-phosphorylated, and separate averages were calculated for sites found in peptides of each type. The resulting data was further filtered for a minimum of 2 quantifications in each respective experiment, a minimum VISTA confidence score of 88 for the best quantification and a minimum signal to noise ratio for the best peptide of 8.SILAC log2 ratios were centered on “0” based on the median SILAC ratio for each dataset.Differentially phosphorylated sites were identified by using the following criteria:a minimum log2 fold change of 0.75 (+ or −), which is ∼1.5 times the standard deviation across the experimental datasets, and a consistent change of phosphorylation site abundance in one or more of the conditions. Note, one mismatch was allowed to capture phosphorylation sites that are just at the threshold, or missing in a single sample due to a bad SILAC quantification, to be included in the dataset. Phosphorylation site quantifications were manually curated by analyzing the MS1 elution profiles and removed if they originated from low-quality quantifications.Protein SILAC ratios were calculated by using the median SILAC ratio for all identified peptides. Pearson correlation was calculated using a two-tailed test with a 95% confidence interval (Prism6). […]

Pipeline specifications

Software tools Comet, Ascore
Databases ToxoDB
Application MS-based untargeted proteomics
Organisms Toxoplasma gondii
Chemicals Calcium