Computational protocol: Proteomic responses of oceanic Synechococcus WH8102 to phosphate and zinc scarcity and cadmium additions

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Protocol publication

[…] The digests were analyzed by LC-MS using a Microhm Paradigm MS4 HPLC system with reverse phase chromatography, Thermo LTQ ion trap mass spectrometer and Microhm ADVANCE source [2 μL/min flow rate; reversed phase Magic C18 AQ column, 0.2 × 150 mm, 3μm particle size, 200Å poresize; 345 min runs; hyperbolic gradient of water to acetonitrile (each containing 0.1% formic acid)]. Each digest was injected three times for a total of 24 mass spectrometry runs; only two runs from each treatment were analyzed. Mass spectra were processed by SEQUEST and PeptideProphet with a fragment tolerance of 1.0 Da (monoisotopic), parent tolerance of 2.0 Da (monoisotopic, fixed modification of +57 on C (carbamidomethyl), variable modification of +16 on M (oxidation) and a maximum of 2 missed trypsin cleavages using a database including reversed proteins and common contaminants.Spectral counts of 16 files were compiled in Scaffold 3 Proteome Software with a peptide false discovery rate of 1.9%, minimum peptide and protein tolerances of 95 and 99%, respectively, with a minimum of 2 peptides (Peng et al., ; Zhang et al., ). A spectral count is the number of times a particular peptide from a protein is sampled during an MS/MS experiment and the normalized spectral count is indicative of protein relative abundance. Protein functions were assigned manually using the Kyoto Encyclopedia of Genes and Genomes (KEGG) unless otherwise noted. […]

Pipeline specifications

Software tools Comet, PeptideProphet
Databases KEGG
Application MS-based untargeted proteomics
Organisms Synechococcus sp.
Chemicals Cadmium, Zinc