Shotgun phosphoproteome of Sulfolobus acidocaldarius using PAciFIC technique. Briefly, proteins were denatured, alkylated, trypsine digestion, SCX separation Fractionated peptides were analysed on Bruker HCTultra. Data processing and bioinformatics: data from mass spectrometry were then extracted to mgf format using Bruker Data Analysis V4.0 with a MRM script, these were then were searched against the Sulfolobus acidocaldarius database (containing 2223 proteins) downloaded from NCBI in March 2010 using Phenyx V 2.6 (Genebio, Geneva). The searches were performed using parameters as follows: carbamidomethylation of cysteine (fixed modification), oxidation of methionine (variation), and phosphorylation of serine, tyrosine, threonine (variation), trypsin with 2 missed cleavages. Furthermore, other parameters such as parent, MS/MS, tolerances were set at 2.0 and 0.8 Da, respectively, whilst minimum peptide length, z-score, p-value and AC score were set at 5, 5.5, 10-5, and 5.5 respectively.