In this chapter, we introduce the bioinformatics methods associated with studying histidine phosphorylation (pHis) by LC-MS/MS. We describe methods for converting and preprocessing raw data from MS instruments, the method of searching MS data against a sequence database and scoring the confidence associated with localizing the modification site on the peptide sequence. We also describe methods for performing pathway enrichment once a set of pHis-containing proteins have been identified to understand the putative functions of modified proteins. Several of the methods are relatively straightforward to run but require some theoretical knowledge to optimize parameters and correctly interpret outputs. We also describe some of the theory underpinning statistical considerations, to assist correct usage and interpretation of these bioinformatics methods.
Keywords: Bioinformatics; Database searching; Phosphohistidine; Phosphoproteomics; Proteomics; Site localization.