Phosphopeptide compositions and anti-phosphopeptide antibody compositions and methods of detecting phosphorylated peptides

A method of identifying phosphorylation sites within a plurality of proteins or peptides or mixture thereof comprising:

• subjecting the protein or peptide to phosphate enrichment and analyzing the phosphate enriched protein or peptide using microcapillary liquid chromatography coupled online to a tandem mass spectrometer (LC-MS/MS) to produce a raw data set of spectra comprising amino acids and applying at least one filtering criterion to the raw data set of spectra to obtain a refined data set of spectra;
• comparing the refined data set of spectra to a protein database of amino acid sequences in a forward direction of the sequences, to identify at least one phosphorylated amino acid residue in each of the phosphorylation sites within the sequences;
• evaluating correct location of the phosphorylation sites by determining site-determining ions and computing the likelihood of detection in the refined data set of spectra by chance alone, wherein the site-determining ions comprise ions whose detection distinguishes between at least two adjacent acceptor residues, wherein the residues are at least one selected from the group of:
• serine, threonine, and tyrosine, wherein computing the likelihood of detection comprises identifying absence or presence of the site-determining b- and y-type fragment ions in the refined data set of spectra using an algorithm, wherein the algorithm comprises a cumulative binomial probability P calculated using number of trials N, number of successes n, and probability of success p:
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• such that P represents the probability of randomly matching at least a given number of fragment ions to the spectra, the total number of trials (N) equals total number of the fragment ions for the protein or peptide, and the total number of successes (n) equaled the number of ions matched to the refined data set of spectra, and such that identifying further includes multiplying −10 by the log(P)
• and;
• comparing the intrinsic amino acid sequence alignment of phospho-residues to a dynamic statistical background to extract phosphorylation motifs from the phosphorylation sites, wherein comparing comprises using the phosphorylation sites having an Ascore of more than 19, and correlating the phosphorylation motifs with cellular localization and protein function, thereby identifying phosphorylation sites.