Mass spectrometry (MS) is a broadly applicable analytical method that is gaining wide use in protein biochemistry and cell biology. Tandem MS (MS2) fragmentation of analyte ions can be utilized for structural elucidation of proteins and peptides, often determining their partial or completed amino acid sequence and identification of post-translational modifications. Presented here is a study to investigate the behavior of gas phase peptide ions generated mass spectrometrically, focusing specifically on how modification of peptides by phosphorylation on serine/threonine amino acid residues affects their fragmentation chemistry under tandem MS conditions, utilizing a representative library of di-, tri, and tetrapeptides phosphorylated either on a serine or tyrosine residue, and des-phospho counterparts.

Some general traits of mass spectrometric phosphopeptide ion behavior are already characterized, but this investigation aims for a more detailed and sequence-specific understanding of these behaviors. For example, it is known that serine/threonine-phosphorylated, and some tyrosine-phosphorylated) peptide ions often undergo the neutral loss of the elements H3PO4 upon collisionally induced dissociation (CID) MS2. While this neutral loss can be diagnostic of the presence of a phosphorylated species, it can (if it dominates the spectrum) result in the absence of other structural data. The degree to which neutral loss occurs likely depends on chemistry that occurs proximal to the phosphate group. Results have been obtained thus far on a series of serine-phosphorylated tripeptides with the N-terminal residue varied. Differences in energy required for fragmentation are correlated with the chemical nature of the side chain of the N-terminal amino acid.