The Confounding Effects of Concurrent Metaphosphate Neutral Loss and Peptide Backbone Fragmentation on Phosphorylation Site Localization

Abstract

Phosphorylation is a critical post-translational modification (PTM) involved in many cellular signaling processes. Tandem liquid-chromatography mass spectrometry (LC-MS) based experiments are commonly used to analyze levels of protein expression and their PTMs. To accurately identify phosphorylation sites through LC-MS experiments, the amino acid sequence of a given peptide identification must be correctly assigned, and the phosphate localized to the appropriate residue. A variety of localization algorithms are utilized to control the false localization rate (FLR) in LC-MS experiments. Some localization algorithms rely on the identification of site determining ions which discriminate between potential phosphorylated residues. These algorithms often interpret fragment ions which lack a phosphate as positive evidence to suggest the phosphate must reside on the complementary fragment. However concurrent HPO3 neutral loss and peptide backbone fragmentation is a possible alternative explanation. Using a phosphopeptide library containing over 2,000 phosphopeptides, we show that concurrent HPO3 neutral loss and peptide backbone fragmentation correlates with incorrect localization assignments. These confounding fragments can be more problematic in some higher energy collision dissociation (HCD) based datasets as opposed to collision- induced dissociation (CID) based datasets. When restricting a test localization algorithm to only consider phosphate harboring site determining ions, we see a consistent decrease in FLR’s across a variety of fragmentation types. Our results implicate HPO3 neutral loss as a problematic neutral loss for common phosphorylation localization algorithms.