Phosphopeptide fragmentation and analysis by mass spectrometry

Abstract

Reversible phosphorylation is a key event in many biological processes and is therefore a much studied phenomenon. The mass spectrometric (MS) analysis of phosphorylation is challenged by the substoichiometric levels of phosphorylation and the lability of the phosphate group in collision-induced dissociation (CID). Here, we review the fragmentation behaviour of phosphorylated peptides in MS and discuss several MS approaches that have been developed to improve and facilitate the analysis of phosphorylated peptides. CID of phosphopeptides typically results in spectra dominated by a neutral loss of the phosphate group. Several proposed mechanisms for this neutral loss and several factors affecting the extent at which this occurs are discussed. Approaches are described to interpret such neutral loss-dominated spectra to identify the phosphopeptide and localize the phosphorylation site. Methods using additional activation, such as MS(3) and multistage activation (MSA), have been designed to generate more sequence-informative fragments from the ion produced by the neutral loss. The characteristics and benefits of these methods are reviewed together with approaches using phosphopeptide derivatization or specific MS scan modes. Additionally, electron-driven dissociation methods by electron capture dissociation (ECD) or electron transfer dissociation (ETD) and their application in phosphopeptide analysis are evaluated. Finally, these techniques are put into perspective for their use in large-scale phosphoproteomics studies.

Analysis of human serum phosphopeptidome by a focused database searching strategy

“…For the two CID fragmentation methods, MSA led to slightly more identification than that of MS2 because of its high spectrum quality. This is because the strong neutral loss peak was further fragmented [26,27]. The high quality of the spectra was confirmed by the higher Mascot peptide scores obtained (Fig.…”

Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography

“…The choice of the MS/MS fragmentation method(s) can also affect phosphopeptide identification and site localization. Certain fragmentation regimes are more appropriate than others, but it depends on the sequence of the peptide 65 . For example, electron-transfer/capture dissociation is most appropriate for peptides containing three charges 66,67 or more, whereas all modes of collision-induced dissociation perform sufficiently for peptides containing two charges 68,69 .…”

Dipole-Guided Electron Capture Causes Abnormal Dissociations of Phosphorylated Pentapeptides

“…This mechanism has been shown to be inconsistent with newer labeling data and CID-MS 3 investigation of product ions. A more likely mechanism involves proton-induced elimination with neighboring participation of amino [56] or amide groups [57] from the N-terminal site of the phosphoserine residue, as recently reviewed [58]. Obviously, none of these mechanisms can apply to the elimination of phosphoric acid from deaminated serine residues in the z ions, which have vinylic α-hydrogen atoms and are missing the N-terminal functional groups.…”