Phosphopeptide fragmentation and analysis by mass spectrometry

• Published in: Journal of mass spectrometry. Vol. 44; no. 6; pp. 861 - 878
• Main Authors: Boersema, Paul J, Mohammed, Shabaz, Heck, Albert J.R
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.06.2009; Wiley
• Subjects: Activation; Analytical, structural and metabolic biochemistry; Biological and medical sciences; CID; ECD; Electron capture; ETD; Fragmentation; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Mass spectrometry; Mass Spectrometry - instrumentation; Mass Spectrometry - methods; Mass Spectrometry - trends; MS3; MSA; multistage activation; neutral loss; Peptides; Phosphates; Phosphopeptides - analysis; Phosphopeptides - chemistry; Phosphorylation; Proteins; Spectra; Tandem mass spectrometry; Phosphorylation; Collisional activation; Peptides; MS; Phosphopeptide; ECD; Electron transfer dissociation; Fragmentation pattern; Review; Protein; fragmentation; Electron capture dissociation; Ion trap; Analysis method; ETD; Peptide fragment; Proteomics; MSA; neutral loss; multistage activation; Mass spectrometry; Structural analysis; CID
• ISSN: 1076-5174; 1096-9888; 1096-9888
• DOI: 10.1002/jms.1599

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 MS³ 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. Copyright © 2009 John Wiley & Sons, Ltd.