Tandem mass spectrometry strategies for phosphoproteome analysis

Abstract

Protein phosphorylation is involved in nearly all essential biochemical pathways and the deregulation of phosphorylation events has been associated with the onset of numerous diseases. A multitude of tandem mass spectrometry (MS/MS) and multistage MS/MS (i.e., MS(n) ) strategies have been developed in recent years and have been applied toward comprehensive phosphoproteomic analysis, based on the interrogation of proteolytically derived phosphopeptides. However, the utility of each of these MS/MS and MS(n) approaches for phosphopeptide identification and characterization, including phosphorylation site localization, is critically dependant on the properties of the precursor ion (e.g., polarity and charge state), the specific ion activation method that is employed, and the underlying gas-phase ion chemistries, mechanisms and other factors that influence the gas-phase fragmentation behavior of phosphopeptide ions. This review therefore provides an overview of recent studies aimed at developing an improved understanding of these issues, and highlights the advantages and limitations of both established (e.g., CID) and newly maturing (e.g., ECD, ETD, photodissociation, etc.) yet complementary, ion activation techniques. This understanding is expected to facilitate the continued refinement of existing MS/MS strategies, and the development of novel MS/MS techniques for phosphopeptide analysis, with great promise in providing new insights into the role of protein phosphorylation on normal biological function, and in the onset and progression of disease. © 2011 Wiley Periodicals, Inc., Mass Spec Rev 30:600-625, 2011.

Gas Phase Chemistry of Li+with Amides: the Observation of LiOH Loss in Mass Spectrometry

“…Introduction E SI-MS/MS has assumed increasing importance as an invaluable tool for the study of the gas phase behaviors of biomolecules [1][2][3]. In the past few decades, continuous efforts have been devoted to the investigation of the dissociation reactions of charged compounds derived from protonation [4][5][6][7][8] or deprotonation [9][10][11][12][13].…”

Peptide Identification by Tandem Mass Spectrometry with Alternate Fragmentation Modes

“…One of the earlier such approaches (73) is the still a popular phosphoproteomics protocol (74) in which MS 3 acquisition is triggered by the dominant loss of phosphate from precursor ions observed in MS 2 spectra. Alternating MS 2 modes further improve identification of phosphorylated peptides (35,45,74) and, in addition, enable otherwise challenging experiments such as co-identification of glycans and peptides from glycosylated peptides using ETD (75) or alternating CID/ETD acquisition (37,76). The complementarity of CID and ETD fragmentation modes is especially useful for PTMs such as glycosylation because CID leads to preferential fragmentation of the more labile glycosidic bonds and generally poor peptide fragmentation, thus facilitating glycan identification but complicating peptide identification.…”

“…Accurate localization of PTM sites is also an important area that stands to gain from alternate (30,32,74) and alternative (77) peptide fragmentation modes. As recently reviewed by Chalkley and Clauser (78), the problem of PTM site localization was first addressed with the AScore approach (79), which assigns a probabilistic score to a site assignment based on the number of observed ions distinguishing the top-scoring site assignment from the runner-up site assignment.…”

A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses

“…The protonated amide linkages are weakened and favored to create a series of homologous products ions upon collision-activation. Fragmentation of peptides by CID creates the complementary b (in case the charge is retained in the C-terminus) and y (in case the charge is retained in the N-terminus) ions [64].…”