Tandem mass spectrometry strategies for phosphoproteome analysis.

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.

Identification of p65-associated phosphoproteins by mass spectrometry after on-plate phosphopeptide enrichment using polymer-oxotitanium films.

Stimuli-induced protein phosphorylation plays a vital role in signal transduction and transcriptional activities in eukaryotic cells. This work aims to develop analysis techniques that rapidly detect stimulus-specific intracellular protein phosphorylation and association, with specific emphasis on identifying phosphoproteins associated with p65, a nuclear regulatory factor. The analytical strategy includes immunoprecipitation of the target protein along with its associated proteins, tryptic digestion directly on the antibody beads, on-plate phosphopeptide enrichment for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) to identify phosphopeptides and phosphorylation sites. Enrichment of the phosphopeptides from the tryptic digest occurs on a polymer-oxotitanium-modified gold wafer (Au-P-oxoTi) and consumes as little as 1 microL of digest solution. The Au-P-oxoTi wafers can capture both mono- and multiphosphorylated peptides from model protein digests containing high concentrations of nonphosphopeptides, urea, and salts. When combined with MALDI-MS/MS, the enrichment technique reveals nine phosphopeptides from p65-associated proteins immunoprecipitated from human acute monocytic leukemia (THP-1) cell nuclear extracts. Semiquantitative MALDI-MS shows that the levels of these proteins increase dramatically after treatment with tumor necrosis factor (TNF)-alpha. Overall, these techniques facilitated the identification of five p65-associated proteins, two of which were not previously reported to interact with p65.

Mass spectrometric characterization and activity of zinc-activated proinsulin C-peptide and C-peptide mutants.

Numerous reports have demonstrated an active role for proinsulin C-peptide in ameliorating chronic complications associated with diabetes mellitus. It has been recently reported that some of these activities are dependent upon activation of C-peptide with certain metal ions, such as Fe(II), Cr(III) or Zn(II). In an effort to gain a greater understanding of the structure/function dependence of the peptide-metal interactions responsible for this activity, a series of experiments involving the use of electrospray ionization (ESI), matrix assisted laser desorption/ionization (MALDI) and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of C-peptide in the presence or absence of Zn(II) have been carried out. Additionally, various C-peptide mutants with alanine substitution at individual aspartic acid or glutamic acid residues throughout the C-peptide sequence were analyzed. CID-MS/MS of wild type C-peptide in the presence of Zn(II) indicated multiple sites for metal binding, localized at acidic residues within the peptide sequence. Mutations of individual acidic residues did not significantly affect this fragmentation behavior, suggesting that no single acidic residue is critical for binding. However, ESI-MS analysis revealed an approximately 50% decrease in relative Zn(II) binding for each of the mutants compared to the wild type sequence. Furthermore, a significant decrease in activity was observed for each of the Zn(II)-activated mutant peptides compared to the wild type C-peptide, indicated by measurement of ATP released from erythrocytes, with a 75% decrease observed for the Glu27 mutant. Additional studies on the C-terminal pentapeptide of C-peptide EGSLQ, as well as a mutant C-terminal pentapeptide sequence AGSLQ, revealed that substitution of the glutamic acid residue resulted in a complete loss of activity, implicating a central role for Glu27 in Zn(II)-mediated C-peptide activity.

Evaluation of gas-phase rearrangement and competing fragmentation reactions on protein phosphorylation site assignment using collision induced dissociation-MS/MS and MS3.

The development of strategies directed toward comprehensive analysis of the phosphoproteome have undoubtedly been facilitated by recent advances in the application of ion trap tandem mass spectrometry-based techniques for routine phosphopeptide identification. However, when multiple potential sites of phosphorylation exist within a phosphorylated peptide sequence, unambiguous characterization of the site of phosphorylation remains a significant challenge. Here, the gas-phase fragmentation reactions of a series of 33 synthetic phospho-serine, -threonine, and -tyrosine peptides containing multiple potential phosphorylation sites have been examined using collision induced dissociation (CID) and multistage tandem mass spectrometry (MS/MS and MS(3)) in a linear quadrupole ion trap. From this study, 15 of the peptides (45%) gave rise to product ions that were formed following initial transfer of a phosphate group from the phosphorylated residue to an unmodified hydroxyl-containing amino acid residue upon CID-MS/MS. The propensity for this rearrangement was found to be highly dependent on the precursor ion charge state and amino acid composition (i.e, proton mobility) of the peptide and was observed predominantly for peptides under "nonmobile" or "partially mobile" protonation conditions. The observation of these rearrangement reactions and/or the lack of product ions that provided definitive evidence for the correct site of phosphorylation, limited the ability to unambiguously assign the correct site of phosphorylation to only 12 of the 33 peptides (36%). Furthermore, the observation of competing fragmentation reactions for the neutral loss of 98 Da from these precursor ions (i.e., the loss of H(3)PO(4) versus the combined losses of HPO(3) and H(2)O) indicates that CID-MS(3) of [M + nH - 98](n+) ions may not be used for unambiguous phosphorylation site localization.

Phosphopeptide enrichment using MALDI plates modified with high-capacity polymer brushes.

Matrix-assisted laser desorption/ionization plates coated with poly(2-hydroxyethyl methacrylate) (PHEMA) brushes that are derivatized with Fe(III)-nitrilotriacetate (NTA) complexes allow selective, efficient phosphopeptide enrichment prior to analysis by mass spectrometry (MS). Fe(III)-NTA-PHEMA brushes (60 nm thick) have a phosphopeptide binding capacity of 0.6 microg/cm(2) and exhibit phosphopeptide recoveries of over 70%, whereas much thinner polymer films containing Fe(III)-NTA afford a recovery of only 20%, and a monolayer of Fe(III)-NTA shows a recovery of just 10%. Recoveries are determined by comparing signals from enriched unlabeled phosphopeptides with those of their deuterium-labeled analogues that were added to the plate just prior to addition of matrix. Mass spectra of phosphopeptide-containing samples enriched using Fe(III)-NTA-PHEMA-modified plates also demonstrate higher recoveries or fewer interfering peaks than corresponding spectra obtained with enrichment using several commercially available Fe(III)-containing films and resins or metal oxide materials. When analyzing tryptic digests of beta-casein, the Fe(III)-NTA-PHEMA brushes allow detection of as little as 15 fmol of phosphopeptide. Moreover, with both ovalbumin and beta-casein digests, phosphopeptide signals dominate the mass spectra obtained using these modified plates.

Mechanistic insights into the multistage gas-phase fragmentation behavior of phosphoserine- and phosphothreonine-containing peptides.

The increasing use of multistage tandem mass spectrometry (MS/MS and MS (3)) methods for comprehensive phosphoproteome analysis studies, as well as the emerging application of in silico spectral intensity prediction algorithms in enhanced database search analysis strategies, necessitate the development of an improved understanding of the mechanisms and other factors that affect the gas-phase fragmentation reactions of phosphorylated peptide ions. To address this need, we have examined the multistage collision-induced dissociation (CID) behavior of a set of singly and doubly charged phosphoserine- and phosphothreonine-containing peptide ions, as well as their regioselectively or uniformly deuterated derivatives, in a quadrupole ion trap mass spectrometer. Consistent with previous reports, the neutral loss of phosphoric acid (H 3PO 4) was observed as a dominant reaction pathway upon MS/MS. The magnitude of this loss was found to be highly dependent on the proton mobility of the precursor ion for both phosphoserine- and phosphothreonine-containing peptides. In contrast to that currently accepted in the literature, however, the results obtained in this study unequivocally demonstrate that the loss of H 3PO 4 does not predominantly occur via a "charge-remote" beta-elimination reaction. The observation of product ions corresponding to the loss of formaldehyde (CH 2O, 30 Da, or CD 2O, 32 Da) or acetaldehyde (CH 3CHO, 44 Da) upon MS (3) dissociation of the [M+ nH-H 3PO 4] ( n+ ) product ions from phosphoserine- and phosphothreonine-containing peptide ions, respectively, provide experimental evidence for a "charge-directed" mechanism involving an S N2 neighboring group participation reaction, resulting in the formation of a cyclic product ion. Potentially, these "diagnostic" MS (3) product ions may provide additional information to facilitate the characterization of phosphopeptides containing multiple potential phosphorylation sites.