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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.sup.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.sub.3]P[O.sub.4] versus the combined losses of HP[O.sub.3] and [H.sub.2]O) indicates that CID-[MS.sub.3] of [[M + nH - 98].sup.n+] ions may not be used for unambiguous phosphorylation site localization.

Chemical cross-linking combined with proteolytic digestion and mass spectrometry (MS) is a promising approach to provide inter-and intramolecular distance constraints for the structural characterization of protein topologies and functional multiprotein complexes. Despite the relative straightforwardness of these methodologies, the identification and characterization of cross-linked proteins presents a significant analytical challenge, due to the complexity of the resultant peptide mixtures, as well as the array of inter-, intra-, or 'dead-end'-cross-linked peptides that may be generated from a single cross-linking experiment. To address these issues, we describe here the synthesis, characterization, and initial evaluation of a novel 'fixed charge' sulfonium ion-containing cross-linking reagent, S-methyl 5,5'-thiodipentanoylhydroxysuccinimide. The peptide products obtained by reaction with this reagent are all shown to fragment exclusively via facile cleavage of the C-S bond directly adjacent to the fixed charge during CID-MS/MS, resulting in the formation of characteristic product ions that enable the presence and type (i.e., inter, intra, or dead-end) of the cross-linked products to be readily determined, independently of the 'proton mobility' of the precursor ion. Subsequent isolation and dissociation of these products by M[S.sup.3] provides additional structural information required for identification of the peptide sequences involved in the cross-linking reactions, as well as for characterization of the specific site(s) at which cross-linking has occurred. The specificity of these gas-phase fragmentation reactions, as well as the solubility and stability of the cross-linking reagent under aqueous conditions, suggests that this strategy holds great promise for use in future studies aimed at the structural analysis of large proteins or multiprotein assemblies.

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 [micro]g/[cm.sup.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.

Immonium ions have been largely overlooked during the rapid expansion of mass spectrometry-based proteomics largely due to the dominance of ion trap instruments in the field. However, immonium ions are visible in hybrid quadrupole-time-of-flight (QTOF) mass spectrometers, which are now widely available. We have created the largest database to date of high-confidence sequence assignments to characterize the appearance of immonium ions in CID spectra using a QTOF instrument under 'typical' operating conditions. With these data, we are able to demonstrate excellent correlation between immonium ion peak intensity and the likelihood of the appearance of the expected amino acid in the assigned sequence for phenylalanine, tyrosine, tryptophan, proline, histidine, valine, and the indistinguishable leucine and isoleucine residues. In addition, we have clearly demonstrated a positional effect whereby the proximity of the amino acid generating the immonium ion to the amino terminal of the peptide correlates with the strength of the immonium ion peak. This compositional information provided by the immonium ion peaks could substantially improve algorithms used for spectral assignment in mass spectrometry analysis using QTOF platforms.

A database of 5500 unique peptide tandem mass spectra acquired in an ion trap mass spectrometer was assembled for peptides derived from proteins digested with trypsin. Peptides were identified initially from their tandem mass spectra by the SEQUEST algorithm and subsequently validated manually. Two different statistical methods were used to identify sequence-dependent fragmentation patterns that could be used to improve fragmentation models incorporated into current peptide sequencing and database search algorithms. The currently accepted 'mobile proton' model was expanded to derive a new classification scheme for peptide mass spectra, the 'relative proton mobility' scale, which considers peptide ion charge state and amino acid composition to categorize peptide mass spectra into peptide ions containing 'nonmobile', 'partially mobile', or 'mobile' protons. Quantitation of amide bond fragmentation, both N- and C-terminal to any given amino acid, as well as the positional effect of an amino acid in a peptide and peptide length on such fragmentation, has been determined. Peptide bond cleavage propensities, both positive (i.e., enhanced) and negative (i.e., suppressed), were determined and ranked in order of their cleavage preferences as primary, secondary, or tertiary cleavage effects. For example, primary positive cleavage effects were observed for Xaa-Pro and Asp-Xaa bond cleavage for mobile and nonmobile peptide ion categories, respectively. We also report specific pairwise interactions (e.g., Asn-Gly) that result in enhanced amide bond cleavages analogous to those observed in solutionphase chemistry. Peptides classified as nonmobile gave low or insignificant scores, below reported MS/MS score thresholds (cutoff filters), indicating that incorporation of the relative proton mobility scale classification would lead to improvements in current MS/MS scoring functions.

The formation of a range of precursor ion charge states from a single concentrated and purified charge state, followed by activation of each charge state, is introduced as a means to obtain more protein structural information than is available from dissociation of a single charge state alone. This approach is illustrated using off-resonance collisional activation of the [[M + 8H].sup.8+] to [[M + 6H].sup.6+] precursor ions of the bacteriophage MS2 viral coat protein following concentration and purification of the [[M + 8H].sup.8+] charge state. This range of charge states was selected on the basis of an ion trap collisional activation study of the effects of precursor ion charge state on the dissociation of the [[M + 12H].sup.12+] to [[M + 5H].sup.5+] ions. Gas-phase ion/ ion proton-transfer reactions and the ion parking technique were applied to purify and concentrate selected precursor ion charge states as well as to simplify the product ion spectra. The high-charge-state ions fragment preferentially at the N-terminal side of proline residues while the product ion spectra of the lowest charge states investigated are dominated by C-terminal aspartic acid cleavages. Maximum structural information is obtained by fragmentation of the intermediate-charge states.

A 'top-down' protein separation and identification methodology makes use of ion parking approach to facilitate the gas-phase concentration and purification of precursor ions from a complex protein mixture. Dissociation of the ions using a quadrupole ion trap and matching up with the ion spectra database identify the proteins in the mixture.

Recently, an approach for the 'top down' sequence analysis of whole protein ions has been developed, employing electrospray ionization, collision-induced dissociation, and ion/ion proton-transfer reactions in a quadrupole ion trap mass spectrometer. This approach has now been extended to an analysis of the [[M + 12H].sup.12+] to [[M + 5H].sup.5+] ions of ribonuclease A and its N-linked glycosylated analogue, ribonuclease B, to determine the influence of the posttranslational modification on protein fragmentation. In agreement with previous studies on the fragmentation of a range of protein ions, facile gas-phase fragmentation was observed to occur along the protein backbone at the C-terminal of aspartic acid residues, and at the N-terminal of proline, depending on the precursor ion charge state. Interestingly, no evidence was found for gas-phase deglycosylation of the N-linked sugar in ribonuclease B, presumably due to effective competition from the facile amide bond cleavage channels that 'protect' the N-linked glycosidic bond from cleavage. Thus, localization of the posttranslational modification site may be determined by analysis of the 'protein fragment ion mass fingerprint'.

Under appropriate ion density conditions, it is possible to selectively inhibit rates of ion/ion reactions in a quadrupole ion trap via the application of oscillatory voltages to one or more electrodes of the ion trap. The phenomenon is demonstrated using dipolar resonance excitation applied to the end-cap electrodes of a three-dimensional quadrupole ion trap. The application of a resonance excitation voltage tuned to inhibit the ion/ion reaction rate of a specific range of ion mass-to-charge ratios is referred to as 'ion parking'. The bases for rate inhibition are (i) an increase in the relative velocity of the ion/ion reaction pair, which reduces the cross section for ion/ion capture and, at least in some cases, (ii) reduction in the time of physical overlap of positively charged and negatively charged ion clouds. The efficiency and specificity of the ion parking experiment is highly dependent upon ion densities, trapping conditions, ion charge states, and resonance excitation conditions. The ion parking experiment is illustrated herein along with applications to the concentration of ions originally present over a range of charge states into a selected charge state and in the selection of a particular ion from a set of ions derived from a simple protein mixture.

An examination of the gas-phase ion-molecule reactions of neutral alanylglycine with the acylium ions RCO+ (R=CH3, CD3, C6H5, C6F5 and (CH3)2N) is presented.