The effect of phosphorylation on the electron capture dissociation of peptide ions.
ABSTRACT: The effect of site and frequency of phosphorylation on the electron capture dissociation of peptide ions has been investigated. The ECD of a suite of synthetic peptides (APLSFRGSLPKSYVK; one unmodified, three singly-phosphorylated, three-doubly phosphorylated, and one triply-phosphorylated); two tryptic phosphopeptides (YKVPQLEIVPN(p)SAEER, alpha-casein and FQ(p)SEEQQQTEDELQDK, beta-casein) and their unmodified counterparts, were determined over a range of ECD cathode potentials. The results show that, for doubly-charged precursor ions, the presence of phosphorylation has a deleterious effect on ECD sequence coverage. The fragmentation patterns observed suggest that for peptides with multiple basic residues, the phospho-groups exist in their deprotonated form and form salt-bridges with protonated amino acid side chains. The fragmentation observed for the acidic tryptic peptides suggested the presence of noncovalent interactions, which were perturbed on phosphorylation. Increasing the ECD electron energy significantly improves sequence coverage. Alternatively, improved sequence coverage can be achieved by performing ECD on triply-charged precursor ions. The findings are important for the understanding of gas-phase fragmentation of phosphopeptides.
Project description:Electron capture dissociation (ECD) is well suited for the characterization of phosphoproteins, with which labile phosphate groups are generally preserved during the fragmentation process. However, the impact of phosphorylation on ECD fragmentation of intact proteins remains unclear. Here, we have performed a systematic investigation of the phosphorylation effect on ECD of intact proteins by comparing the ECD cleavages of mono-phosphorylated ?-casein, multi-phosphorylated ?-casein, and immunoaffinity-purified phosphorylated cardiac troponin I with those of their unphosphorylated counterparts, respectively. In contrast to phosphopeptides, phosphorylation has significantly reduced deleterious effects on the fragmentation of intact proteins during ECD. On a global scale, the fragmentation patterns are highly comparable between unphosphorylated and phosphorylated precursors under the same ECD conditions, despite a slight decrease in the number of fragment ions observed for the phosphorylated forms. On a local scale, single phosphorylation of intact proteins imposes minimal effects on fragmentation near the phosphorylation sites, but multiple phosphorylations in close proximity result in a significant reduction of ECD bond cleavages. Graphical Abstract ?.
Project description:We report a new method called metal affinity capture that when coupled with tandem mass spectrometry (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with Ga(III) or Fe(III) and N(alpha),N(alpha)-bis(carboxymethyl)lysine (LysNTA) in solution and electrosprayed as doubly or triply charged positive ions. The gas-phase complexes uniformly dissociate to produce a common (LysNTA + H)+ ion that is used as a specific marker in precursor ion scans. The advantages of MAC-MSMS over the current methods of phosphopeptide detection are as follows. (1) MAC-MSMS uses metal complexes that self-assemble in solution at pH <5, which is favorable for the production of positive ions by electrospray. (2) Phosphorylation at tyrosine, serine, and threonine is detected by MAC-MSMS. (3) The phosphopeptide peaks in the mass spectra are encoded with the 69Ga-71Ga isotope pattern for selective recognition in mixtures. Detection by MAC-MSMS of singly and multiply phosphorylated peptides in tryptic digests is demonstrated at low-nanomolar protein concentrations.
Project description:Electron capture dissociation (ECD), which generally preserves the position of labile post-translational modifications, can be a powerful method for de novo sequencing of proteins and peptides. In this report, ECD product-ion mass spectra of singly and doubly sodiated, nonphosphorylated, and phosphorylated peptides are presented and compared with the ECD mass spectra of their protonated counterparts. ECD of doubly charged, singly sodiated peptides yielded essentially the same sequence information as was produced by the corresponding doubly protonated peptides. The presence of several sodium binding sites on the polypeptide backbone, however, resulted in more complicated spectra. This situation is aggravated by the zwitterionic equilibrium of the free acid peptide precursors. The product-ion spectra of doubly and triply charged peptides possessing two sodium ions were further complicated by the existence of isomers created by the differential distribution of sodium binding sites. Triply charged, phosphorylated precursors containing one sodium, wherein the sodium is attached exclusively to the PO4 group, were found to be as useful for sequence analysis as the fully protonated species. Although sodium adducts are generally minimized during sample preparation, it appears that they can nonetheless provide useful sequence information. Additionally, they enable straightforward identification of a peptide's charge state, even on low-resolution instruments. The experiments were carried out using a radio frequency-free electromagnetostatic cell retrofitted into the collision-induced dissociation (CID) section of a hybrid quadrupole/time-of-flight tandem mass spectrometer. Graphical Abstract ?.
Project description:A radio frequency-free electromagnetostatic (EMS) cell devised for electron-capture dissociation (ECD) of ions has been retrofitted into the collision-induced dissociation (CID) section of a triple quadrupole mass spectrometer to enable recording of ECD product-ion mass spectra and simultaneous recording of ECD-CID product-ion mass spectra. This modified instrument can be used to produce easily interpretable ECD and ECD-CID product-ion mass spectra of tyrosine-phosphorylated peptides that cover over 50% of their respective amino-acid sequences and readily identify their respective sites of phosphorylation. ECD fragmentation of doubly protonated, tyrosine-phosphorylated peptides, which was difficult to observe with FT-ICR instruments, occurs efficiently in the EMS cell.
Project description:Top down proteomics in a TOF-TOF instrument was further explored by examining the fragmentation of multiply charged precursors ions generated by matrix-assisted laser desorption ionization. Evaluation of sample preparation conditions allowed selection of solvent/matrix conditions and sample deposition methods to produce sufficiently abundant doubly and triply charged precursor ions for subsequent CID experiments. As previously reported, preferential cleavage was observed at sites C-terminal to acidic residues and N-terminal to proline residues for all ions examined. An increase in nonpreferential fragmentation as well as additional low mass product ions was observed in the spectra from multiply charged precursor ions providing increased sequence coverage. This enhanced fragmentation from multiply charged precursor ions became increasingly important with increasing protein molecular weight and facilitates protein identification using database searching algorithms. The useable mass range for MALDI TOF-TOF analysis of intact proteins has been expanded to 18.2 kDa using this approach.
Project description:Advances in liquid chromatography tandem mass spectrometry (LC-MS/MS) have permitted phosphoproteomic analysis on a grand scale, but ongoing challenges specifically associated with confident phosphate localization continue to motivate the development of new fragmentation techniques. In the present study, ultraviolet photodissociation (UVPD) at 193 nm is evaluated for the characterization of phosphopeptides in both positive and negative ion modes. Compared to the more standard higher energy collisional dissociation (HCD), UVPD provided more extensive fragmentation with improved phosphate retention on product ions. Negative mode UVPD showed particular merit for detecting and sequencing highly acidic phosphopeptides from alpha and beta casein, but was not as robust for larger scale analysis because of lower ionization efficiencies in the negative mode. HeLa and HCC70 cell lysates were analyzed by both UVPD and HCD. While HCD identified more phosphopeptides and proteins compared to UVPD, the unique matches from UVPD analysis could be combined with the HCD data set to improve the overall depth of coverage compared to either method alone.
Project description:Ion-ion reactions between a variety of peptide cations (doubly and triply charged) and SO2 anions have been studied in a 3-D quadrupole ion trap, resulting in proton and electron transfer. Electron transfer dissociation (ETD) gives many c- and z-type fragments, resulting in extensive sequence coverage in the case of triply protonated peptides with SO2*-. For triply charged neurotensin, in which a direct comparison can be made between 3-D and linear ion trap results, abundances of ETD fragments relative to one another appear to be similar. Reactions of doubly protonated peptides with SO2*- give much less structural information from ETD than triply protonated peptides. Collision-induced dissociation (CID) of singly charged ions formed in reactions with SO2*- shows a combination of proton and electron transfer products. CID of the singly charged species gives more structural information than ETD of the doubly protonated peptide, but not as much information as ETD of the triply protonated peptide.
Project description:The fragmentation characteristics of peptides derivatized at the side-chain epsilon-amino group of lysyl residues via reductive amination with benzaldehyde have been examined using collision-induced dissociation (CID) tandem mass spectrometry. The resulting MS/MS spectra exhibit peaks representing product ions formed from two independent fragmentation pathways. One pathway results in backbone fragmentation and commonly observed sequence ion peaks. The other pathway corresponds to the unsymmetrical, heterolytic cleavage of the C(zeta)-N(epsilon) bond that links the benzyl derivative to the side-chain lysyl residue. This results in the elimination of the derivative as a benzylic or tropylium carbocation and a (n - 1)(+)-charged peptide product (where n is the precursor ion charge state). The frequency of occurrence of the elimination pathway increases with increasing charge of the precursor ion. For the benzyl-modified tryptic peptides analyzed in this study, peaks representing products from both of these pathways are observed in the MS/MS spectra of doubly-charged precursor ions, but the carbocation elimination pathway occurs almost exclusively for triply-charged precursor ions. The experimental evidence presented herein, combined with molecular orbital calculations, suggests that the elimination pathway is a charge-directed reaction contingent upon protonation of the secondary epsilon-amino group of the benzyl-derivatized lysyl side chain. If the secondary epsilon-amine is protonated, the elimination of the carbocation is observed. If the precursor is not protonated at the secondary epsilon-amine, backbone fragmentation persists. The application of appropriately substituted benzyl analogs may allow for selective control over the relative abundance of product ions generated from the two pathways.
Project description:The investigation of site-specific glycosylation is essential for further understanding the many biological roles that glycoproteins play; however, existing methods for characterizing site-specific glycosylation either are slow or yield incomplete information. Mass spectrometry (MS) is being applied to investigate site-specific glycosylation with bottom-up proteomic type strategies. When using these approaches, tandem mass spectrometry techniques are often essential to verify glycopeptide composition, minimize false positives, and investigate structure. The fragmentation behavior of glycopeptide ions has previously been investigated with multiple techniques including collision induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD); however, due to the almost exclusive analysis of multiply protonated tryptic glycopeptide ions, some dissociation behaviors of N-linked glycopeptide ions have not been fully elucidated. In this study, IRMPD of N-linked glycopeptides has been investigated with a focus on the effects of charge state, charge carrier, glycan composition, and peptide composition. Each of these parameters was shown to influence the fragmentation behavior of N-linked glycopeptide ions. For example, in contrast to previously reported accounts that IRMPD results only in glycosidic bond cleavage, the fragmentation of singly protonated glycopeptide ions containing a basic amino acid residue almost exclusively resulted in peptide backbone cleavage. The fragmentation of the doubly protonated glycopeptide ion exhibited fragmentation similar to that previously reported; however, when the same glycopeptide was sodium coordinated, a previously inaccessible series of glycan fragments were observed. Molecular modeling calculations suggest that differences in the site of protonation and metal ion coordination may direct glycopeptide ion fragmentation.
Project description:The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR 300 domain at Ser20 and at a non-identified site, neither of which are phosphorylated in vivo, and that (ii) the two sites phosphorylated by CK II in vivo and in vitro are Ser82 and Ser157. The results indicate that the human MPR 300 is a physiological substrate of either CK II or a related kinase which may play a role in the transport function of MPR 300 and/or interaction with other proteins.