Electron Capture Dissociation of Sodium-Adducted Peptides on a Modified Quadrupole/Time-of-Flight Mass Spectrometer (original) (raw)
Related papers
Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry
Proceedings of the National Academy of Sciences, 2004
Peptide sequence analysis using a combination of gas-phase ion͞ion chemistry and tandem mass spectrometry (MS͞MS) is demonstrated. Singly charged anthracene anions transfer an electron to multiply protonated peptides in a radio frequency quadrupole linear ion trap (QLT) and induce fragmentation of the peptide backbone along pathways that are analogous to those observed in electron capture dissociation. Modifications to the QLT that enable this ion͞ion chemistry are presented, and automated acquisition of high-quality, single-scan electron transfer dissociation MS͞MS spectra of phosphopeptides separated by nanoflow HPLC is described. electron capture dissociation ͉ fragmentation ͉ ion͞ion reactions ͉ charge transfer ͉ ion trap S ix years ago, McLafferty and coworkers (1) introduced a unique method for peptide͞protein ion fragmentation: electron capture dissociation (ECD). In this method, multiply protonated peptides or proteins are confined in the Penning trap of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer and exposed to electrons with near-thermal energies. Capture of a thermal electron by a protonated peptide is exothermic by Ϸ6 eV (1 eV ϭ 1.602 ϫ 10 Ϫ19 J) and causes the peptide backbone to fragment by a nonergodic process, e.g., one that does not involve intramolecular vibrational energy redistribution (2-5). One pathway for this process involves generation of an odd-electron hypervalent species (RNH 3 • ) that dissociates to produce RNH 2 and a hydrogen radical (6). As shown in , addition of H • to the carbonyl groups of the peptide backbone leads to a homologous series of complementary fragment ions of types c and z. Addition of H • to an amide nitrogen, a secondary pathway, leads to the formation of carbon monoxide plus a homologous series of complementary fragment ions of types a and y. Subtraction of the m͞z values for the fragments within a given ion series that differ by a single amino acid affords the mass and thus the identity of the extra residue in the larger of the two fragments. The complete amino acid sequence of a peptide is deduced by extending this process to all homologous pairs of fragments within a particular ion series.
Analytical Chemistry, 2007
We have tested the effect of m-nitrobenzyl alcohol (m-NBA) as a method to increase the average charge state of protonated gas-phase molecular ions generated by ESI from tryptic peptides and phosphopeptides. Various concentrations of m-NBA were added to the mobile phases of a liquid chromatography system coupled to an ESI tandem mass spectrometer. Addition of just 0.1% m-NBA changed the average charge state for the identified tryptic BSA peptides from 2.2+ to 2.6+. As a result, the predominant charge states for BSA peptides were changed from 2+ to g3+. To evaluate the benefits of peptide charge enhancement, the ETD fragmentation efficiency and Mascot peptide score were compared for BSA peptides in charge states 2+ and 3+. In all cases but one, triply charged peptides fragmented more efficiently than the analogues 2+ peptide ions. On average, triply charged peptides received a 68% higher Mascot score (24 units) than doubly charged peptides. m-NBA also increased the average charge state of phosphopeptides by up to 0.5 charge unit. The ease of implementation and the analytical benefits of charge enhancement of tryptic peptides by addition of m-NBA to the LC solvents suggest the general application of this reagent in proteomic studies that employ ETD-MS/MS and related techniques.
Journal of the American Society for Mass Spectrometry, 2008
Electron detachment dissociation (EDD) of peptide poly-anions is gentle towards posttranslational modifications (PTMs) and produces predictable and interpretable fragment ion types (a·, x ions). However, EDD is considered an inefficient fragmentation technique and has not yet been implemented in large-scale peptide characterization strategies. We successfully increased the EDD fragmentation efficiency (up to 9%), and demonstrate for the first time the utility of EDD-MS/MS in liquid chromatography time-scale experiments. Peptides and phosphopeptides were analyzed in both positive-and negative-ion mode using electron capture/transfer dissociation (ECD/ETD) and EDD in comparison. Using approximately 1 pmol of a BSA tryptic digest, LC-EDD-MS/MS sequenced 14 peptides (27% aa sequence coverage) and LC-ECD-MS/MS sequenced 19 peptides (39% aa sequence coverage). Seven peptides (18% aa sequence coverage) were sequenced by both EDD and ECD. The relative small overlap of identified BSA peptides demonstrates the complementarity of the two dissociation modes. Phosphopeptide mixtures from three trypsin-digested phosphoproteins were subjected to LC-EDD-MS/MS resulting in the identification of five phospho-peptides. Of those, one was not found in a previous study using a similar sample and LC-ETD-MS/MS in the positive-ion mode. In this study, the ECD fragmentation efficiency (15.7% av.) was superior to the EDD fragmentation efficiency (3.6% av.). However, given the increase in amino acid sequence coverage and extended PTM characterization the new regime of EDD in combination with other ion-electron fragmentation techniques in the positive-ion mode is a step towards a
Electron capture dissociation mass spectrometric analysis of lysine-phosphorylated peptides
Bioscience Reports, 2010
Phosphorylation of proteins is an essential signalling mechanism in eukaryotic and prokaryotic cells. Although Nphosphorylation of basic amino acid is known for its importance in biological systems, it is still poorly explored in terms of products and mechanisms. In the present study, two MS fragmentation methods, ECD (electron-capture dissociation) and CID (collision-induced dissociation), were tested as tools for analysis of N-phosphorylation of three model peptides, RKRSRAE, RKRARKE and PLSRTLSVAAKK. The peptides were phosphorylated by reaction with monopotassium phosphoramidate. The results were confirmed by 1 H NMR and 31 P NMR studies. The ECD method was found useful for the localization of phosphorylation sites in unstable lysine-phosphorylated peptides. Its main advantage is a significant reduction of the neutral losses related to the phosphoramidate moiety. Moreover, the results indicate that the ECD-MS may be useful for analysis of regioselectivity of the N-phosphorylation reaction. Stabilities of the obtained lysine-phosphorylated peptides under various conditions were also tested.
Organic Mass Spectrometry, 1994
The neutral products arising during the collisionally activated dissociation of protonated oligopeptides (MH+) are post-ionized by collision and detected in neutral fragment-reionizatioo (+N,R+) mass spectra. For the isomeric tripeptides Ala-Gly-Gly, Gly-Ala-Gly and Gly-Gly-Ala, the amino acid and dipeptide losses from the C-terminus and the diketopiperazine losses from the N-terminus allow for differentiation. These neutral fragments are identified in the corresponding +N,R+ spectra by comparison to reference collision-induced dissociative ionization (CIDI) mass spectra of individual amino acids, dipeptides and diketopiperazines. Peptides with distinct C-termini but otherwise identical sequences are found to yield +N,R+ products that are characteristic of the respective C-terminal amino acid. This is demonstrated for several peptide pairs, including leucine-and methionineenkephalin. In general, +NfR+ spectra are dominated by the heavier neutral losses; further, +NfR+ and ClDI cause extensive dissociation, indicating that the collisional ionization process imparts high average internal energies. 0 CH3
The software Peptide Fragment Ion Analyser (PFIA) aids in the analysis and interpretation of tandem mass spectrometric data of peptides. The software package has been designed to facilitate the analysis of product ions derived from acyclic and cyclic peptide natural products that possess unusual amino acid residues and are heavily post-translationally modified. The software consists of two programmes: (a) PFIA-I lists the amino acid compositions and their corresponding product ion types for 'a queried m/z value' (z = +1) and (b) PFIA-II displays fragmentation pattern diagram(s) and lists all sequence-specific product ion types for the protonated adduct of 'a queried sequence'. The unique feature of PFIA-II is its ability to handle cyclic peptides. The two programmes used in combination can prove helpful for deriving peak assignments in the de novo sequencing of novel peptides.
Journal of Mass Spectrometry, 2014
Improving the sensitivity of detection and fragmentation of peptides to provide reliable sequencing of peptides is an important goal of mass spectrometric analysis. Peptides derivatized by bicyclic quaternary ammonium ionization tags: 1-azabicyclo[2.2.2]octane (ABCO) or 1,4-diazabicyclo[2.2.2]octane (DABCO), are characterized by an increased detection sensitivity in electrospray ionization mass spectrometry (ESI-MS) and longer retention times on the reverse-phase (RP) chromatography columns. The improvement of the detection limit was observed even for peptides dissolved in 10 mM NaCl. Collision-induced dissociation tandem mass spectrometry of quaternary ammonium salts derivatives of peptides showed dominant a- and b-type ions, allowing facile sequencing of peptides. The bicyclic ionization tags are stable in collision-induced dissociation experiments, and the resulted fragmentation pattern is not significantly influenced by either acidic or basic amino acid residues in the peptide sequence. Obtained results indicate the general usefulness of the bicyclic quaternary ammonium ionization tags for ESI-MS/MS sequencing of peptides.