Understanding and Exploiting Peptide Fragment Ion Intensities Using Experimental and Informatic Approaches (original) (raw)
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Peptide Identification by Tandem Mass Spectrometry with Alternate Fragmentation Modes
Molecular & Cellular Proteomics, 2012
The high-throughput nature of proteomics mass spectrometry is enabled by a productive combination of data acquisition protocols and the computational tools used to interpret the resulting spectra. One of the key components in mainstream protocols is the generation of tandem mass (MS/MS) spectra by peptide fragmentation using collision induced dissociation, the approach currently used in the large majority of proteomics experiments to routinely identify hundreds to thousands of proteins from single mass spectrometry runs. Complementary to these, alternative peptide fragmentation methods such as electron capture/transfer dissociation and higher-energy collision dissociation have consistently achieved significant improvements in the identification of certain classes of peptides, proteins, and post-translational modifications. Recognizing these advantages, mass spectrometry instruments now conveniently support fine-tuned methods that automatically alternate between peptide fragmentation modes for either different types of peptides or for acquisition of multiple MS/MS spectra from each peptide. But although these developments have the potential to substantially improve peptide identification, their routine application requires corresponding adjustments to the software tools and procedures used for automated downstream processing. This review discusses the computational implications of alternative and alternate modes of MS/MS peptide fragmentation and addresses some practical aspects of using such protocols for identification of peptides and post-translational modifications. Molecular & Cellular
PROTEOMICS, 2005
In MS/MS experiments with automated precursor ion, selection only a fraction of sequencing attempts lead to the successful identification of a peptide. A number of reasons may contribute to this situation. They include poor fragmentation of the selected precursor ion, the presence of modified residues in the peptide, mismatches with sequence databases, and frequently, the concurrent fragmentation of multiple precursors in the same CID attempt. Current database search engines are incapable of correctly assigning the sequences of multiple precursors to such spectra. We have developed a search engine, ProbIDtree, which can identify multiple peptides from a CID spectrum generated by the concurrent fragmentation of multiple precursor ions. This is achieved by iterative database searching in which the submitted spectra are generated by subtracting the fragment ions assigned to a tentatively matched peptide from the acquired spectrum and in which each match is assigned a tentative probability score. Tentatively matched peptides are organized in a tree structure from which their adjusted probability scores are calculated and used to determine the correct identifications. The results using MALDI-TOF-TOF MS/MS data demonstrate that multiple peptides can be effectively identified simultaneously with high confidence using ProbIDtree.
Analytical Chemistry, 2003
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. Proteomics is playing a pivotal role in the postgenome era in helping to define the functional role of genes. 1,2 Mass spectrometry (MS), coupled with a range of electrophoretic and multidimensional chromatographic separation techniques, has emerged as a key platform technology in proteomics for the rapid and highthroughput identification, characterization, and quantitation of proteins. 3 Typically, proteins are digested using trypsin and the resultant peptides then subjected to MS analysis. The tryptic peptide masses provide a characteristic "mass fingerprint", which can be used to identify proteins. 4-10 Although this approach is useful for identifying proteins in simple mixtures (e.g., 2-DE gel spots), peptide sequence information, obtained by tandem mass spectrometry (MS/MS), is required for identifying individual proteins in more complex mixtures (e.g., 1-DE gels). To this end, sophisticated algorithms (e.g., SEQUEST, Mascot) have been developed for identifying proteins from peptide MS/MS data, 11-14 whereby peptides are identified by correlating the uninterpreted
Analytical Chemistry, 1993
Collisionally activated dissociation (CAD) of the protonated polyalanines Ala-Ala, Ala-Ala-Ala, and Ala-Ala-Ala-Ala causes breakup of the peptide bonds leading to sequence-indicative fragment ions. The neutral molecules eliminated during these reactions are identified here using neutralization-reionization mass spectrometry (NRMS). N-terminal acylium ions (b,) arise after the Cterminus is lost as an intact amino acid or peptide; further loss of CO leads to immonium ions (a,).
Mass spectrometry of peptides and proteins
Methods, 2005
This tutorial article introduces mass spectrometry (MS) for peptide fragmentation and protein identification. The current approaches being used for protein identification include top-down and bottom-up sequencing. Top-down sequencing, a relatively new approach that involves fragmenting intact proteins directly, is briefly introduced. Bottom-up sequencing, a traditional approach that fragments peptides in the gas phase after protein digestion, is discussed in more detail. The most widely used ion activation and dissociation process, gas-phase collision-activated dissociation (CAD), is discussed from a practical point of view. Infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) are introduced as two alternative dissociation methods. For spectral interpretation, the common fragment ion types in peptide fragmentation and their structures are introduced; the influence of instrumental methods on the fragmentation pathways and final spectra are discussed. A discussion is also provided on the complications in sample preparation for MS analysis. The final section of this article provides a brief review of recent research efforts on different algorithmic approaches being developed to improve protein identification searches.
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
Analytical Chemistry, 2012
Increasing peptide sequence coverage by tandem mass spectrometry improves confidence in database search-based peptide identification and facilitates mapping of post-translational modifications and de novo sequencing. Inducing 2-fold fragmentation by combining electrontransfer and higher-energy collision dissociation (EThcD) generates dual fragment ion series and facilitates extensive peptide backbone fragmentation. After an initial electron-transfer dissociation step, all ions including the unreacted precursor ions are subjected to collision induced dissociation which yields b/y-and c/z-type fragment ions in a single spectrum. This new fragmentation scheme provides richer spectra and substantially increases the peptide sequence coverage and confidence in peptide identification.
Molecular & Cellular Proteomics, 2014
Quantifying the similarity of spectra is an important task in various areas of spectroscopy, for example, to identify a compound by comparing sample spectra to those of reference standards. In mass spectrometry based discovery proteomics, spectral comparisons are used to infer the amino acid sequence of peptides. In targeted proteomics by selected reaction monitoring (SRM) or SWATH MS, predetermined sets of fragment ion signals integrated over chromatographic time are used to identify target peptides in complex samples. In both cases, confidence in peptide identification is directly related to the quality of spectral matches. In this study, we used sets of simulated spectra of well-controlled dissimilarity to benchmark different spectral comparison measures and to develop a robust scoring scheme that quantifies the similarity of fragment ion spectra. We applied the normalized spectral contrast angle score to quantify the similarity of spectra to objectively assess fragment ion variability of tandem mass spectrometric datasets, to evaluate portability of peptide fragment ion spectra for targeted mass spectrometry across different types of mass spectrometers and to discriminate target assays from decoys in targeted proteomics. Altogether, this study validates the use of the normalized spectral contrast angle as a sensitive spectral similarity measure for targeted proteomics, and more generally provides a methodology to assess the performance of spectral comparisons and to support the rational selection of the most appropriate similarity measure. The algorithms used in this study are made publicly available as an open source toolset with a graphical user interface.
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.