Protein primary structure using orthogonal fragmentation techniques in Fourier transform mass spectrometry

Proteomics analysis using tandem mass spectrometry requires informative backbone fragmentation of peptide ions. Collision-activated dissociation (CAD) of cations alone is not sufficiently informative to satisfy all requirements. Thus, there is a need to supplement CAD with a complementary fragmentation technique. Electron capture dissociation (ECD) is complementary to collisional excitation in terms of the cleavage of a different bond (N-C_α versus C-N bond) and other properties. CAD-ECD combination improves protein identification and enables high-throughput de novo sequencing of peptides. ECD and its variants are also useful in mapping labile post-translational modifications in proteins and isomer differentiation; for example, distinguishing Ile from Leu, iso-Asp from Asp and even D- from L-amino acid residues.     

Application of different fragmentation techniques for the analysis of phosphopeptides using a hybrid linear ion trap-FTICR mass spectrometer

Electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) present complementary techniques for the fragmentation of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) in addition to the commonly used collisionally activated dissociation (CAD). Both IRMPD and ECD have been shown to be applicable for an efficient sequencing of peptides and proteins, whereas ECD has proven especially valuable for mapping labile posttranslational modifications (PTMs), such as phosphorylations. In this work, we compare the different fragmentation techniques and MS detection in a linear ion trap and the ICR cell with respect to their abilities to efficiently identify and characterize phosphorylated peptides. For optimizing fragmentation parameters, sets of synthetic peptides with molecular weights ranging from approximately 1 to 4 kDa and different levels of phosphorylation were analyzed. The influence of spectrum averaging for obtaining high-quality spectra was investigated. Our results show that the fragmentation methods CAD and ECD allow for a facilitated analysis of phosphopeptides; however, their general applicability for analyzing phosphopeptides has to be evaluated in each specific case with respect to the given analytical task. The major advantage of complementary peptide cleavages by combining different fragmentation methods is the increased amount of information that is obtained during MS/MS analysis of modified peptides. On the basis of the obtained results, we are planning to design LC time-scale compatible, data-dependent MS/MS methods using the different fragmentation techniques in order to improve the identification and characterization of phosphopeptides.     

Improving protein identification using complementary fragmentation techniques in fourier transform mass spectrometry

Identification of proteins by MS/MS is performed by matching experimental mass spectra against calculated spectra of all possible peptides in a protein data base. The search engine assigns each spectrum a score indicating how well the experimental data complies with the expected one; a higher score means increased confidence in the identification. One problem is the false-positive identifications, which arise from incomplete data as well as from the presence of misleading ions in experimental mass spectra due to gas-phase reactions, stray ions, contaminants, and electronic noise. We employed a novel technique of reduction of false positives that is based on a combined use of orthogonal fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD). Since ECD and CAD exhibit many complementary properties, their combined use greatly increased the analysis specificity, which was further strengthened by the high mass accuracy (approximately 1 ppm) afforded by Fourier transform mass spectrometry. The utility of this approach is demonstrated on a whole cell lysate from Escherichia coli. Analysis was made using the data-dependent acquisition mode. Extraction of complementary sequence information was performed prior to data base search using in-house written software. Only masses involved in complementary pairs in the MS/MS spectrum from the same or orthogonal fragmentation techniques were submitted to the data base search. ECD/CAD identified twice as many proteins at a fixed statistically significant confidence level with on average a 64% higher Mascot score. The confidence in protein identification was hereby increased by more than 1 order of magnitude. The combined ECD/CAD searches were on average 20% faster than CAD-only searches. A specially developed test with scrambled MS/MS data revealed that the amount of false-positive identifications was dramatically reduced by the combined use of CAD and ECD.     

Electron-capture dissociation tandem mass spectrometry

Electron capture dissociation (ECD) is a new fragmentation technique used in Fourier transform ion cyclotron resonance mass spectrometry and is complementary to traditional tandem mass spectrometry techniques. Disulfide bonds, normally stable to vibrational excitation, are preferentially cleaved in ECD. Fragmentation is fast and specific and labile post-translational modifications and non-covalent bonds often remain intact after backbone bond dissociation. ECD provides more extensive sequence coverage in polypeptides, and at higher electron energies even isoleucine and leucine are distinguishable. In biotechnology, the main area of ECD application is expected to be the top-down verification of DNA-predicted protein sequences, de novo sequencing, disulfide bond analysis and the combined top-down/bottom-up analysis of post-translational modifications.     

Metal oxide-based enrichment combined with gas-phase ion-electron reactions for improved mass spectrometric characterization of protein phosphorylation

Gas-phase ion-electron reactions, including electron capture dissociation (ECD) and electron detachment dissociation (EDD), are advantageous for characterization of protein posttranslational modifications (PTMs), because labile modifications are not lost during the fragmentation process. However, at least two positive charges and relatively abundant precursor ions are required for ECD due to charge reduction and lower fragmentation efficiency compared to conventional gas-phase fragmentation techniques. Both these criteria are difficult to fulfill for phosphopeptides due to their acidic character. The negative ion mode operation of EDD is more compatible with phosphopeptide ionization, but EDD suffers from a fragmentation efficiency even lower than that of ECD. Recently, metal oxides such as ZrO 2 and TiO 2 have been shown to provide selective enrichment of phosphopeptides from proteolytic digests. Here, we utilize this enrichment strategy to improve ECD and EDD of phosphopeptides. This approach allowed determination of the locations of phosphorylation sites in highly acidic, multiply phosphorylated peptides from complex peptide mixtures by ECD. For singly phosphorylated peptides, EDD provided complementary sequence information compared to ECD.     

Increased coverage in the transmembrane domain with activated-ion electron capture dissociation for top-down Fourier-transform mass spectrometry of integral membrane proteins

The c-subunit of ATP synthase (AtpH) is an 8 kD integral membrane protein with two transmembrane domains; we set out to demonstrate it amenable to top-down electrospray-ionization Fourier-transform mass spectrometry (FT-MS) using both collision activated and electron capture dissociation (CAD/ECD). Thermal activation concomitant with electron delivery was necessary for efficient ECD (activated-ion ECD; aiECD), yielding complementary information and greater sequence coverage in the transmembrane domains in comparison with CAD.     

New data base-independent, sequence tag-based scoring of peptide MS/MS data validates Mowse scores, recovers below threshold data, singles out modified peptides, and assesses the quality of MS/MS techniques

The Mascot score (M-score) is one of the conventional validity measures in data base identification of peptides and proteins by MS/MS data. Although tremendously useful, M-score has a number of limitations. For the same MS/MS data, M-score may change if the protein data base is expanded. A low M-value may not necessarily mean poor match but rather poor MS/MS quality. In addition M-score does not fully utilize the advantage of combined use of complementary fragmentation techniques collisionally activated dissociation (CAD) and electron capture dissociation (ECD). To address these issues, a new data base-independent scoring method (S-score) was designed that is based on the maximum length of the peptide sequence tag provided by the combined CAD and ECD data. The quality of MS/MS spectra assessed by S-score allows poor data (39% of all MS/MS spectra) to be filtered out before the data base search, speeding up the data analysis and eliminating a major source of false positive identifications. Spectra with below threshold M-scores (poor matches) but high S-scores are validated. Spectra with zero M-score (no data base match) but high S-score are classified as belonging to modified sequences. As an extension of S-score, an extremely reliable sequence tag was developed based on complementary fragments simultaneously appearing in CAD and ECD spectra. Comparison of this tag with the data base-derived sequence gives the most reliable peptide identification validation to date. The combined use of M- and S-scoring provides positive sequence identification from >25% of all MS/MS data, a 40% improvement over traditional M-scoring performed on the same Fourier transform MS instrumentation. The number of proteins reliably identified from Escherichia coli cell lysate hereby increased by 29% compared with the traditional M-score approach. Finally S-scoring provides a quantitative measure of the quality of fragmentation techniques such as the minimum abundance of the precursor ion, the MS/MS of which gives the threshold S-score value of 2.     

PhosTShunter: a fast and reliable tool to detect phosphorylated peptides in liquid chromatography Fourier transform tandem mass spectrometry data sets

A database independent search algorithm for the detection of phosphopeptides is described. The program interrogates the tandem mass spectra of LC-MS/MS data sets regarding the presence of phosphorylation specific signatures. To achieve maximum informational content, the complementary fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD) are used independently for peptide fragmentation. Several criteria characteristic for peptides phosphorylated on either serine or threonine residues were evaluated. The final algorithm searches for product ions generated by either the neutral loss of phosphoric acid or the combined neutral loss of phosphoric acid and water. Various peptide mixtures were used to evaluate the program. False positive results were not observed because the program utilizes the parts-per-million mass accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Additionally, false negative results were not generated owing to the high sensitivity of the chosen criteria. The limitations of database dependent data interpretation tools are discussed and the potential of the novel algorithm to overcome these limitations is illustrated.     

Electron Capture Dissociation Mass Spectrometry in Characterization of Peptides and Proteins

Electron capture dissociation (ECD) represents one of the most recent and significant advancements in tandem mass spectrometry (MS/MS) for the identification and characterization of polypeptides. In comparison with the conventional fragmentation techniques, such as collisionally activated dissociation (CAD), ECD provides more extensive sequence fragments, while allowing the labile modifications to remain intact during backbone fragmentation—an important attribute for characterizing post-translational modifications. Herein, we present a brief overview of the ECD technique as well as selected applications in characterization of peptides and proteins. Case studies including characterization and localization of amino acid glycosylation, methionine oxidation, acylation, and “top–down” protein mass spectrometry using ECD will be presented. A recent technique, coined as electron transfer dissociation (ETD), will be also discussed briefly.     

Mass spectrometry of peptides and proteins

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.     

Decision tree-driven tandem mass spectrometry for shotgun proteomics

Mass spectrometry has become a key technology for modern large-scale protein sequencing. Tandem mass spectrometry, the process of peptide ion dissociation followed by mass-to-charge ratio (m/z) analysis, is the critical component for peptide identification. Recent advances in mass spectrometry now permit two discrete, and complementary, types of peptide ion fragmentation: collision-activated dissociation (CAD) and electron transfer dissociation (ETD) on a single instrument. To exploit this complementarity and increase sequencing success rates, we designed and embedded a data-dependent decision tree algorithm (DT) to make unsupervised, real-time decisions of which fragmentation method to use based on precursor charge and m/z. Applying the DT to large-scale proteome analyses of Saccharomyces cerevisiae and human embryonic stem cells, we identified 53,055 peptides in total, which was greater than by using CAD (38,293) or ETD (39,507) alone. In addition, the DT method also identified 7,422 phosphopeptides, compared to either 2,801 (CAD) or 5,874 (ETD) phosphopeptides.     

Comparison of the electron capture dissociation fragmentation behavior of doubly and triply protonated peptides from trypsin, Glu-C, and chymotrypsin digestion

In bottom-up proteomics, proteolytically derived peptides from proteins of interest are analyzed to provide sequence information for protein identification and characterization. Electron capture dissociation (ECD), which provides more random cleavages compared to "slow heating" techniques such as collisional activation, can result in greater sequence coverage for peptides and proteins. Most bottom-up proteomics approaches rely on tryptic doubly protonated peptides for generating sequence information. However, the effectiveness, in terms of peptide sequence coverage, of tryptic doubly protonated peptides in ECD remains to be characterized. Herein, we examine the ECD fragmentation behavior of 64 doubly- and 64 triply protonated peptides (i.e., a total of 128 peptide ions) from trypsin, Glu-C, and chymotrypsin digestion in a Fourier transform ion cyclotron resonance mass spectrometer. Our findings indicate that when triply protonated peptides are fragmented in ECD, independent of which proteolytic enzyme was used for protein digestion, more c- and z-type product ions are observed, and the number of complementary fragment pairs increases dramatically (44%). In addition, triply protonated peptides provide an increase (26%) in peptide sequence coverage. ECD of tryptic peptides, in both charge states, resulted in higher sequence coverage compared to chymotryptic and Glu-C digest peptides. The peptide sequence coverage we obtained in ECD of tryptic doubly protonated peptides (64%) is very similar to that reported for electron transfer dissociation of the same peptide type (63%).     

Electron capture dissociation mass spectrometry in characterization of post-translational modifications

Electron capture dissociation (ECD) represents a significant advance in tandem mass spectrometry for the identification and characterization of post-translational modifications (PTMs) of polypeptides. In comparison with the conventional fragmentation techniques, such as collisionally induced dissociation and infrared multi-photon dissociation, ECD provides more extensive sequence fragments, while allowing the labile modifications to remain intact during backbone fragmentation. This unique attribute offers ECD as an attractive alternative for detection and localization of PTMs. The success and rapid adoption of ECD recently led to the culmination of The 1st International Uppsala Symposium on Electron Capture Dissociation of Biomolecules and Related Phenomena (October 19-22, 2003, Stockholm, Sweden). Herein, we present a general overview of the ECD technique as well as selected applications in characterization of post-translationally modified polypeptides.     

Characterization of the iron-binding properties of pyoverdine using electron-capture dissociation-tandem mass spectrometry

Pyoverdines (PVD) are a group of siderophores produced by fluorescent Pseudomonads. Identification of PVD variants mostly relies on liquid chromatography-tandem mass spectrometry (LC–MS/MS) using collision-induced dissociation (CID). Here, both CID and the novel dissociation technique electron-capture dissociation (ECD) were applied to characterize PVD succinamide and its Fe(III)-chelated complex. The results clearly showed that ECD produced diagnostic side chain fragmentation of the PVD peptide chain and preserved the labile Fe(III) binding to the chromophore in contrast to CID. The ECD technique is therefore expected to support the understanding of strain-specific Fe(III) transport processes of PVDs.     

Performance characteristics of electron transfer dissociation mass spectrometry

We performed a large scale study of electron transfer dissociation (ETD) performance, as compared with ion trap collision-activated dissociation (CAD), for peptides ranging from approximately 1000 to 5000 Da (n approximately 4000). These data indicate relatively little overlap in peptide identifications between the two methods ( approximately 12%). ETD outperformed CAD for all charge states greater than 2; however, regardless of precursor charge a linear decrease in percent fragmentation, as a function of increasing precursor m/z, was observed with ETD fragmentation. We postulate that several precursor cation attributes, including peptide length, charge distribution, and total mass, could be relevant players. To examine these parameters unique ETD-identified peptides were sorted by length, and the ratio of amino acid residues per precursor charge (residues/charge) was calculated. We observed excellent correlation between the ratio of residues/charge and percent fragmentation. For peptides of a given residue/charge ratio, there is no correlation between peptide mass and percent fragmentation; instead we conclude that the ratio of residues/charge is the main factor in determining a successful ETD outcome. As charge density decreases so does the probability of non-covalent interactions that can bind a newly formed c/z-type ion pair. Recently we have described a supplemental activation approach (ETcaD) to convert these non-dissociative electron transfer product ions to useful c- and z-type ions. Automated implementation of such methods should remove this apparent precursor m/z ceiling. Finally, we evaluated the role of ion density (both anionic and cationic) and reaction duration for an ETD experiment. These data indicate that the best performance is achieved when the ion trap is filled to its space charge limit with anionic reagents. In this largest scale study of ETD to date, ETD continues to show great promise to propel the field of proteomics and, for small- to medium-sized peptides, is highly complementary to ion trap CAD.     

Proteomics-grade de novo sequencing approach

The conventional approach in modern proteomics to identify proteins from limited information provided by molecular and fragment masses of their enzymatic degradation products carries an inherent risk of both false positive and false negative identifications. For reliable identification of even known proteins, complete de novo sequencing of their peptides is desired. The main problems of conventional sequencing based on tandem mass spectrometry are incomplete backbone fragmentation and the frequent overlap of fragment masses. In this work, the first proteomics-grade de novo approach is presented, where the above problems are alleviated by the use of complementary fragmentation techniques CAD and ECD. Implementation of a high-current, large-area dispenser cathode as a source of low-energy electrons provided efficient ECD of doubly charged peptides, the most abundant species (65-80%), in a typical trypsin-based proteomics experiment. A new linear de novo algorithm is developed combining efficiency and speed, processing on a conventional 3 GHz PC, 1000 MS/MS data sets in 60 s. More than 6% of all MS/MS data for doubly charged peptides yielded complete sequences, and another 13% gave nearly complete sequences with a maximum gap of two amino acid residues. These figures are comparable with the typical success rates (5-15%) of database identification. For peptides reliably found in the database (Mowse score > or = 34), the agreement with de novo-derived full sequences was >95%. Full sequences were derived in 67% of the cases when full sequence information was present in MS/MS spectra. Thus the new de novo sequencing approach reached the same level of efficiency and reliability as conventional database-identification strategies.     

The utility of ETD mass spectrometry in proteomic analysis

Mass spectrometry has played an integral role in the identification of proteins and their post-translational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of post-translationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another.     

基于电子捕获裂解/电子转运裂解串联质谱技术的蛋白质组学研究

蛋白质组学的兴起带动了质谱技术的快速发展,而质谱技术的进步则拓宽了蛋白质组学研究问题的广度.最近10年内,肽段或完整蛋白质在质谱仪中的裂解技术——电子捕获裂解(electron capture dissociation,ECD)与电子转运裂解(electron transfer dissociation,ETD)逐渐发展起来.ECD和ETD在蛋白质组学中的应用,特别是在蛋白质的翻译后修饰鉴定和自顶而下(Top-down)的完整蛋白质裂解研究中已经展示出了诱人的前景.对ECD和ETD的基本原理、质谱特点、仪器实现、数据解析算法与软件开发,以及在蛋白质组学中的应用进展等方面进行了比较系统全面的阐述,并对当前的研究问题、面临的技术挑战与未来的发展趋势等方面作了深入剖析.     

De novo peptide sequencing using CID and HCD spectra pairs

In tandem mass spectrometry (MS/MS), there are several different fragmentation techniques possible, including, collision‐induced dissociation (CID) higher energy collisional dissociation (HCD), electron‐capture dissociation (ECD), and electron transfer dissociation (ETD). When using pairs of spectra for de novo peptide sequencing, the most popular methods are designed for CID (or HCD) and ECD (or ETD) spectra because of the complementarity between them. Less attention has been paid to the use of CID and HCD spectra pairs. In this study, a new de novo peptide sequencing method is proposed for these spectra pairs. This method includes a CID and HCD spectra merging criterion and a parent mass correction step, along with improvements to our previously proposed algorithm for sequencing merged spectra. Three pairs of spectral datasets were used to investigate and compare the performance of the proposed method with other existing methods designed for single spectrum (HCD or CID) sequencing. Experimental results showed that full‐length peptide sequencing accuracy was increased significantly by using spectra pairs in the proposed method, with the highest accuracy reaching 81.31%.