Peptide Sequencing and Characterization of Post-Translational Modifications by Enhanced Ion-Charging and Liquid Chromatography Electron-Transfer Dissociation Tandem Mass Spectrometry (original) (raw)
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European Journal of Mass Spectrometry, 2010
We report here a procedure for the independent analysis of two groups of peptides by liquid chromatography–matrix-assisted laser desorption/ionization mass spectrometry (LC-MALDI MS/MS), using a selective isolation–detection procedure. In this procedure all primary amino groups of tryptic peptides derived from mouse liver proteins are blocked, restricting their positive charge, at acidic pH, to the presence of histidine and arginine residues. After strong cation exchange chromatography, multiply charged peptides (R + H > 1) are retained on the column and separated with high selectivity from singly (R + H = 1) and neutral peptides (R + H = 0) which are together collected in the flow-through. Using LC-MALDI-MS/MS analysis, the retained fraction displayed a 94% of enrichment of multiply charged peptides while in the flow-through; peptides with at least one arginine or histidine residue were exclusively identified, which suggests that MS detection in this fraction is restricted only ...
The utility of ETD mass spectrometry in proteomic analysis
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006
Mass spectrometry has played an integral role in the identification of proteins and their posttranslational 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 posttranslationally 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.
Nat Biotechnol, 2003
Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system 1,2 or it is first reduced in complexity by isolating sets of representative peptides 3-8. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis 9 and diagonal chromatography 10. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides 11. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation 12 and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS) 13,14 ; they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins. We used this technique to identify 264 proteins and 78 in vivoacetylated proteins in a cytosolic and membrane skeleton fraction of human thrombocytes. In addition to showing that this method can be used for gel-free proteomics, we demonstrate that it allows one to examine N-terminal protein processing such as removal of signal sequences and modifications. A general scheme depicting the procedure for sorting N-terminal peptides is shown in Figure 1. At the protein level, cysteines are alkylated with iodoacetamide, and free primary amines are blocked by acetylation. Upon trypsin digestion, two types of peptides are generated: internal peptides with a free α-amino group and blocked N-terminal peptides. These peptides are fractionated by RP-high-performance (HP) LC, typically in 12 fractions (primary run, Fig. 2A). The dried fractionated peptides are redissolved in an appropriate buffer for reaction with TNBS. Only internal peptides react with TNBS to form very hydrophobic trinitrophenyl-peptides (TNP-peptides), whereas blocked N-terminal peptides are not affected. On average, this TNBS modification reaction proceeds to more than 98% completion. Each TNBS-treated primary fraction is separately rerun on the same column and under conditions identical to those for the primary run. The TNP-labeled internal peptides now shift to later elution times and separate from the unaltered N-terminal peptides, which do not shift and can be easily collected for further analysis (Fig. 2B). The secondary run and analysis step is repeated for each TNBS-modified fraction. With this procedure we analyzed the proteome of a cytosolic and membrane skeleton fraction of human thrombocytes 15. Sorted N-terminal peptides in all fractions were analyzed using 96 LC-MS/MS runs, during which 5,640 collision-induced dissociation (CID)
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
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.
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.
2 Tandem Mass Spectrometry of Peptides
2020
2. Peptide fragmentation 2.1 Proteins into amino acids Nowadays, peptide fragmentation is the most commonly used MS information for protein identification in proteomic studies. In this type of studies, there are two approaches that can be used: a gel-based approach (Franco et al., 2001a) or a gel-free approach (Washburn et al., 2001), where the former involves an initial separation of a complex mixture of proteins in, www.intechopen.com Tandem Mass Spectrometry-Applications and Principles 36 for example, SDS-or two-dimensional-PAGE. Instead, in the latter, the separation of the complex mixture of previously digested proteins is accomplished using one or two different types of chromatographic separations. In this case, before the separation step, the proteins need to be cleaved into smaller compounds, namely peptides. This is usually accomplished through the use of specific proteases, most commonly trypsin. Different peptides are generated during protein digestion with different proteases, for example trypsin cleaves after lysine or arginine, and hence tryptic peptides will always end in one of these residues. This digestion is necessary as, for most mass spectrometers, proteins are too big to be analysed by tandem MS. This type of approach, where protein digestion is performed before mass spectrometry analysis, is called bottom-up proteomics. Alternatively, intact proteins can be directly analysed in particular mass spectrometers, for example FTICR and Orbitrap mass spectrometers, for protein identification and characterization without the need of proteolytic digestion. In this strategy, named top-down proteomics, proteins are introduced into the mass spectrometer, its mass measured and directly fragmented in the equipment (Reid & McLuckey, 2002). This procedure has several applications related to protein analysis, such as the characterization of post-translational modifications, protein confirmations, protein-ligand and protein-protein complexes, among others (see section 4.2 of this chapter). For more detailed information, we suggest the review by Cui and colleagues (2011). Sample handling for tandem mass spectrometry analysis should always be done with special care due to the fact that contaminations and induced modifications during sample processing can occur. A common contamination that can occur when performing, in particular, protein digestions is with keratin, which can hinder the identification of proteins. The most common keratin contaminations occur from human hair and hands from the operator , indicating the crucial use of clean lab coat, cap and nitrile gloves. The use of an electrostatic eliminator can also be used to reduce this type of contamination, as demonstrated by Xu et al. (2011). Standard protein digestion procedures are time consuming and involve multisteps. Automatization has been introduced, although it is only cost effective and efficient for a large number of samples. Alternatively simplified protocols have been developed. In a study by Ren and co-workers (2009) a quick digestion protocol was implemented in order to minimize the digestion-induced modifications on proteins, such as asparagine deamidation and N-terminal glutamine cyclization, found to be directly proportional to incubation time in reduction or alkylation and depending on digestion buffer composition. The authors tested this modified protocol on immunoglobulin gamma, which allowed reducing the total experimental procedure to a few hours, beside a protein coverage of 98.6% for IgG. Particular attention must be driven to the amount of salts and detergents that protein digests have prior to MS or MS/MS analysis due to the fact that these can interfere with the ionization of peptides and can create strong interference on the mass spectrum signal. This can be bypassed by using a HPLC system prior to the MS analysis due to the fact that salts and detergents do not bind to the typical reverse phase columns used for peptide separation eluting in the first steps of the chromatographic run. If a HPLC system is not used, then alternative simpler methods should be used, such as micro columns packed with reverse-phase resins or graphite (Larsen et al., 2007) and, when using MALDI-TOF-TOF, this desalting step can be done directly onto the MALDI target plate (Jia et al., 2007). www.intechopen.com Tandem Mass Spectrometry of Peptides 37 2.2 The fragmentation process The MS fragmentation process occurs in the mass spectrometer mass analyzer or in a collision cell through the action of collision energy on gas phase ions generated in the mass spectrometer ion source. Several parameters influence this fragmentation process, including amino acid composition, size of the peptide, excitation method, time scale of the instrument used, ion charge state, etc (Paiz & Suhai, 2005). Presently, there are several fragmentation processes available in commercial mass spectrometers, namely collision induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), etc. The CID fragmentation method can be of two types: low-or high-energy, where the former uses up to 100 eV and the latter from hundreds eV up to several keV (Wells & McLuckey, 2005). The low-energy CID can be found in quadrupoles whereas the high-energy is used in, for example, tof-tof instruments. The nomenclature of the daughter ions generated by CID was first established by Roepstorff & Fohlmann (1984) and later reviewed by Biemann (1988), where the b-series ions extend from the N-terminal and the y-series ions extend from the C-terminal. (see Fig. 1). The calculation of the mass difference between consecutive daughter ions belonging to the same ion series (for example b or y ion-series), allows the determination of peptide's primary sequence.
Journal of Mass Spectrometry, 2007
field have been possible owing to novel MS instrumentation, experimental strategies, and bioinformatics tools. Today it is possible to identify and determine relative expression levels of thousands of proteins in a biological system by MS analysis of peptides produced by proteolytic digestion. In some situations, however, the precise characterization of a particular peptide species in a very complex peptide mixture is needed. While single-fragment ion-based scanning modes such as selected ion reaction monitoring (SIRM) or consecutive reaction monitoring (CRM) may be highly sensitive, they do not produce MS/MS information and their actual specificity must be determined in advance, a prerequisite that is not usually met in a basic research context. In such cases, the MS detector may be programmed to perform continuous MS/MS spectra on the peptide ion of interest in order to obtain structural information. This selected MS/MS ion monitoring (SMIM) mode has a number of advantages that are fully exploited by MS detectors that, like the linear ion trap, are characterized by high scanning speeds. In this work, we show some applications of this technique in the context of biological studies. These results were obtained by selecting an appropriate combination of scans according to the purpose of each one of these research scenarios.