Towards liquid chromatography time-scale peptide sequencing and characterization of post-translational modifications in the negative-ion mode using electron detachment dissociation tandem mass spectrometry

Electron detachment dissociation (EDD) of peptide poly-anions is gentle towards post-translational 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 more comprehensive strategy of analysis in proteome research.     

Electron capture/transfer versus collisionally activated/induced dissociations: Solo or duet?

New ion fragmentation technologies--electron capture dissociation (ECD) and electron-transfer dissociation (ETD)--are based on interaction of multiply charged polypeptides with either free electrons (ECD) or anionic species (ETD). After initial difficulties, these ECD/ETD (ExD) technologies are now being increasingly implemented in high-throughput proteomics work. This critical analysis presents arguments for the combined use of ExD with the conventional low-energy collisional excitation CID/CAD (CxD). It is argued that the database search, a key technology in MS/MS-based proteomics, is vulnerable with respect to the incomplete sequence information obtainable with either of the techniques, peptide MS/MS homology being a major complicating factor. De novo sequencing is viewed as the only adequate answer to this challenge and it can be achieved only with combined use of ExD and CxD. The payoff in the form of additional sequence information is projected to exceed the costs of such implementation. The greatest impact of combining ExD and CxD is expected in high-resolution instruments.     

Toward a general mechanism of electron capture dissociation

The effects of positive charge on the properties of ammonium and amide radicals were investigated by ab initio and density functional theory calculations with the goal of elucidating the energetics of electron capture dissociation (ECD) of multiply charged peptide ions. The electronic properties of the amide group in N-methylacetamide (NMA) are greatly affected by the presence of a remote charge in the form of a point charge, methylammonium, or guanidinium cations. The common effect of the remote charge is an increase of the electron affinity of the amide group, resulting in exothermic electron capture. The N-Calpha bond dissociation and transition state energies in charge-stabilized NMA anions are 20-50 kJ mol(-1) greater than in the hydrogen atom adduct. The zwitterions formed by electron capture have proton affinities that were calculated as 1030-1350 kJ mol(-1), and are sufficiently basic for the amide carbonyl to exothermically abstract a proton from the ammonium, guanidinium and imidazolium groups in protonated lysine, arginine, and histidine residues, respectively. A new mechanism is proposed for ECD of multiply charged peptide and protein cations in which the electron enters a charge-stabilized electronic state delocalized over the amide group, which is a superbase that abstracts a proton from a sterically proximate amino acid residue to form a labile aminoketyl radical that dissociates by N-Calpha bond cleavage. This mechanism explains the low selectivity of N-Calpha bond dissociations induced by electron capture, and is applicable to dissociations of peptide ions in which the charge carriers are metal ions or quaternary ammonium groups. The new amide superbase and the previously proposed mechanisms of ECD can be uniformly viewed as being triggered by intramolecular proton transfer in charge-reduced amide cation-radicals. In contrast, remote charge affects N-H bond dissociation in weakly bound ground electronic states of hypervalent ammonium radicals, as represented by methylammonium, CH3NH3*, but has a negligible effect on the N-H bond dissociation in the strongly bound excited electronic states. This refutes previous speculations that loss of "hot hydrogen" can occur from an excited state of an ammonium radical.     

Characterization of Tyrosine Nitration and Cysteine Nitrosylation Modifications by Metastable Atom-Activation Dissociation Mass Spectrometry

The fragmentation behavior of nitrated and S-nitrosylated peptides were studied using collision induced dissociation (CID) and metastable atom-activated dissociation mass spectrometry (MAD-MS). Various charge states, such as 1+, 2+, 3+, 2–, of modified and unmodified peptides were exposed to a beam of high kinetic energy helium (He) metastable atoms resulting in extensive backbone fragmentation with significant retention of the post-translation modifications (PTMs). Whereas the high electron affinity of the nitrotyrosine moiety quenches radical chemistry and fragmentation in electron capture dissociation (ECD) and electron transfer dissociation (ETD), MAD does produce numerous backbone cleavages in the vicinity of the modification. Fragment ions of nitrosylated cysteine modifications typically exhibit more abundant neutral losses than nitrated tyrosine modifications because of the extremely labile nature of the nitrosylated cysteine residues. However, compared with CID, MAD produced between 66% and 86% more fragment ions, which preserved the labile –NO modification. MAD was also able to differentiate I/L residues in the modified peptides. MAD is able to induce radical ion chemistry even in the presence of strong radical traps and therefore offers unique advantages to ECD, ETD, and CID for determination of PTMs such as nitrated and S-nitrosylated peptides.     

Does Electron Capture Dissociation Cleave Protein Disulfide Bonds?

Peptide and protein characterization by mass spectrometry (MS) relies on their dissociation in the gas phase into specific fragments whose mass values can be aligned as ‘mass ladders’ to provide sequence information and to localize possible posttranslational modifications. The most common dissociation method involves slow heating of even-electron (M+n H)ⁿ⁺ ions from electrospray ionization by energetic collisions with inert gas, and cleavage of amide backbone bonds. More recently, dissociation methods based on electron capture or transfer were found to provide far more extensive sequence coverage through unselective cleavage of backbone N–Cα bonds. As another important feature of electron capture dissociation (ECD) and electron transfer dissociation (ETD), their unique unimolecular radical ion chemistry generally preserves labile posttranslational modifications such as glycosylation and phosphorylation. Moreover, it was postulated that disulfide bond cleavage is preferred over backbone cleavage, and that capture of a single electron can break both a backbone and a disulfide bond, or even two disulfide bonds between two peptide chains. However, the proposal of preferential disulfide bond cleavage in ECD or ETD has recently been debated. The experimental data presented here reveal that the mechanism of protein disulfide bond cleavage is much more intricate than previously anticipated.     

An online nano‐LC‐ESI‐FTICR‐MS method for comprehensive characterization of endogenous fragments from amyloid β and amyloid precursor protein in human and cat cerebrospinal fluid

Amyloid precursor protein (APP) is the precursor protein to amyloid β (Aβ), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aβ peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aβ peptides by low‐energy collision‐induced dissociation tandem mass spectrometry (MS/MS), mainly long b‐fragments are observed, limiting the possibility to determine variations such as amino acid variants or post‐translational modifications (PTMs) within the N‐terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aβ peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aβ processing has also been described in the lysosomal storage disease Niemann–Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aβ was identified. Further, sialylated core 1 like O‐glycans at Tyr10, recently discovered in human Aβ (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aβ peptide species in CSF by using an online top‐down MS‐based method. Copyright © 2012 John Wiley & Sons, Ltd.     

The use of ECD/ETD to identify the site of electrostatic interaction in noncovalent complexes

Electrostatic interactions play an important role in the formation of noncovalent complexes. Our previous work has highlighted the role of certain amino acid residues, such as arginine, glutamate, aspartate, and phosphorylated/sulfated residues, in the formation of salt bridges resulting in noncovalent complexes between peptides. Tandem mass spectrometry (MS) studies of these complexes using collision-induced dissociation (CID) have provided information on their relative stability. However, product-ion spectra produced by CID have been unable to assign specifically the site of interaction for the complex. In this work, tandem MS experiments were conducted on noncovalent complexes using both electron capture dissociation (ECD) and electron-transfer dissociation (ETD). The resulting spectra were dominated by intramolecular fragments of the complex with the electrostatic interaction site intact. Based upon these data, we were able to assign the binding site for the peptides forming the noncovalent complex.     

Charge remote fragmentation in electron capture and electron transfer dissociation

Secondary fragmentations of three synthetic peptides (human αA crystallin peptide 1-11, the deamidated form of human βB2 crystallin peptide 4-14, and amyloid β peptide 25-35) were studied in both electron capture dissociation (ECD) and electron-transfer dissociation (ETD) mode. In ECD, in addition to c and z· ion formations, charge remote fragmentations (CRF) of z· ions were abundant, resulting in internal fragment formation or partial/entire side-chain losses from amino acids, sometimes several residues away from the backbone cleavage site, and to some extent multiple side-chain losses. The internal fragments were observed in peptides with basic residues located in the middle of the sequences, which was different from most tryptic peptides with basic residues located at the C-terminus. These secondary cleavages were initiated by hydrogen abstraction at the α-, β-, or γ-position of the amino acid side chain. In comparison, ETD generates fewer CRF fragments than ECD. This secondary cleavage study will facilitate ECD/ETD spectra interpretation, and help de novo sequencing and database searching.     

Cation Recombination Energy/Coulomb Repulsion Effects in ETD/ECD as Revealed by Variation of Charge per Residue at Fixed Total Charge

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) experiments in electrodynamic ion traps operated in the presence of a bath gas in the 1–10 mTorr range have been conducted on a common set of doubly protonated model peptides of the form X(AG)_nX (X = lysine, arginine, or histidine, n?=?1, 2, or 4). The partitioning of reaction products was measured using thermal electrons, anions of azobenzene, and anions of 1,3-dinitrobenzene as reagents. Variation of n alters the charge per residue of the peptide cation, which affects recombination energy. The ECD experiments showed that H-atom loss is greatest for the n?=?1 peptides and decreases as n increases. Proton transfer in ETD, on the other hand, is expected to increase as charge per residue decreases (i.e., as n increases). These opposing tendencies were apparent in the data for the K(AG)_nK peptides. H-atom loss appeared to be more prevalent in ECD than in ETD and is rationalized on the basis of either internal energy differences, differences in angular momentum transfer associated with the electron capture versus electron transfer processes, or a combination of the two. The histidine peptides showed the greatest extent of charge reduction without dissociation, the arginine peptides showed the greatest extent of side-chain cleavages, and the lysine peptides generally showed the greatest extent of partitioning into the c/z?-product ion channels. The fragmentation patterns for the complementary c- and z?-ions for ETD and ECD were found to be remarkably similar, particularly for the peptides with X = lysine.

Structural analysis of O-glycopeptides employing negative- and positive-ion multi-stage mass spectra obtained by collision-induced and electron-capture dissociations in linear ion trap time-of-flight mass spectrometry

Structural analyses of various glycans attached to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on mass spectrometry (MS) combining both collision-induced dissociation (CID) and electron-capture dissociation (ECD) in the positive- and negative-ion modes has been proposed as a simple and direct method of assigning an O-glycan without releasing it from the peptide and of determining the amino acid sequence of the peptide and glycosylation site. The instrument used is an electrospray ionization (ESI) linear ion trap (LIT) time-of-flight (TOF) mass spectrometer with tandem LITs for CID by He gas and ECD. The proposed approach was tested with two synthetic O-glycopeptides binding a sialyl Lewis x (sLe(x)) oligosaccharide and a 3'-sialyl N-acetyllactosamine (3'-SLN) on a serine (S) residue. In the negative-ion mode, the CID MS(2) spectra of O-glycopeptides showed a relatively abundant glycoside-bond cleavage between the core N-acetylglucosamine (GlcNAc) and serine (S) that yields deprotonated C(3)-type fragment ions of O-glycan and deprotonated Z(0)-type peptide ions. The structure of the sLe(x) (3'-SLN) oligosaccharide was simply assigned by comparing the CID MS(3) spectrum derived from the C(3)-type fragment ion with the CID MS(2) spectra of the sLe(x) and sLe(a) (3'- and 6'-SLN) standards (i.e., negative-ion MS(n) spectral matching). The amino acid sequence of the peptide including the glycosylation site was determined from the ECD MS(2) spectrum in the positive-ion mode.     

Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se–S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen (m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se–S cleavage, analogous to the S–S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se–S of the tag to the S–S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se–S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes.     

Electron transfer dissociation of amide nitrogen methylated polypeptide cations

Unmodified and amide nitrogen methylated peptide cations were reacted with azobenzene radical anions to study the utility of electron transfer dissociation (ETD) in analyzing N-methylated peptides. We show that methylation of the amide nitrogen has no deleterious effects on the ETD process. As a result, location of alkylation on amide nitrogens should be straightforward. Such a modification might be expected to affect the ETD process if hydrogen bonding involving the amide hydrogen is important for the ETD mechanism. The partitioning of the ion/ion reaction products into all of the various reaction channels was determined and compared for modified and unmodified peptide cations. While subtle differences in the relative abundances of the various ETD channels were observed, there is no strong evidence that hydrogen bonding involving the amide nitrogen plays an important role in the ETD process.     

Gas-phase peptide fragmentation: how understanding the fundamentals provides a springboard to developing new chemistry and novel proteomic tools

This tutorial provides an overview of the evolution of some of the key concepts in the gas-phase fragmentation of different classes of peptide ions under various conditions [e.g. collision-induced dissociation (CID) and electron transfer dissociation (ETD)], and then demonstrates how these concepts can be used to develop new methods. For example, an understanding of the role of the mobile proton and neighboring group interactions in the fragmentation reactions of protonated peptides has led to the design of the 'SELECT' method. For ETD, a model based on the Landau-Zener theory reveals the role of both thermodynamic and geometric effects in the electron transfer from polyatomic reagent anions to multiply protonated peptides, and this predictive model has facilitated the design of a new strategy to form ETD reagent anions from precursors generated via ESI. Finally, two promising, emerging areas of gas-phase ion chemistry of peptides are also described: (1) the design of new gas-phase radical chemistry to probe peptide structure, and (2) selective cleavage of disulfide bonds of peptides in the gas phase via various physicochemical approaches.     

Electron attachment step in electron capture dissociation (ECD) and electron transfer dissociation (ETD)

We have made use of classical dynamics trajectory simultions and ab initio electronic structure calculations to estimate the cross sections with which electrons are attached (in electron capture dissociation (ECD)) or transferred (in electron transfer dissociation (ETD)) to a model system that contained both an S-S bond that is cleaved and a -NH(3)(+) positively charged site. We used a Landau-Zener-Stueckelberg curve-crossing approximation to estimate the ETD rates for electron transfer from a CH(3)(-) anion to the -NH(3)(+) Rydberg orbital or the S-S sigma* orbital. We draw conclusions about ECD from our ETD results and from known experimental electron-attachment cross sections for cations and sigma-bonds. We predict the cross section for ETD at the positive site of our model compound to be an order of magnitude larger than that for transfer to the Coulomb-stabilized S-S bond site. We also predict that, in ECD, the cross section for electron capture at the positive site will be up to 3 orders of magnitude larger than that for capture at the S-S bond site. These results seem to suggest that attachment to such positive sites should dominate in producing S-S bond cleavage in our compound. However, we also note that cleavage induced by capture at the positive site will be diminished by an amount that is related to the distance from the positive site to the S-S bond. This dimunition can render cleavage through Coulomb-assisted S-S sigma* attachment competitive for our model compound. Implications for ECD and ETD of peptides and proteins in which SS or N-C(alpha) bonds are cleaved are also discussed, and we explain that such events are most likely susceptible to Coulomb-assisted attachment, because the S-S sigma* and C=O pi* orbitals are the lowest-lying antibonding orbitals in most peptides and proteins.     

Sequence analysis of peptide:oligonucleotide heteroconjugates by electron capture dissociation and electron transfer dissociation

Mass spectrometry analysis of protein-nucleic acid cross-links is challenging due to the dramatically different chemical properties of the two components. Identifying specific sites of attachment between proteins and nucleic acids requires methods that enable sequencing of both the peptide and oligonucleotide component of the heteroconjugate cross-link. While collision-induced dissociation (CID) has previously been used for sequencing such heteroconjugates, CID generates fragmentation along the phosphodiester backbone of the oligonucleotide preferentially. The result is a reduction in peptide fragmentation within the heteroconjugate. In this work, we have examined the effectiveness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) for sequencing heteroconjugates. Both methods were found to yield preferential fragmentation of the peptide component of a peptide:oligonucleotide heteroconjugate, with minimal differences in sequence coverage between these two electron-induced dissociation methods. Sequence coverage was found to increase with increasing charge state of the heteroconjugate, but decreases with increasing size of the oligonucleotide component. To overcome potential intermolecular interactions between the two components of the heteroconjugate, supplemental activation with ETD was explored. The addition of a supplemental activation step was found to increase peptide sequence coverage over ETD alone, suggesting that electrostatic interactions between the peptide and oligonucleotide components are one limiting factor in sequence coverage by these two approaches. These results show that ECD/ETD methods can be used for the tandem mass spectrometry sequencing of peptide:oligonucleotide heteroconjugates, and these methods are complementary to existing CID methods already used for sequencing of protein-nucleic acid cross-links.     

Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and radical z-type ions in ECD FT-ICR MS and especially activated ion-ECD, thus providing a new insight into the fundamentals of ECD/ETD.     

Comparison of data analysis parameters and MS/MS fragmentation techniques for quantitative proteome analysis using isobaric peptide termini labeling (IPTL)

Isobaric peptide termini labeling (IPTL) is a quantification method which permits relative quantification using quantification points distributed throughout the whole tandem mass spectrometry (MS/MS) spectrum. It is based on the complementary derivatization of peptide termini with different isotopes resulting in isobaric peptides. Here, we use our recently developed software package IsobariQ to investigate how processing and data analysis parameters can improve IPTL data. Deisotoping provided cleaner MS/MS spectra and improved protein identification and quantification. Denoising should be used with caution because it may remove highly regulated ion pairs. An outlier detection algorithm on the ratios within every individual MS/MS spectrum was beneficial in removing false-positive quantification points. MS/MS spectra using IPTL typically contain two peptide series with complementary labels resulting in lower Mascot ion scores than non-labeled equivalent peptides. To avoid this penalty, the two chemical modifications for IPTL were specified as variables including satellite neutral losses of tetradeuterium with positive loss for the heavy isotopes and negative loss for the light isotopes. Thus, the less dominant complementary ion series were not considered for the scoring, which improved the ion scores significantly. In addition, we showed that IPTL was suitable for fragmentation by electron transfer dissociation (ETD) and higher energy collisionally activated dissociation (HCD) besides the already reported collision-induced dissociation (CID). Notably, ETD and HCD data can be identified and quantified using IsobariQ. ETD outperformed CID and HCD only for charge states ≥4+ but yielded in total fewer protein identifications and quantifications. In contrast, the high-resolution information of HCD fragmented peptides provided most identification and quantification results using the same scan speed.     

Radical Stability Directs Electron Capture and Transfer Dissociation of β‐Amino Acids in Peptides

We report on the characteristics of the radical‐ion‐driven dissociation of a diverse array of β‐amino acids incorporated into α‐peptides, as probed by tandem electron‐capture and electron‐transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β‐amino acids than for their α‐form counterparts. In particular, only aromatic (e.g., β‐Phe), and to a substantially lower extent, carbonyl‐containing (e.g., β‐Glu and β‐Gln) amino acid side chains, lead to N? C_β bond cleavage in the corresponding β‐amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical‐driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α‐amino acids, ECD of peptides composed mainly of β‐amino acids reveals a shift in cleavage priority from the N? C_β to the C_α? C bond. The incorporation of CH₂ groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical‐driven β‐amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.     

Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry

When localizing protein post-translational modifications (PTMs) using liquid-chromatography (LC)-tandem mass spectrometry (MS/MS), existing implementations are limited by inefficient selection of PTM-carrying peptides for MS/MS, particularly when PTM site occupancy is sub-stoichiometric. The present contribution describes a method by which peptides carrying specific PTMs of interest-in this study, methylarginines-may be selectively targeted for MS/MS: peptide features are extracted from high mass accuracy single-stage MS data, searched against theoretical PTM-carrying peptide masses, and matching features are subjected to targeted data acquisition LC-MS/MS. Using trypsin digested Saccharomyces cerevisiae Npl3, in which evidence is presented for 18 methylarginine sites-17 of which fall within a glycine-arginine-rich (GAR) domain spanning <120 amino acids-it is shown that this approach outperforms conventional data dependent acquisition (DDA): when applied to a complex protein mixture featuring in vivo methylated Npl3, 95% more (P=0.030) methylarginine-carrying peptides are selected for MS/MS than DDA, leading to an 86% increase (P=0.044) in the number of methylated peptides producing Mascot ion scores ≥20 following electron-transfer dissociation (ETD). Notably, significantly more low abundance arginine methylated peptides (maximum ion intensities <6×10(4) cps) are selected for MS/MS using this approach relative to DDA (50% more in a digest of purified in vitro methylated Npl3). It is also demonstrated that relative to collision-induced dissociation (CID), ETD facilitates a 586% increase (P=0.016) in average Mascot ion scores of methylarginine-carrying peptides. The present PTM-specific targeted data acquisition approach, though described using methylarginine, is applicable to any ionizable PTM of known mass.     

Effects of Electron-Transfer Coupled with Collision-Induced Dissociation (ET/CID) on Doubly Charged Peptides and Phosphopeptides

Electron-transfer dissociation (ETD) is a useful peptide fragmentation technique that can be applied to investigate post-translational modifications (PTMs), the sequencing of highly hydrophilic peptides, and the identification of large peptides and even intact proteins. In contrast to traditional fragmentation methods, such as collision-induced dissociation (CID), ETD produces c- and z^·-type product ions by randomly cleaving the N–Cα bonds. The disappointing fragmentation efficiency of ETD for doubly charged peptides and phosphopeptide ions has been improved by ETcaD (supplemental activation). However, the ETD data derived from most database search algorithms yield low confidence scores due to the presence of unreacted precursors and charge-reduced ions within MS/MS spectra. In this work, we demonstrate that eight out of ten standard doubly charged peptides and phosphopeptides can be effortlessly identified by electron-transfer coupled with collision-induced dissociation (ET/CID) using the SEQUEST algorithm without further spectral processing. ET/CID was performed with the further dissociation of the charge-reduced ions isolated from ETD ion/ion reactions. ET/CID had high fragmentation efficiency, which elevated the confidence scores of doubly charged peptide and phosphospeptide sequencing. ET/CID was found to be an effective fragmentation strategy in “bottom-up” proteomic analysis.