Ion Trap CID Research Articles

Negative Electron Transfer Collision-Induced Dissociation of G-Quadruplexes: Uncovering the Guanine Radical Anion Loss Pathway.

G-quadruplex (G4) DNA can form highly stable secondary structures in the presence of metal cations, and research has shown its potential as a transcriptional regulator for oncogenes in the human genome. In order to explore the interactions of DNA with metal cations using mass spectrometry, employing complementary fragmentation methods can enhance structural information. This study explores the use of ion-ion reactions for sequential negative electron transfer collision-induced dissociation (nET-CID) as a complement to traditional ion-trap CID (IT-CID). The resulting nET-CID data for G4 anions with and without metal cations show an increase in fragment ion type diversity and yield of structurally informative ions relative to IT-CID. The nET-CID yields greater sequence coverage by virtue of fragmentation at the 3'-side of thymine residues, which is lacking with IT-CID. Potassium adductions to backbone fragments in IT-CID and nET-CID spectra were nearly identical. Of note is a prominent fragment resulting from a loss of a 149 Da anion seen in nET-CID of large, G-rich sequences, proposed to be radical anion guanine loss. Neutral loss of neutral guanine (151 Da) and deprotonated nucleobase loss (150 Da) have been previously reported, but this is the first report of radical anion guanine loss (149 Da). Confirmation of the identity of the 149 Da anion results from the examination of the homonucleobase sequence 5'-GGGGGGGG-3'. Loss of a charged adenine radical anion at much lower relative abundance was also noted for the sequence 5'-AAAAAAAA-3'. DFT modeling indicates that the loss of a nucleobase as a radical anion from odd-electron nucleic acid anions is a thermodynamically favorable fragmentation pathway for G.

Enhancing structural characterisation of glucuronidated O-linked glycans using negative mode ion trap higher energy collision-induced dissociation mass spectrometry.

High protein production and secretion with eukaryotic glycosylation machinery make T. reesei RUT-C30 a suitable expression host for recombinant proteins. The N-glycosylation of secreted proteins of RUT-C30 is known to vary depending on culture nutrients but O-glycosylation has been less extensively studied. O-Glycans and glycopeptides from secreted proteins were separated by porous graphitised carbon and C-18 liquid chromatography, respectively. O-Glycans were analysed in negative ion mode by electrospray ionisation linear ion trap mass spectrometry and glycopeptides in positive ion mode by electrospray ionisation hybrid quadrupole-orbitrap mass spectrometry. Tandem mass spectrometry was used on O-glycans and glycopeptides including ion trap higher energy collision-induced dissociation (tHCD) to detect glycan fragments not detectable with standard ion trap fragmentation. tHCD allowed targeted MS3 experiments to be performed on structures containing hexuronic acid, which was not possible with ion trap CID, validating this novel O-glycan composition. Positive mode C18-LC/ESI-MS/MS was used to identify and characterise glycopeptides found to be modified with this class of O-glycans, identifying cellobiohydrolase I as a carrier of these novel O-glycans. Negative mode ion trap higher energy collision-induced dissociation allowed detection and targeted MS3 experiments to be performed on the hexuronic acid substituent of O-glycan structures, which was not possible with ion trap CID, validating the novel O-glycan composition to include hexuronic acid. Using glycopeptide analysis, this novel O-glycan composition was found to be present on the catalytic domain of cellobiohydrolase I, the most abundant secreted protein by T. reesei. These are the first reported O-glycans to contain acidic sugars in fungi and they could have significant implications for cellobiohydrolase I structure and activity as well as the activity of recombinant proteins expressed in this host system. Copyright © 2017 John Wiley & Sons, Ltd.

On the inter‐instrument and the inter‐laboratory transferability of a tandem mass spectral reference library. 3. Focus on ion trap and upfront CID

Mass spectral libraries represent versatile tools for the identification of small bioorganic molecules. Libraries based on electron impact spectra are rated robust and transferable. Tandem mass spectral libraries are often considered to work properly only on the instrument that has been used to build the library. An exception from that rule is the 'Wiley Registry of Tandem Mass Spectral Data, MSforID'. In various studies with data sets from different kinds of tandem mass spectrometric instruments, the outstanding sensitivity and robustness of this tandem mass spectral library search approach was demonstrated. The instrumental platforms tested, however, mainly included various tandem-in-space instruments. Herein, the results of a multicenter study with a focus on upfront and tandem-in-time fragmentation are presented. Five laboratories participated and provided fragment ion mass spectra from the following types of mass spectrometers: time-of-flight (TOF), quadrupole-hexapole-TOF, linear ion trap (LIT), 3-D ion trap and LIT-Orbitrap. A total number of 1231 fragment ion mass spectra were collected from 20 test compounds (amiloride, buphenin, cinchocaine, cyclizine, desipramine, dihydroergotamine, dyxirazine, dosulepin, ergotamine, ethambutol, etofylline, mefruside, metoclopramide, phenazone, phentermine, phenytoin, sulfamethoxazole, sulfamoxole, sulthiame and tetracycline) on seven electrospray ionization instruments using 18 different instrumental configurations for fragmentation. For 1222 spectra (99.3%), the correct compound was retrieved as the best matching compound. Classified matches (matches with 'relative average match probability' >40.0) were obtained for 1207 spectra (98.1%). This high percentage of correct identifications clearly supports the hypothesis that the tandem mass spectral library approach tested is a robust and universal identification tool.

Using Electron Induced Dissociation (EID) on an LC Time-Scale to Characterize a Mixture of Analogous Small Organic Molecules

LC ESI FTICR MS of a sample of cediranib identified this pharmaceutical target molecule plus an additional 10 compounds of interest, all of which were less than 10% total ion current (TIC) peak intensity relative to cediranib. LC FTICR tandem mass spectrometry using electron induced dissociation (EID) has been achieved and has proven to be the best way to generate useful product ion information for all of these singly protonated molecules. Cediranib [M + H](+) fragmented by EID to give 29 product ions whereas QTOF-CID generated only one very intense product ion, and linear ion trap-CID, which generated 10 product ions, but all with poor S/N. Twenty-six of the EID product ions were unique to this fragmentation technique alone. By considering the complementary LC-EID and LC-CID data together, all 10 unknown compounds were structurally characterized and proven to be analogous to cediranib. Of particular importance, EID produced unique product ion information for one of the low level cediranib analogues that enabled full characterization of the molecule such that the presence of an extra propylpyrrolidine group was discovered and proven to be located on the pyrrolidine ring of cediranib, solving an analytical problem that could not be solved by collision induced dissociation (CID). Thus, it has been demonstrated that EID is in harmony with the chromatography duty-cycle and the dynamic concentration range of synthetic compounds containing trace impurities, providing crucial analytical information that cannot be obtained by more traditional methodologies.

18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.

Implementation of dipolar direct current (DDC) collision‐induced dissociation in storage and transmission modes on a quadrupole/time‐of‐flight tandem mass spectrometer

Means for effecting dipolar direct current collision-induced dissociation (DDC CID) on a quadrupole/time-of-flight in a mass spectrometer have been implemented for the broadband dissociation of a wide range of analyte ions. The DDC fragmentation method in electrodynamic storage and transmission devices provides a means for inducing fragmentation of ions over a large mass-to-charge range simultaneously. It can be effected within an ion storage step in a quadrupole collision cell that is operated as a linear ion trap or as ions are continuously transmitted through the collision cell. A DDC potential is applied across one pair of rods in the quadrupole collision cell of a QqTOF hybrid mass spectrometer to effect fragmentation. In this study, ions derived from a small drug molecule, a model peptide, a small protein, and an oligonucleotide were subjected to the DDC CID method in either an ion trapping or an ion transmission mode (or both). Several key experimental parameters that affect DDC CID results, such as time, voltage, low mass cutoff, and bath gas pressure, are illustrated with protonated leucine enkephalin. The DDC CID dissociation method gives a readily tunable, broadband tool for probing the primary structures of a wide range of analyte ions. The method provides an alternative to the narrow resonance conditions of conventional ion trap CID and it can access more extensive sequential fragmentation, depending upon conditions. The DDC CID approach constitutes a collision analog to infrared multiphoton dissociation (IRMPD).

Improving SRM Assay Development: A Global Comparison between Triple Quadrupole, Ion Trap, and Higher Energy CID Peptide Fragmentation Spectra

In proteomics, selected reaction monitoring (SRM) is rapidly gaining importance for targeted protein quantification. The triple quadrupole mass analyzers used in SRM assays allow for levels of specificity and sensitivity hard to accomplish by more standard shotgun proteomics experiments. Often, an SRM assay is built by in silico prediction of transitions and/or extraction of peptide precursor and fragment ions from a spectral library. Spectral libraries are typically generated from nonideal ion trap based shotgun proteomics experiments or synthetic peptide libraries, consuming considerable time and effort. Here, we investigate the usability of beam type CID (or "higher energy CID" (HCD)) peptide fragmentation spectra, as acquired using an Orbitrap Velos, to facilitate SRM assay development. Therefore, peptide fragmentation spectra, obtained by ion-trap CID, triple-quadrupole CID (QqQ-CID) and Orbitrap HCD, originating from digested cellular lysates, were compared. Spectral comparison and a dedicated correlation algorithm indicated significantly higher similarity between QqQ-CID and HCD fragmentation spectra than between QqQ-CID and ion trap-CID spectra. SRM transitions generated using a constructed HCD spectral library increased SRM assay sensitivity up to 2-fold, when compared to the use of a library created from more conventionally used ion trap-CID spectra, showing that HCD spectra can assist SRM assay development.

Top-Down Tandem Mass Spectrometry of tRNA Via Ion Trap Collision-Induced Dissociation

Transfer RNA is a class of highly modified and structured non-coding RNA molecules generally comprised of 74-95 nucleotides. In this study, tandem mass spectrometry of intact multiply charged tRNA anions of roughly 25 kDa in mass has been demonstrated using a quadrupole/time-of-flight tandem mass spectrometer adapted for ion/ion reaction studies. The sample proved to be a mixture of tRNA molecules. The mass of the most abundant component of the mixture was not consistent with that of the nominal identity of the tRNA from the supplier, viz., tRNA(Phe); rather, the mass was consistent with tRNA(Phe) bearing an incomplete 3'-terminus. Multiply-charged anions from the major components were isolated in the gas phase and subjected to ion trap collision-induced dissociation without subsequent ion/ion reactions. Abundant fragments from the 5'- and 3'-termini of the molecule could be used to identify the major component as tRNA(Phe)-3'adenosine (without 3'-phosphorylation). Roughly 15% of the primary sequence of the intact tRNA was unambiguously reflected in the product ion spectrum. The existence of a possible tRNA(Phe) variant and the intact tRNA(Phe) was also supported by ion trap CID data. The multiply-charged fragment ions derived from tRNA(Phe)-3'adenosine were further charge-reduced to mostly singly- and doubly-charged species via proton transfer ion/ion reactions with benzoquinoline cations. The resulting reduction in spectral overlap and charge state ambiguity simplified interpretation of the product ion spectrum and allowed for the identification of product ions from roughly 60% of the sequence.

Evaluation of the utility of neutral‐loss‐dependent MS3 strategies in large‐scale phosphorylation analysis

Phosphopeptide identification and site determination are major challenges in biomedical MS. Both are affected by frequent and often overwhelming losses of phosphoric acid in ion trap CID fragmentation spectra. These losses are thought to translate into reduced intensities of sequence informative ions and a general decline in the quality of MS/MS spectra. To address this issue, several methods have been proposed, which rely on extended fragmentation schemes including collecting MS3 scans from neutral loss-containing ions and multi-stage activation to further fragment these same ions. Here, we have evaluated the utility of these methods in the context of a large-scale phosphopeptide analysis strategy with current instrumentation capable of accurate precursor mass determination. Remarkably, we found that MS3-based schemes did not increase the overall number of confidently identified peptides and had only limited value in site localization. We conclude that the collection of MS3 or pseudo-MS3 scans in large-scale proteomics studies is not worthwhile when high-mass accuracy instrumentation is used.

Simplifying the Hunt for Optimal SRM Transitions: Utilizing Discovery Data to Expedite Targeted Peptide Quantitation

Greater emphasis has been placed on advancing proteomics studies from discovery and/or relative quantitation to validated quantitative methods in an effort to establish clinical assays. The typical workflow involves first performing discovery based experiments to identify protein expression levels that are confidently changing between a control and treated samples and generate product ion information used to sequence the precursor peptide. The difficulty arises in transferring discovery based methods directly over to validated quantitation methods since each is generally performed on separate mass spectral platforms. Low confidence has been placed on relating relative product ion abundance obtained from ion trap CID to that observed using a triple quadrupole mass spectrometer due to the difference in ion activation mechanisms and the timescale of the excitation. Thus, the only information transferred from one method to the other is protein id, peptide sequence, and the most abundant charge state resulting in further method development to complete the SRM assay. Common approaches to determine SRM transitions are based on a set of accepted rules to determine the best possible ion pair(s), which are then searched against the matrix database to determine the uniqueness of each mass pair. We contend that the relative abundance of product ions originating from ion trap CID can be a used to directly assign the most sensitive ion pairs for the targeted SRM methods.

Colander: A Probability-Based Support Vector Machine Algorithm for Automatic Screening for CID Spectra of Phosphopeptides Prior to Database Search

We developed a probability-based machine-learning program, Colander, to identify tandem mass spectra that are highly likely to represent phosphopeptides prior to database search. We identified statistically significant diagnostic features of phosphopeptide tandem mass spectra based on ion trap CID MS/MS experiments. Statistics for the features are calculated from 376 validated phosphopeptide spectra and 376 nonphosphopeptide spectra. A probability-based support vector machine (SVM) program, Colander, was then trained on five selected features. Data sets were assembled both from LC/LC-MS/MS analyses of large-scale phosphopeptide enrichments from proteolyzed cells, tissues and synthetic phosphopeptides. These data sets were used to evaluate the capability of Colander to select pS/pT-containing phosphopeptide tandem mass spectra. When applied to unknown tandem mass spectra, Colander can routinely remove 80% of tandem mass spectra while retaining 95% of phosphopeptide tandem mass spectra. The program significantly reduced computational time spent on database search by 60-90%. Furthermore, prefiltering tandem mass spectra representing phosphopeptides can increase the number of phosphopeptide identifications under a predefined false positive rate.

A mass spectrometric and molecular orbital study of H2O loss from protonated tryptophan and oxidized tryptophan derivatives.

Protonated N-acetyltryptophan, oxindolylalanine (a mono-oxidized derivative of tryptophan), and N-acetyloxindolylalanine, as well as several di- and tripeptide derivatives containing oxindolylalanine, undergo a range of fragmentation reactions in the gas phase, including the loss of water. In order to elucidate the sites of water loss within these ions, and to determine the mechanisms associated with these processes, we have conducted a series of experiments employing multistage tandem mass spectrometry (MS/MS and MS(3)) in a quadrupole ion trap mass spectrometer, regiospecific structural labeling, and independent solution-phase syntheses of proposed product ion structures, coupled with the use of molecular orbital calculations at the B3LYP/6-31G* level of theory. We demonstrate that the loss of H(2)O from the amide carbonyl group of protonated N-acetyltryptophan O-methyl ester occurs via a "side-chain-backbone" neighboring group reaction to yield a protonated carboline derivative. In contrast, the loss of water from the O-methyl ester of protonated oxindolylalanine results in the formation of a tricyclic structure by "backbone-side-chain" nucleophilic attack from the amino nitrogen to the C2 position of the indole ring. The O-methyl ester of protonated N-acetyloxindolylalanine was found to dissociate via the loss of water from both possible sites, i.e. from the side-chain indolyl oxygen and the backbone amide carbonyl group. An estimate of the relative preference for water loss from each site was obtained from the abundances of product ions formed from MS(3) analysis of regiospecifically labeled derivatives of N-acetyloxindolylalanine, and from the results of molecular orbital calculations. These studies indicate the absence of a characteristic 'signature' ion or neutral loss for peptides containing oxindolylalanine residues under low-energy ion trap CID conditions.

Efficient polyatomic interference reduction in plasma-source mass spectrometry via collision induced dissociation

Evidence is provided that illustrates quadrupole ion traps can be used to selectively attenuate strongly bound diatomic ions occurring at the same nominal mass as an analyte ion of interest. Dissociation rates for TaO+ (D0 ∼ 750 kJ mol−1) are found to be at least an order of magnitude larger than the loss rate of Au+ due to scattering under “slow heating” resonance excitation conditions at qz = 0.67 and using neon as the bath gas. This rate difference is sufficient for the selective removal of this strongly-bound diatomic ion over the loss of the Au+ at the same mass-to-charge ratio. Other examples of quadrupole ion trap CID for the selective reduction of common plasma-generated species are also evaluated by examining the dissociation of GdO+ in the presence of Yb+, and Cu2+ in the presence of Te+. In each case, a different method of applying the excitation signals is presented, and the attenuation rates for the diatomic species due to CID are substantially larger than scattering losses for the bare metal ions. Evidence is also presented that demonstrates CID can be accomplished in concert with a slow mass analysis scan, thereby providing a means of (1) eliminating polyatomic ions (formed in the plasma or reaction cell) over an extended mass range, (2) recovering metal ion signal from the metal-containing polyatomic ions, and (3) minimizing deleterious secondary reactions of product ions.

The influence of histidine on cleavage C-terminal to acidic residues in doubly protonated tryptic peptides

An ion-trap CID MS/MS spectral database of 505 doubly protonated tryptic peptides was used to investigate the influence of an internal basic residue on preferential fragmentation C-terminal to the acidic amino acid residues, aspartic acid (Asp) and glutamic acid (Glu). Because tryptic peptides, which contain C-terminal Lys or Arg, were selected for analysis, the majority of the peptides contain His as the internal basic residue. A comparison between spectra for peptides that do and do not contain an internal basic residue shows that cleavage is more prominent at Asp-Xxx bonds for peptides that do contain the internal basic residue. This result corroborates a previously published mechanism for Asp-Xxx cleavage that states that cleavage at Asp-Xxx is enhanced when protons are sequestered at basic sites, allowing the acidic hydrogen of the Asp side chain to initiate cleavage. The data suggest that in doubly-charged His-containing tryptic peptides, one proton is typically located at the C-terminal Arg or Lys while the mobility of the second proton is hindered by the His side chain’s relatively high basicity. The same investigation was performed for cleavage at Glu-Xxx amide bonds, but in this case there is only a marginal difference between peptides that do and do not contain the internal basic residue.

Nanospray-ESI low-energy CID and MALDI post-source decay for determination of O-glycosylation sites in MUC4 peptides

Mucins are glycoproteins containing from one to several hundreds of carbohydrate chains attached to the peptide by O-glycosidic linkages between N-acetylgalactosamine (GalNAc) and either serine (Ser) or threonine (Thr). Apomucin proteins are encoded by different mucin genes (MUC1–8) and show in general tandem repeats in which the carbohydrate chains can be highly clustered. The glycosylation density of human mucins seems to be related to some type of cancer. Fast atom-bombardment mass spectrometry (FAB-MS) has previously been used for the determination of O-glycosylation sites in combination with β-elimination. In this contribution, a comparative study by electrospray (ESI)-MS/MS and post-source decay (PSD)-MALDI-MS is presented. Nanospray-ESI-MS/MS experiments were performed using two analyzers, a triple quadrupole and a quadrupole ion trap. A series of O-GalNAc substituted synthetic MUC4 fragments with blocked N- and C-termini, and their nonglycosylated counterparts were investigated. In the sequence analysis of differently glycosylated and nonglycosylated MUC4 decapeptides by MALDI-PSD-MS, the spectra were dominated by b- and y-type ions. Substantial and structure-unrelated loss of the sugar from molecular [M+H]+ and fragment ions was generally observed. The single GalNAc attachment site out of three to five potential substitution Ser/Thr sites was identified by attribution of N- and C-terminal fragment ions still carrying the GalNAc moiety. Similar fragmentation patterns were produced by nanospray ESI–low-energy CID on a Q3 instrument using Ar as a collision gas or on ion trap CID using He as a collision gas. Less abundant sequence ions were obtained from CID experiments on sodiated molecular [M+Na]+ ions, which required much higher energy. © 1998 John Wiley & Sons, Ltd.