Ion Trap Collision-induced Dissociation Research Articles

Pentafluorobenzylpyridinium: new thermometer ion for characterizing the ions produced by collisional activation during tandem mass spectrometry.

In this study, pentafluorobenzylpyridinium (F5-BnPy+), which has the highest dissociation energy among the reported benzylpyridinium thermometer ion, is proposed to characterize the internal energy distributions of ions activated by higher energy collisional dissociation (HCD) and ion-trap collision-induced dissociation (CID) during tandem mass spectrometry. The dissociation threshold energies of F5-BnPy+ was determined using quantum chemistry calculations at the CCSD(T)/6-311++G(d,p)//M06-2X-D3/6-311++G(d,p) level of theory, and the appearance energies for ion dissociation in HCD and ion-trap CID were estimated using Rice-Ramsperger-Kassel-Marcus theory. The main differences between HCD and ion-trap CID are the collision energies used and the timescales of collisional activation. For both HCD and ion-trap CID, the average internal energy of the ions increased with increasing collision energy. In contrast, the average value for the internal energy of the ions activated by ion-trap CID was lower than that of ions activated by HCD, probably because of the smaller collisional energy and longer activation time of the ion-trap CID experiments. The reported method will aid in the determination of the optimum tandem mass spectrometry parameters for the analysis of small molecules such as metabolites.

RDD-HCD Provides Variable Fragmentation Routes Dictated by Radical Stability.

Radical-directed dissociation (RDD) is a fragmentation technique in which a radical created by selective 213/266 nm photodissociation of a carbon-iodine bond is reisolated and collisionally activated. In previous RDD experiments, collisional activation was effected by ion-trap collision-induced dissociation (CID). Higher-energy collisional dissociation (HCD) differs from CID both in terms of how ions are excited and in the number, type, or abundance of fragments that are observed. In this paper, we explore the use of HCD for activation in RDD experiments. While RDD-CID favors fragments produced from radical-directed pathways such as a/z-ions and side chain losses regardless of the activation energy employed, RDD-HCD spectra vary considerably as a function of activation energy, with lower energies favoring RDD while higher energies favor products resulting from cleavage directed by mobile protons (b/y-ions). RDD-HCD therefore affords more tunable fragmentation based on the HCD energy provided. Importantly, the abundance of radical products decreases as a function of increasing HCD energy, confirming that RDD generally proceeds via lower-energy barriers relative to mobile-proton-driven dissociation. The dominance of b/y-ions at higher energies for RDD-HCD can therefore be explained by the higher survivability of fragments not containing the radical after the initial or subsequent dissociation events. Furthermore, these results confirm previous suspicions that HCD spectra differ from CID spectra due to multiple dissociation events.

A Frequency-Based Approach to Predict the Low-Energy Collision-Induced Dissociation Fragmentation Spectra.

Peptide identification algorithms rely on the comparison between the experimental tandem mass spectrometry spectrum and the theoretical spectrum to identify a peptide from the tandem mass spectra. Hence, it is important to understand the fragmentation process and predict the tandem mass spectra for high-throughput proteomics research. In this study, a novel method was developed to predict the theoretical ion trap collision-induced dissociation (CID) tandem mass spectra of the singly, doubly, and triply charged tryptic peptides. The fragmentation statistics of the ion trap CID spectra were used to predict the theoretical tandem mass spectra of the peptide sequence. The study estimated the relative cleavage frequency for each pair of adjacent amino acids along the peptide length. The study showed that the cleavage frequency can be directly used to predict the tandem mass spectra. The predicted spectra show a high correlation with the experimental spectra used in this study; 99.73% of the high-quality reference spectra have correlation scores greater than 0.8. The new method predicts the theoretical spectrum and correlates significantly better with the experimental spectrum as compared to the existing spectrum prediction tools OpenMS_Simulator, MS2PIP, and MS2PBPI, where only 80, 85.76, and 85.80% of the spectral count, respectively, has a correlation score greater than 0.8.

Coupling Headgroup and Alkene Specific Solution Modifications with Gas-Phase Ion/Ion Reactions for Sensitive Glycerophospholipid Identification and Characterization.

Shotgun lipidomics provides sensitive and fast lipid identification without the need for chromatographic separation. Challenges faced by shotgun analysis of glycerophospholipids (GPs) include the lack of signal uniformity across GP classes and the inability to determine the carbon-carbon double bond (C═C) location within the fatty acyl chains of an unsaturated species. Two distinct derivatization strategies were employed to both enhance the ionization of GPs, via trimethylation enhancement using 13C-diazomethane (13C-TrEnDi), as well as determine location of double bonds within fatty acyl chains, employing an in-solution photochemical reaction with acetone (via the Paternò-Büchi reaction). The modified GPs were then subjected to positive ion mode ionization via electrospray ionization, producing uniform ionization efficiencies for different classes of GP species. The GPs were charge inverted via gas-phase ion/ion reactions and sequentially fragmented using ion trap collision-induced dissociation (CID). The CID of the species led to fragmentation producing diagnostic ions indicative of C═C bond location. The approach enabled enhanced ionization and the identification of phosphatidylcholine and phosphatidylethanolamine species at the C═C level in a bovine lipid extract.

Characterization of neutral loss peaks in the fragmentation pattern of peptide in Collision Induced Dissociation spectra

The fragmentation statistics were analyzed to study the fragmentation characteristics of quadrupole ion trap CID (Collision-Induced Dissociation) spectra extracted from the human peptide library in the NIST library. Instead of using intensity information, usually considered earlier, here the frequency of occurrence of the fragment ion peaks and its neutral loss events were analyzed with respect to the cleavage position along the peptide backbone and residue at the cleavage site. The analysis demonstrated the significance of the residues and the position of the cleavage site associated with neutral loss events. The analysis shows that the frequency of occurrence of neutral loss peaks has nearly similar proportion along the length of the peptide, but it increases towards the middle of the peptide. Cleavage sites H–H/R/K triggers the generation of the fragment, bn+18 peaks. Neutral loss fragment, y-17 is observed at higher frequencies than its corresponding y-ion peaks, when arginine is on the C- terminal side of the cleavage site. In the case of consecutive loss of neutral fragments, acidic amino acids-aspartic acid and glutamic acid cause decrease in the b-45 & y-45 peaks and increase in b-44, y-44 and b-46, y-46 peaks, while its amide-asparagine and glutamine show the inverse behavior for both b-type and y-type ion.

Characterization of collision-induced dissociation of deprotonated peptides of 4–16 amino acids using high-resolution mass spectrometry

Mass spectrometry (MS) analysis of peptides has traditionally been conducted in the positive ion mode on protonated peptides, although negative ion-mode analysis of deprotonated peptides can provide complementary and sometimes critical structural information. This is partly due to insufficient understanding of the fragmentation behaviors of deprotonated peptides. Here, using ion-trap collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD, a beam-type CID), we characterized the fragmentation patterns of 36 deprotonated peptides of 4–16 amino acids on a high-resolution, high-mass accuracy instrument. Our study finds that among the backbone cleavage products, y-, c- and z-type ions (using a nomenclature similar to that of positive ions) are the most dominant species in both CID and HCD spectra of deprotonated peptides, accompanied by abundant neutral loss (NL) peaks. Similar to the charge-dependency of collisional energy of protonated peptides, we find that singly charged deprotonated peptides require significantly higher collisional energy than their doubly or multiply charged counterparts to reach 50% fragmentation. HCD is generally better than CID for peptide sequencing in the negative ion mode, since HCD generates more backbone cleavage products whereas CID produces predominately NL peaks of precursors. For disulfide-bonded peptides and C-terminally amidated peptides, unusual fragmentation is observed in the negative ion mode. The fragmentation behaviors of deprotonated peptides reported in this study will promote further investigation of the fundamental mechanisms and facilitate algorithm development for peptide sequencing in the negative mode.

Generating Fatty Acid Profiles in the Gas Phase: Fatty Acid Identification and Relative Quantitation Using Ion/Ion Charge Inversion Chemistry.

Representing the most fundamental lipid class, fatty acids (FA) play vital biological roles serving as energy sources, cellular signaling molecules, and key architectural components of complex lipids. Direct infusion electrospray ionization spectrometry, also known as shotgun lipidomics, has emerged as a rapid and powerful toolbox for lipid analysis. While shotgun lipidomics can be a sensitive approach to FA detection, the diverse molecular structure of FA presents challenges for unambiguous identification and the relative quantification of isomeric contributors. In particular, pinpointing double bond position(s) in unsaturated FA and determining the relative contribution of double bond isomers has limited the application of the shotgun approach. Recently, we reported the use of gas-phase ion/ion reactions to facilitate the identification of FA. Briefly, singly deprotonated FA anions undergo charge inversion when reacted in the gas phase with tris-phenanthroline magnesium dications by forming [FA - H + MgPhen]+ complex ions. These charge-inverted FA complex cations fragment upon ion-trap collision-induced dissociation (CID) to generate product ion spectra unique to individual FA isomers. Herein, we report the development of a mass spectral library comprised of [FA - H + MgPhen]+ product ion spectra. The developed FA library permits confident FA identification, including polyunsaturated FA isomers. Furthermore, we demonstrate the ability to determine relative contributions of isomeric FA using multiple linear regression analysis paired with gas-phase ion/ion reactions. We successfully applied the presented method to generate a FA profile for bovine liver phospholipidome based entirely on gas-phase chemistries.

Cross-Ring Fragmentation Patterns in the Tandem Mass Spectra of Underivatized Sialylated Oligosaccharides and Their Special Suitability for Spectrum Library Searching

Reference spectral library searching, while widely used to identify compounds in other areas of mass spectrometry, is not commonly used in glycomics. Building on a study by Cotter and coworkers on analysis of sialylated oligosaccharides using atmospheric pressure-matrix-assisted laser-induced tandem mass spectrometry (MS/MS), we show that library search methods enable the automated differentiation of such sialylated oligosaccharide isomers using MS/MS derived from electrospray collision-induced dissociation in ion trap and beam-type fragmentation mass spectrometers. We compare MS/MS spectra of five sets of native sialylated oligosaccharide isomers and show a spectral library search method that can distinguish between these isomers using the precursor ion [M+2X-H]+, where X=Li, Na, or K. Sialic acid linkage (α2,3 vs. α2,6) is known to have a dramatic effect on the fragmentation of the sialylated compounds. We found that 2,4A3 cross-ring fragment at the terminal monosaccharide in sialyllactoses, sialyllactosamines, and sialyl pentasaccharides is highly abundant in the MS/MS spectra of [M+2X-H]+ species of α2,6-NeuAc glycans, while (2,4A3-H2O) fragment is highly abundant in α2,3-NeuAc moiety. The 2,4A3-H2O peak is specific to NeuAc-α2,3-Gal-β1,4-Y (Y=GlcNAc or Glc). To our knowledge, this observation was not reported previously. Theoretical calculations reveal major conformational differences between α2,6-NeuAc and α2,3-NeuAc structures that provide reasonable explanations for the observed fragmentation patterns. Other singly-charged ions ([M+X]+) do not show similar cross-ring cleavages. Implemented in a searchable library, these spectral differences provide a facile method to distinguish sialyl isomers without derivatization. We also found good spectral matching across instruments. MS/MS spectra and tools are available at http://chemdata.nist.gov/glycan/spectra.Graphical

Structural Characterization and Absolute Quantification of Microcystin Peptides Using Collision-Induced and Ultraviolet Photo-Dissociation Tandem Mass Spectrometry.

Microcystin (MC) peptides produced by cyanobacteria pose a hepatotoxic threat to human health upon ingestion from contaminated drinking water. While rapid MC identification and quantification in contaminated body fluids or tissue samples is important for patient treatment and outcomes, conventional immunoassay-based measurement strategies typically lack the specificity required for unambiguous determination of specific MC variants, whose toxicity can significantly vary depending on their structures. Furthermore, the unambiguous identification and accurate quantitation of MC variants using tandem mass spectrometry (MS/MS)-based methods can be limited due to a current lack of appropriate stable isotope-labeled internal standards. To address these limitations, we have systematically examined here the sequence and charge state dependence to the formation and absolute abundance of both "global" and "variant-specific" product ions from representative MC-LR, MC-YR, MC-RR, and MC-LA peptides, using higher-energy collisional dissociation (HCD)-MS/MS, ion-trap collision-induced dissociation (CID)-MS/MS and CID-MS3, and 193nm ultraviolet photodissociation (UPVD)-MS/MS. HCD-MS/MS was found to provide the greatest detection sensitivity for both global and variant-specific product ions in each of the MC variants, except for MC-YR where a variant-specific product uniquely formed via UPVD-MS/MS was observed with the greatest absolute abundance. A simple methodology for the preparation and characterization of 18O-stable isotope-labeled MC reference materials for use as internal standards was also developed. Finally, we have demonstrated the applicability of the methods developed herein for absolute quantification of MC-LR present in human urine samples, using capillary scale liquid chromatography coupled with ultra-high resolution / accurate mass spectrometry and HCD-MS/MS. Graphical abstract ᅟ.

Redox behavior of gas phase Pu(IV)-monodentate ligand complexes: an investigation by electrospray ionization mass spectrometry

This study reports the gas phase complexation behavior of Pu(IV) with N,N-dialkylamide ligands and tributylphosphate by electrospray ionization mass spectrometry. In-source collision induced dissociation (CID) and ion trap collision induced dissociation were used to investigate the kinetic stability of Pu species in the gas phase. With both class of ligands, N,N-dialkylamide and trialkylphosphate, oxidation of Pu from the tetravalent state to the hexavalent state was observed at both high skimmer voltages and during ion trap CID. The results suggest that oxidation is due to the presence of a nitrate group in the coordination sphere of the metal and is independent of the ligand.

Gas-Phase Chemical Separation of Phosphatidylcholine and Phosphatidylethanolamine Cations via Charge Inversion Ion/Ion Chemistry.

The [M + H](+) cations formed upon electrospray ionization of the glycerophospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) show distinct reactivities upon gas-phase reactions with doubly deprotonated 1,4-phenylenedipropionic acid (PDPA). PC cations undergo charge inversion via adduct formation with subsequent methyl cation and proton transfer to the acid to yield [PC - CH3](-) anions. These demethylated PC anions fragment upon ion trap collision-induced dissociation (CID) to yield products that reveal fatty acid chain lengths and degrees of unsaturation. PE cations, on the other hand, undergo charge inversion via double proton transfer to the two carboxylate moieties in doubly deprotonated PDPA to yield [PE - H](-) anions. These anions also fragment upon ion trap CID to yield product ions indicative of chain lengths and degrees of unsaturation in the fatty acyl moieties. Advantage is taken of this distinct reactivity to separate isomeric and isobaric PC and PE cations present in mass spectra of lipid mixtures. A cation precursor ion population containing a mixture of PE and PC cations is mass-selected and subjected to ion/ion charge inversion reactions that result in separation of PC and PE anions into different mass-to-charge ratios. Mass selection and subsequent ion trap CID of the lipid anions allows for the characterization of the isomeric lipids within each subclass. The charge inversion approach described here is demonstrated to provide increased signal-to-noise ratios for detection of PCs and PEs relative to the standard negative ionization approach as well as improved mixture analysis performance.

Assignment of saccharide identities through analysis of oxonium ion fragmentation profiles in LC-MS/MS of glycopeptides.

Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.

The dissociation behaviour of a singly and doubly charged (cationised) aza-crown ether/metalloporphyrin-conjugate: Metal-induced reactivity and Coulomb explosions

Fe(III) and Mn(III) complexes of an aza-crown ether/porphyrin conjugate, 52-N-(4-aza-18-crown-6)methyl-54,104,154,204-tetra-tert-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin, with Cl− and Br− as respective counter ions, are studied by electrospray ionisation (ESI)-mass spectrometry (MS). Fragmentation pathways are established by MS2 and MS3 collision-induced dissociation (CID) experiments performed with a quadrupole ion trap (QIT) and energy-dependent CID is studied with a quadrupole/time-of-flight (q-ToF) instrument. Singly charged ions are produced by selective protonation, sodiation or potassiation of the crown ether moiety, and dications possess one additional charge on the halide-released porphyrin metal cation. Singly charged parent ions undergo a two-step decay. Firstly, an “ion pair” is lost, composed of the cationic charge carrier of the crown ether and the anionic counter ion at the porphyrin. Secondly, the intact crown ether is released. The threshold of this process is dependent on the nature of the central porphyrin metal, with Fe(III) dramatically facilitating the reaction. The dications undergo two different Coulomb explosions: crown/porphyrin-fission and cation loss from the crown ether, the ratio of which is dependent on the affinity of the cation to the crown ether.

Top-Down Interrogation of Chemically Modified Oligonucleotides by Negative Electron Transfer and Collision Induced Dissociation

Two sets of synthetic 21-23mer oligonucleotides with various types of 2'-position modifications have been studied with tandem mass spectrometry using ion trap collision-induced dissociation (IT-CID) and negative electron transfer (NET)-CID. A systematic study has been conducted to define the limitations of IT-CID in sequencing such 2'-chemically modified oligonucleotides. We found that IT-CID is sufficient in characterizing oligonucleotide sequences that do not contain DNA residues, where high sequence coverage can be achieved by performing IT-CID on multiple charge states. However, oligonucleotides containing DNA residues gave limited backbone fragmentation with IT-CID, largely due to dominant fragmentation at the DNA residue sites. To overcome this limitation, we employed the negative electron transfer to strip an electron from the multiply charged oligonucleotide anion. Then, the radical anion species formed in this reaction can fragment via an alternative radical-directed dissociation mechanism. Unlike IT-CID, NET-CID mainly generates a noncomplementary d/w ion series. Furthermore, we found that NET-CID did not show preferential dissociations at the DNA residue sites and thus generated higher sequence coverage for the studied oligonucleotide. Information from NET-CID of different charge states is not fully redundant such that the examination of multiple charge states can lead to more extensive sequence confirmation. This work demonstrates that the NET-CID is a valuable tool to provide high sequence coverage for chemically modified oligonucleotides, and such detailed characterization can serve as an important assay to control the quality of therapeutic oligonucleotides that are produced under the good manufacture practice (GMP) regulations.

Gas-Phase Intramolecular Protein Crosslinking via Ion/Ion Reactions: Ubiquitin and a Homobifunctional sulfo-NHS Ester

Gas-phase intra-molecular crosslinking of protein ubiquitin cations has been demonstrated via ion/ion reactions with anions of a homobifunctional N-hydroxysulfosuccinimide (sulfo-NHS) ester reagent. The ion/ion reaction between multiply-protonated ubiquitin and crosslinker monoanions produces a stable, charge-reduced complex. Covalent crosslinking is indicated by the consecutive loss of 2 molecules of sulfo-NHS under ion trap collisional activation conditions. Covalent modification is verified by the presence of covalently crosslinked sequence ions produced by ion-trap collision-induced dissociation of the ion generated from the losses of sulfo-NHS. Analysis of the crosslinked sequence fragments allows for the localization of crosslinked primary amines, enabling proximity mapping of the gas-phase 3-D structures. The presence of two unprotonated reactive sites within the distance constraint of the crosslinker is required for successful crosslinking. The ability to covalently crosslink is, therefore, sensitive to protein charge state. As the charge state increases, fewer reactive sites are available and protein structure is more likely to become extended because of intramolecular electrostatic repulsion. At high charge states, the reagent shows little evidence for covalent crosslinking but does show evidence for 'electrostatic crosslinking' in that the binding of the sulfonate groups to the protein is sufficiently strong that backbone cleavages are favored over reagent detachment under ion trap collisional activation conditions.

Electron transfer followed by collision‐induced dissociation (NET‐CID) for generating sequence information from backbone‐modified oligonucleotide anions

Oligonucleotides with 2'-modifications and/or phosphorothioate (PS) backbones are prone to undergo limited backbone fragmentation upon ion trap collision-induced dissociation (CID). For better identification and characterization of chemically modified oligonucleotides, a more universal fragmentation method is desirable. Gas-phase dissociation of various 2'-position-modified oligonucleotides and mixed-backbone oligonucleotides (MBOs) has been studied by ion trap CID of the radical anion species formed via electron transfer ion/ion reactions. For 2'-modified mix-mer radical anions, complete sequence information was generated with non-complementary d/w-ion series, while a/z-ions were observed randomly with relatively low intensity. The 2'-position modification, which has been observed to affect CID patterns of oligonucleotide anions, did not exhibit any observable influence on the dissociation patterns of oligonucleotide radical anions. For MBOs comprised of DNA nucleotides, ion trap CID of even-electron species generated complementary a-B/w-type ions and multiple fragment types at the phosphorothioate (PS) linkages. For MBOs comprised of 2'-OMe-modified nucleotides, only PS bond cleavage was observed for ion trap CID of doubly deprotonated precursor ions. Negative electron transfer reaction with or without supplemental activation of MBOs gave rise to a/d/w-type fragments similar to those of the 2'-modified mix-mers. PS bonds were observed to be more fragile under the electron detachment process, and phosphodiester (PO) bond cleavages were noted upon further collisional activation. NET-CID proved to be an efficient method of generating full sequence information for 2'-modifications and/or mixed-backbone oligonucleotides.

Higher Energy Collision Dissociation (HCD) Product Ion-Triggered Electron Transfer Dissociation (ETD) Mass Spectrometry for the Analysis of N-Linked Glycoproteins

Large scale mass spectrometry analysis of N-linked glycopeptides is complicated by the inherent complexity of the glycan structures. Here, we evaluate a mass spectrometry approach for the targeted analysis of N-linked glycopeptides in complex mixtures that does not require prior knowledge of the glycan structures or pre-enrichment of the glycopeptides. Despite the complexity of N-glycans, the core of the glycan remains constant, comprising two N-acetylglucosamine and three mannose units. Collision-induced dissociation (CID) mass spectrometry of N-glycopeptides results in the formation of the N-acetylglucosamine (GlcNAc) oxonium ion and a [mannose+GlcNAc] fragment (in addition to other fragments resulting from cleavage within the glycan). In ion-trap CID, those ions are not detected due to the low m/z cutoff; however, they are detected following the beam-type CID known as higher energy collision dissociation (HCD) on the orbitrap mass spectrometer. The presence of these product ions following HCD can be used as triggers for subsequent electron transfer dissociation (ETD) mass spectrometry analysis of the precursor ion. The ETD mass spectrum provides peptide sequence information, which is unobtainable from HCD. A Lys-C digest of ribonuclease B and trypsin digest of immunoglobulin G were separated by ZIC-HILIC liquid chromatography and analyzed by HCD product ion-triggered ETD. The data were analyzed both manually and by search against protein databases by commonly used algorithms. The results show that the product ion-triggered approach shows promise for the field of glycoproteomics and highlight the requirement for more sophisticated data mining tools.

Collision‐induced dissociation of oligonucleotide anions fully modified at the 2'‐position of the ribose: 2'‐F/‐H and 2'‐F/‐H/‐OMe mix‐mers

Gas-phase dissociation of various 2'-position modified oligonucleotide anions has been studied as a function of precursor ion charge state using ion trap and low energy beam-type collision-induced dissociation (CID). For a completely 2'-O-methyl modified 6-mer, all possible dissociation channels along the phosphodiester linkage, generating complementary (a-B)/w-, b/x-, c/y-, d/z-ion series, were observed with no single dominant type of dissociation pathway. Full sequence information was generated from each charge state via ion trap CID. More sequential fragmentation was noted under beam-type CID conditions. Comparison with model DNA, in which all 2'-OH groups are converted to 2'-H, and RNA anions suggests that the 2'-OMe substitution stabilizes the phosphodiester linkage with respect to fragmentation relative to both DNA and RNA oligomers. For modified mix-mer anions, comprised of DNA nucleotides and 2'-F substituted nucleotides or a mixture of DNA nucleotides and 2'-O-methyl (2'-OMe) and 2'-F substituted nucleotides, 3'-side backbone cleavage was found to be inhibited by the 2'-OMe or 2'-F modification on the nucleotides under ion trap CID conditions. Thus, the sequence information was limited to the a-Base/w-fragments from the cleavage of the 3' C-O bond of the 2'-H (DNA) nucleotides. Under beam-type CID conditions, limited additional cleavage adjacent to 2'-OMe substituted nucleotides was noted but 2'-F modified residues remained resistant to cleavage.

Partial De Novo Sequencing and Unusual CID Fragmentation of a 7 kDa, Disulfide-Bridged Toxin

A 7 kDa toxin isolated from the venom of the Texas coral snake (Micrurus tener tener) was subjected to collision-induced dissociation (CID) and electron-transfer dissociation (ETD) analyses both before and after reduction at low pH. Manual and automated approaches to de novo sequencing are compared in detail. Manual de novo sequencing utilizing the combination of high accuracy CID and ETD data and an acid-related cleavage yielded the N-terminal half of the sequence from the reduced species. The intact polypeptide, containing 3 disulfide bridges produced a series of unusual fragments in ion trap CID experiments: abundant internal amino acid losses were detected, and also one of the disulfide-linkage positions could be determined from fragments formed by the cleavage of two bonds. In addition, internal and c-type fragments were also observed.

Dipolar DC Collisional Activation in a “Stretched” 3-D Ion Trap: The Effect of Higher Order Fields on rf-Heating

Applying dipolar DC (DDC) to the end-cap electrodes of a 3-D ion trap operated with a bath gas at roughly 1 mTorr gives rise to ‘rf-heating’ and can result in collision-induced dissociation (CID). This approach to ion trap CID differs from the conventional single-frequency resonance excitation approach in that it does not rely on tuning a supplementary frequency to coincide with the fundamental secular frequeny of the precursor ion of interest. Simulations using the program ITSIM 5.0 indicate that application of DDC physically displaces ions solely in the axial (inter end-cap) dimension whereupon ion acceleration occurs via power absorption from the drive rf. Experimental data shows that the degree of rf-heating in a stretched 3-D ion trap is not dependent solely on the ratio of the dipolar DC voltage/radio frequency (rf) amplitude, as a model based on a pure quadrupole field suggests. Rather, ion temperatures are shown to increase as the absolute values of the dipolar DC and rf amplitude both decrease. Simulations indicate that the presence of higher order multi-pole fields underlies this unexpected behavior. These findings have important implications for the use of DDC as a broad-band activation approach in multi-pole traps.