Collision-induced Dissociation Fragmentation Patterns Research Articles

Charge site manipulation to enhance top-down fragmentation efficiency.

In recent years, top-down mass spectrometry has become a widely used approach to study proteoforms; however, improving sequence coverage remains an important goal. Here, two different proteins, α-synuclein and bovine carbonic anhydrase, were subjected to top-down collision-induced dissociation (CID) after electrospray ionisation. Two high-boiling solvents, DMSO and propylene carbonate, were added to the protein solution in low concentration (2%) and the effects on the top-down fragmentation patterns of the proteins were systematically investigated. Each sample was measured in triplicate, which revealed highly reproducible differences in the top-down CID fragmentation patterns in the presence of a solution additive, even if the same precursor charge state was isolated in the quadrupole of the instrument. Further investigation supports the solution condition-dependent selective formation of different protonation site isomers as the underlying cause of these differences. Higher sequence coverage was often observed in the presence of additives, and the benefits of this approach became even more evident when datasets from different solution conditions were combined, as increases up to 35% in cleavage coverage were obtained. Overall, this approach therefore represents a promising opportunity to increase top-down fragmentation efficiency.

Collision-Induced Dissociation of α-Isomaltose and α-Maltose.

Determination of carbohydrate structures remains a considerable challenge. Collision-induced dissociation (CID) tandem mass spectroscopy (MS/MS) is widely used for carbohydrate structure determination. Structural information derived from MS/MS relies on an understanding of the carbohydrate dissociation mechanism. Among various hexose disaccharides, the major dissociation channels (dehydration, glycosidic bond cleavage, and cross-ring dissociation) of 1→2-, 1→3-, and 1→4-linked disaccharide sodium ion adducts can be explained by the dissociation mechanism derived from hexose monosaccharides. However, 1→6-linked disaccharides, which have low branching ratios for dehydration and glycosidic bond cleavage, cannot be explained by the same dissociation mechanism. In this study, we performed high-level quantum chemistry calculations to examine the CID mechanism of the α-isomaltose sodium ion adduct, a 1→6-linked glucose disaccharide. For comparison, we examined the CID dissociation mechanism of the α-maltose sodium ion adduct, a 1→4-linked glucose-disaccharide. Calculations revealed that although α-isomaltose and α-maltose had similar dissociation mechanisms, energy differences between the lowest transition states of various dissociation channels led to different CID fragmentation patterns. The dissociation barriers of dehydration and glycosidic bond cleavage were similar for the two disaccharides, but the cross-ring dissociation, which has the lowest dissociation barrier, exhibited differences in barriers between the disaccharides. The cross-ring dissociation barrier for α-maltose was only slightly lower than those of dehydration and glycosidic bond cleavage. However, the cross-ring dissociation barrier for α-isomaltose was substantially lower than those of dehydration and glycosidic bond cleavage. In addition, most of the α-isomaltose conformers that led to dehydration also led to cross-ring dissociation, resulting in suppression of dehydration by cross-ring dissociation. The findings can explain the low branching ratios for dehydration and glycosidic bond cleavage observed in α-isomaltose CID spectra.

Establishment of a Charge Reversal Derivatization Strategy to Improve the Ionization Efficiency of Limaprost and Investigation of the Fragmentation Patterns of Limaprost Derivatives Via Exclusive Neutral Loss and Survival Yield Method.

Sensitivity is generally an issue in bioassays of prostaglandins and their synthetic analogs due to their extremely low concentration in vivo. To improve the ionization efficiency of limaprost, an oral prostaglandin E1 (PGE1) synthetic analog, we investigated a charge reversal derivatization strategy in electrospray ionization mass spectrometry (ESI-MS). We established that the cholamine derivative exhibits much greater signal intensity in the positive-ion mode compared with limaprost in the negative ion mode. Collision-induced dissociation (CID) involved exclusive neutral mass loss and positive charge migration to form stable cationic product ions with the positive charge on the limaprost residue rather than on the modifying group. This has the effect of maintaining the efficiency and specificity of multiple reaction monitoring (MRM) and avoiding cross talk. CID fragmentation patterns of other limaprost derivatives allowed us to relate the dissociation tendency of different neutral leaving groups to an internal energy distribution scale based on the survival yield method. Knowledge of the energy involved in the production of stabilized positive ions will potentially assist the selection of suitable derivatization reagents for the analysis of a wide variety of lipid acids. Graphical Abstract ᅟ.

Collision induced dissociation of N-(pyridin-2-yl)-substituted benzo(thio)amides and N-(isoquinolin-1-yl)furan(thiophene)-2-carboxamides and their difluoroboranyl derivatives

The effect of the properties of sulfur and oxygen atoms and of the presence/absence of the BF2 group on the collision induced dissociation (CID) behavior of singly charged ions generated by electrospray ionization (ESI) of N-(pyridin-2-yl)-substituted benzo(thio)amides and N-(isoquinolin-1-yl)furan(thiophene)-2-carboxamides and their difluoroboranyl derivatives has been investigated. The results show that O/S replacement (both in the amide/thioamide and the furan/thiophene moieties) has a strong influence on the CID fragmentation patterns. The loss of HOBF2 (or HSBF2) has been observed in ESI-CID MS/MS experiments performed for all difluoroboranyl derivatives. It can serve as a signature tag for the presence of the OBF2 (SBF2) moiety.

Tandem mass spectrometry of isomeric aniline-labeled N-glycans separated on porous graphitic carbon: Revealing the attachment position of terminal sialic acids and structures of neutral glycans.

Quantitative monitoring of changes in the N-glycome upon disease has gained significance in the context of biomarker discovery. Separation and quantification of isobaric glycan isomers can be attained by using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Collision-induced dissociation (CID)-based fragmentation of separated isobaric glycans is evaluated in respect to its potential of providing fragment ions specific for the linkage positions of terminal sialic acids and the presence of intersecting GlcNAc moieties, respectively. N-Glycans were labeled via reductive amination using (12)C6-aniline and (13)C6-aniline as isotope-coded labeling reagents. The differently labeled glycans were merged and separated into various species using a porous graphitic carbon (PGC) stationary phase. Identification of structural features of separated isobaric isomers was performed by CID-based tandem mass spectrometry (MS/MS) carried out in a quadrupole time-of-flight (QqTOF) or a quadrupole ion-trap (IT) mass spectrometer. Working in the negative ion mode, new diagnostic CID fragment ions could be found that are indicative for the α2,6-type linkage of sialic acids. Other diagnostic ions, identified before as being indicative for the substitution of the 6-antenna, could be confirmed as being of relevance also in the case of aniline labeling. In the positive ion mode, CID fragment ions indicative for the structure of short neutral N-glycans were identified. One new diagnostic ion specific for the linkage position of the terminal sialic acids and one for the presence of bisecting GlcNAc in N-glycans were identified. The aniline label introduced for improved relative quantitation in MS(1) was found not to significantly alter the CID fragmentation patterns that were reported previously by other authors for unlabeled/reduced glycans or for glycans with more polar labels.

Populations of metal-glycan structures influence MS fragmentation patterns.

The structures and collision-induced dissociation (CID) fragmentation patterns of the permethylated glycan Man5GlcNAc2 are investigated by a combination of hybrid ion mobility spectrometry (IMS), mass spectrometry (MS), and MS/MS techniques. IMS analysis of eight metal-adducted glycans ([Man5GlcNAc2 + M](2+), where M = Mn, Fe, Co, Ni, Cu, Mg, Ca, and Ba) shows distinct conformer patterns. These conformers appear to arise from individual metals binding at different sites on the glycan. Fragmentation studies suggest that these different binding sites influence the CID fragmentation patterns. This paper describes a series of separation, activation, and fragmentation studies that assess which fragments arise from each of the different gas-phase conformer states. Comparison of the glycan distributions formed under gentle ionization conditions with those obtained after activation of the gas-phase ions suggests that these conformer binding states also appear to exist in solution.

The detection of iron protoporphyrin (heme b) in phytoplankton and marine particulate material by electrospray ionisation mass spectrometry – comparison with diode array detection

A mass spectrometric (MS) method for the identification of iron protoporphyrin (IX) (FePTP, heme b) in marine particulate material and phytoplankton is described. Electrospray ionisation of FePTP produced the molecular Fe(III)PTP+ ion (m/z=616) or the pseudomolecular [Fe(II)PTP + H]+ ion (m/z=617), depending on the oxidation state of the central iron ion. Collision induced dissociation (CID) in the ion trap mass spectrometer resulted in a single detected product ion (m/z=557) indicative of loss of ethanoic acid from a carboxylic acid side chain. Widening the isolation width to 616±3 resulted in production of a mass spectrum demonstrating the distinctive isotopic ratio of the iron containing fragment, further increasing the specificity of the analysis. Selective reactant monitoring (SRM) of the fragment ion (m/z=557) was applied to the detection of FePTP after chromatography of ammoniacal OGP extracts of marine samples. The detection limit for FePTP analysed by SRM after chromatography was 1.2±0.5fmol. For phytoplankton samples, reasonably good agreement was achieved between results obtained with SRM and those obtained by monitoring absorbance at λ=400nm using a diode array detector (DAD). Use of SRM for analysis of particulate material obtained from the high latitude North Atlantic allowed for the analysis of FePTP in the presence of a co-eluting compound that interfered with detection by DAD. Simultaneous collection of mass spectra from m/z=300 to 1500 resulted in identification of the pseudomolecular ion for the interfering compound. The CID fragmentation pattern and UV–visible mass spectra indicated that the interfering compound was a previously unidentified chlorin type compound. Comparison of FePTP determined by SRM and DAD on samples where this compound could not be detected showed that results collected using the two methods correlated. The use of both MS and DAD results in a powerful tool for quantifying this important biogenic component of the particulate iron pool.

Matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight collision-induced dissociation study of poly(p-phenylenediamine terephthalamide) fragmentation reactions

MALDI-TOF/TOF collision-induced dissociation (CID) experiments are reported on model poly(p-phenylenediamine terephthalamide) (PPD-T) polymers, revealing a variety of synthesis reaction products. Diamine-terminated oligomers were the major product of synthesis using excess amine, and di-carboxylic acid oligomers were the major product for excess acid. Structures of major reaction products were confirmed by CID fragmentation studies, along with detailed studies of MS/MS decomposition pathways. Apparent fracture of the phenylcarbonyl bond was the major fragmentation pathway (independent of end groups), resulting from initial NHCO bond cleavage with subsequent CO loss. Hydrogen-transfer reactions play an important role in fragmentation, involving both cross-chain abstraction of NH hydrogen and long-range H-transfer. End-group and main-chain modifications produce fingerprint CID fragmentation patterns that can be used to identify end groups and branching patterns; the structure of an unanticipated synthesis product was established using CID. The effect of synthesis conditions on polymer composition was studied using the analysis of variance, specifically, the amine-to-acid ratio used and post-synthesis addition of CaO. Of particular interest is oligomer end-group modification by the solvent (N-methyl pyrrolidone) induced by addition of CaO.

Shotgun Lipidomic Analysis of Human Meibomian Gland Secretions with Electrospray Ionization Tandem Mass Spectrometry

The purpose of this investigation was to determine the major molecular components of the lipids in normal human meibomian gland secretions (meibum). The meibum samples were studied by direct infusion electrospray ionization (ESI), quadrupole time-of-flight mass spectrometry, and tandem mass spectrometry (MS/MS) analysis, in both positive and negative detection modes. Hundreds of peaks were detected, among which the molecular compositions and subclasses of approximately 160 major peaks were confidently identified. The compositions and subclasses of these peaks were determined from collision-induced dissociation fragmentation patterns, high-resolution and high-mass-accuracy spectra, and references of literature reports. The major peaks detected in positive mode were those of nonpolar lipids, including wax esters, cholesteryl esters, triacylglycerols, and diesters, whereas in negative mode, the major peaks detected were those of polar lipids, including free fatty acids and (O-acyl)-ω-hydroxy fatty acids. The analysis of intact lipids in meibum with direct infusion ESI-MS/MS analysis has the advantages of minimal sample preparation (no chromatography or pre-separation needed), mild experimental conditions, high throughput, and high sensitivity.

Red cell membrane and plasma linoleic acid nitration products: synthesis, clinical identification, and quantitation.

Nitric oxide (*NO) and its reactive metabolites mediate the oxidation, nitration, and nitrosation of DNA bases, amino acids, and lipids. Here, we report the structural characterization and quantitation of two allylic nitro derivatives of linoleic acid (LNO(2)), present as both free and esterified species in human red cell membranes and plasma lipids. The LNO(2) isomers 10-nitro-9-cis, 12-cis-octadecadienoic acid and 12-nitro-9-cis, 12-cis-octadecadienoic acid were synthesized and compared with red cell and plasma LNO(2) species based on chromatographic elution and mass spectral properties. Collision-induced dissociation fragmentation patterns from synthetic LNO(2) isomers were identical to those of the two most prevalent LNO(2) positional isomers found in red cells and plasma. By using [(13)C]LNO(2) as an internal standard, red cell free and esterified LNO(2) content was 50 +/- 17 and 249 +/- 104 nM, respectively. The free and esterified LNO(2) content of plasma was 79 +/- 35 and 550 +/- 275 nM, respectively. Nitrated fatty acids, thus, represent the single largest pool of bioactive oxides of nitrogen in the vasculature, with a net LNO(2) concentration of 477 +/- 128 nM, excluding buffy coat cells. These observations affirm that basal oxidative and nitrating conditions occur in healthy humans to an extent that is sufficient to induce abundant membrane and lipoprotein-fatty acid nitration. Given that LNO(2) is capable of mediating cGMP and non-cGMP-dependent signaling reactions, fatty acid nitration products are species representing the convergence of ()NO and oxygenated lipid cell-signaling pathways.

Solution Phase Cationic Niobium and Tantalum Alkoxide Clusters: Insights into Formation and Structure

Electrospray mass spectrometry (ESMS) is used to investigate cationic niobium and tantalum alkoxide clusters generated in solution by adding equal molar amounts of B(C6F5)3 to the metal ethoxide, M(OEt)5. The dominant metal ethoxide cations that were observed in the electrospray mass spectrum correspond to M(OEt)4+, M2O(OEt)7+, M2(OEt)9+, and M3O(OEt)12+, where M is niobium or tantalum. In separate experiments heterometallic Nb/Ta ethoxide clusters were produced, and the heterometallic cations present in the electrospray mass spectrum are NbTaO(OEt)7+, NbTa(OEt)9+, Nb2TaO(OEt)12+, and NbTa2O(OEt)12+. Collision-induced dissociation (CID) experiments were completed to provide structural information about the observed metal ethoxide cations. At low collision energy (ECM < 2 eV), the CID fragmentation pattern of Nb3O(OEt)12+ favors loss of Nb2(OEt)9+ and neutral NbO(OEt)3, whereas the fragmentation pattern of Ta3O(OEt)12+ favors loss of Ta2O(OEt)7+ and neutral Ta(OEt)5. In addition, the major CID product of M2O(OEt)7+ and M2(OEt)9+ at low collision energy is M(OEt)4+. During CID experiments of M(OEt)4+, four consecutive ethylene molecules were released leaving M(OH)4+, which at higher collision energies (ECM > 2 eV) liberates two water molecules resulting in MO2+. The CID fragmentation pattern of crotyl alcohol (CH3CHCHCH2OH) substituted and n-butanol (CH3CH2CH2CH2OH) substituted metal alkoxides resembles the fragmentation pattern proposed for M(OEt)4+, in which four consecutive organic molecules are shed leaving M(OH)4+.

Elucidation of isomeric structures for ubiquitin [M + 12H]12+ ions produced by electrospray ionization mass spectrometry.

Gas-phase deprotonation reactions, hydrogen-deuterium exchange reactions and collision-induced dissociation (CID) were used to distinguish between two isomeric forms of [M + 12H]12+ produced from the protein ubiquitin. Ions were generated by electrospray ionization and studied in a Fourier transform ion cyclotron resonance mass spectrometer. For [M + 12H]12+ formed directly from the electrospray process, deprotonation reactions with ammonia and 2-fluoropyridine yield non-linear pseudo-first-order kinetic behavior that indicates the presence of two ion structures. The fraction of ions that undergo the fastest deprotonation reactions, and is presumably the least energetically stable isomer, accounts for approximately 60% of the [M + 12H]12+ produced by electrospray. In reactions with D2O and CD3OD, the [M + 12H]12+ which are deprotonated faster exchange the first 11 +/- 1 hydrogens more readily that the remaining [M + 12H]12+ population. Results from CID experiments, obtained as a function of reaction time with the amines, also indicate the existence of more than one [M + 12H]12+ structure. The CID fragmentation patterns provide information about the general locations of the charge sites. Surprisingly, evidence for only one structure (the slow-reacting, more stable species) is found for [M + 12H]12+ that is produced by gas-phase deprotonation of [M + 13H]13+, which is the "fully protonated' form of ubiquitin. These results are discussed in terms of ubiquitin isomers related to protonation site and three-dimensional conformation.

Internal energy and angular momentum effects on collision induced dissociation fragmentation patterns

AbstractData are reported on the effects of internal energy and angular momentum on the collision induced dissociation CID fragmentation pattern. For the ions studied changes in the relative intensities of the fragment ions as internal energy varied were found to be larger than suggested by McLafferty and coworkers. Possible effects of angular momentum on the CID fragmentation pattern are discussed. The charge stripping spectra of the ions studied were found to be strongly dependent on initial energy and/or angular momentum. Hence care must be taken if charge stripping spectra are used to distinguish ion structures.

Internal energy effects in collision induced dissociation spectra

AbstractThe effect of the precursor ion internal energy on the branching ratios obtained from collision induced dissociation fragmentation patterns was examined for [NH3]+ and [C2H4N]+. The ion internal energy was changed by varying both the chemical ionization reagent gas and the ion source pressure. Effects observed in the collision induced dissociation fragmentation patterns as a function of the ion source pressure are explained by the reaction exothermicities and by collisional deactivation of internally excited ions (at high pressure).