Travelling‐wave ion mobility and negative ion fragmentation of high‐mannose N‐glycans

The isomeric structure of high‐mannose N‐glycans can significantly impact biological recognition events. Here, the utility of travelling‐wave ion mobility mass spectrometry for isomer separation of high‐mannose N‐glycans is investigated. Negative ion fragmentation using collision‐induced dissociation gave more informative spectra than positive ion spectra with mass‐different fragment ions characterizing many of the isomers. Isomer separation by ion mobility in both ionization modes was generally limited, with the arrival time distributions (ATD) often showing little sign of isomers. However, isomers could be partially resolved by plotting extracted fragment ATDs of the diagnostic fragment ions from the negative ion spectra, and the fragmentation spectra of the isomers could be extracted by using ions from limited areas of the ATD peak. In some cases, asymmetric ATDs were observed, but no isomers could be detected by fragmentation. In these cases, it was assumed that conformers or anomers were being separated. Collision cross sections of the isomers in positive and negative fragmentation mode were estimated from travelling‐wave ion mobility mass spectrometry data using dextran glycans as calibrant. More complete collision cross section data were achieved in negative ion mode by utilizing the diagnostic fragment ions. Examples of isomer separations are shown for N‐glycans released from the well‐characterized glycoproteins chicken ovalbumin, porcine thyroglobulin and gp120 from the human immunodeficiency virus. In addition to the cross‐sectional data, details of the negative ion collision‐induced dissociation spectra of all resolved isomers are discussed. Copyright © 2016 John Wiley & Sons, Ltd.     

The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap MS

Thirteen high mannose isomers have been structurally characterized within three glycomers, Man₅GlcNAc₂, Man₇GlcNAc₂, and Man₈GlcNAc₂ released from bovine ribonuclease B, six previously unreported. The study was carried out with a single ion trap instrument involving no chromatography. Three previously characterized isomers from Man₇ and Man₈ (three each) have been identified plus one unreported Man₇ isomer. Incomplete α-glucosidase activity on the Man₆ and Man₇ glycoproteins appears to account for two additional isomeric structures. The preeminence of ion traps for detail analysis was further demonstrated by resolving three new isomers within the Man₅ glycomer summing to the six previously unreported structures in this glycoprotein. All reported structures represent a distribution of Golgi processing remnants that fall within the Man₉GlcNAc₂ footprint. Topologies were defined by ion compositions along a disassembly pathway while linkage and branching were aided by spectral identity in a small oligomer fragment library. Isomers from this glycoprotein appear to represent a distribution of Golgi processing remnants, and an alphanumeric classification scheme has been devised to identify all products. Although numerous analytical strategies have been introduced to identify selected components of structure, it has been the continued focus of this and previous reports to only build upon protocols that can be integrated into a high throughput strategy consistent with automation. Duplication of these and results from comparable standards could bring an important analytical focus to carbohydrate sequencing that is greatly lacking.     

Mannose7 Glycan Isomer Characterization by IMS-MS/MS Analysis

The isomers of the Man₇GlcNAc₂ glycan obtained from bovine ribonuclease B have been characterized by ion mobility spectrometry-tandem mass spectrometry (IMS-MS/MS). In these experiments, [Man7 + 2Na]²⁺ precursors having different mobilities are selected by ion mobility spectrometry and analyzed by MS/MS techniques in an ion trap. The fragmentation spectra obtained for various precursor ions are specific, suggesting the isolation or enrichment of different glycan isomers. One fragment ion with a mass-to-charge ratio (m/z) of 903.8 is found to correspond to the loss of an internal mannose residue of a specific isomer. Extracted fragment ion drift time distributions (XFIDTDs) yield distinctive precursor ion drift time profiles indicating the existence of four separate isomers as proposed previously.     

Glycopeptide Mimetics Recapitulate High‐Mannose‐Type Oligosaccharide Binding and Function

High‐mannose‐type glycans (HMTGs) decorating viral spike proteins are targets for virus neutralization. For carbohydrate‐binding proteins, multivalency is important for high avidity binding and potent inhibition. To define the chemical determinants controlling multivalent interactions we designed glycopeptide HMTG mimetics with systematically varied mannose valency and spacing. Using the potent antiviral lectin griffithsin (GRFT) as a model, we identified by NMR spectroscopy, SPR, analytical ultracentrifugation, and microcalorimetry glycopeptides that fully recapitulate the specificity and kinetics of binding to Man₉GlcNAc₂Asn and a synthetic nonamannoside. We find that mannose spacing and valency dictate whether glycopeptides engage GRFT in a face‐to‐face or an intermolecular binding mode. Surprisingly, although face‐to‐face interactions are of higher affinity, intermolecular interactions are longer lived. These findings yield key insights into mechanisms involved in glycan‐mediated viral inhibition.     

Fragmentation of negative ions from N‐linked carbohydrates,Part 4. Fragmentation of complex glycans lacking substitution on the 6‐antenna

Negative ion CID spectra of N‐linked glycans released from glycoproteins contain many ions that are diagnostic for specific structural features such as the detailed arrangement of antennae and the location of fucose residues. Identification of such ions requires reference glycans that are often difficult to acquire in a pure state. The recent acquisition of a sample of N‐glycans from a patient lacking the enzyme N‐acetylglucosaminyltransferase‐2 provided an opportunity to investigate fragmentation of glycans lacking a 6‐antenna. These glycans contained one or two galactose‐N‐acetylglucosamine‐chains attached to the 3‐linked mannose residue of the trimannosyl‐chitobiose core with and without fucose substitution. The spectra from the patient sample clearly defined the antenna distribution and showed striking differences from the spectra of isomeric compounds obtained from normal subjects. Furthermore, they provided additional information on previously identified antenna‐specific fragment ions and indicated the presence of additional ions that were diagnostic of fucose substitution. Glycans obtained from such enzyme‐deficient patients can, thus, be a valuable way of obtaining spectra of specific isomers in a relatively pure state for interpretation of mass spectra. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of a tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source

A new tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source ‘ESI‐TOF/quadTOF’ was designed and constructed to achieve the desired aim of structural elucidation via high‐energy collision‐induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration‐type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic‐field ion mirror and a microchannel plate detector. High‐energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a‐, d‐, v‐ and w‐type ions, characteristic of high‐energy CID, were clearly observed in these spectra. These high‐energy CID fragment ions enabled confirmation of the complete sequence, including leucine–isoleucine determinations. It was demonstrated that high‐energy CID of multiply protonated peptides could be achieved in the ESI‐TOF/quadTOF. Copyright © 2010 John Wiley & Sons, Ltd.     

An experimental and computational study on the dissociation behavior of hydroxypyridine N‐oxides in atmospheric pressure ionization mass spectrometry

A tandem mass spectrometric study of protonated isomeric hydroxypyridine N‐oxides was carried out with a hybrid quadrupole/time‐of‐flight mass spectrometer coupled with different atmospheric pressure ionization sources. The behavior observed in the collision‐induced dissociation (CID) mass spectra of the parent cations, was similar irrespective of the source employed. However, there were intrinsic differences in the intensities of the two fragments observed for each isomer. The major fragment because of elimination of a hydroxyl radical, dominated the CID spectra (in contrast with weaker water loss) at different energy thresholds. Therefore, it was possible to differentiate both isomers at collision energies above 13 eV by comparing the ratio of intensities of the major fragment relative to the precursor cation. In addition, quantum chemical calculations at the B3LYP/6‐31 + + G(d,p) level of theory were performed for the protonated isomers of hydroxypyridine N‐oxide and their radical cation products in order to gain insight into the major routes of dissociation. The results suggest that dissociation from the lowest triplet excited state of the protonated species would provide a reasonable rationalization for the difference in behavior of both isomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Construction of a High‐Mannose‐Type Glycan Library by a Renewed Top‐Down Chemo‐Enzymatic Approach

A comprehensive method for the construction of a high‐mannose‐type glycan library by systematic chemo‐enzymatic trimming of a single Man9‐based precursor was developed. It consists of the chemical synthesis of a non‐natural tridecasaccharide precursor, the orthogonal demasking of the non‐reducing ends, and trimming by glycosidases, which enabled a comprehensive synthesis of high‐mannose‐type glycans in their mono‐ or non‐glucosylated forms. It employed glucose, isopropylidene, and N‐acetylglucosamine groups for blocking the A‐, B‐, and C‐arms, respectively. After systematic trimming of the precursor, thirty‐seven high‐mannose‐type glycans were obtained. The power of the methodology was demonstrated by the enzymatic activity of human recombinant N‐acetylglucosaminyltransferase‐I toward M7–M3 glycans, clarifying the substrate specificity in the context of high‐mannose‐type glycans.     

Gold Manno‐Glyconanoparticles: Multivalent Systems to Block HIV‐1 gp120 Binding to the Lectin DC‐SIGN

The HIV envelope glycoprotein gp120 takes advantage of the high‐mannose clusters on its surface to target the C‐type lectin dendritic cell‐specific intracellular adhesion molecule‐3‐grabbing non‐integrin (DC‐SIGN) on dendritic cells. Mimicking the cluster presentation of oligomannosides on the virus surface is a strategy for designing carbohydrate‐based antiviral agents. Bio‐inspired by the cluster presentation of gp120, we have designed and prepared a small library of multivalent water‐soluble gold glyconanoparticles (manno‐GNPs) presenting truncated (oligo)mannosides of the high‐mannose undecasaccharide Man₉GlcNAc₂ and have tested them as inhibitors of DC‐SIGN binding to gp120. These glyconanoparticles are ligands for DC‐SIGN, which also interacts in the early steps of infection with a large number of pathogens through specific recognition of associated glycans. (Oligo)mannosides endowed with different spacers ending in thiol groups, which enable attachment of the glycoconjugates to the gold surface, have been prepared. manno‐GNPs with different spacers and variable density of mannose (oligo)saccharides have been obtained and characterized. Surface plasmon resonance (SPR) experiments with selected manno‐GNPs have been performed to study their inhibition potency towards DC‐SIGN binding to gp120. The tested manno‐GNPs completely inhibit the binding from the micro‐ to the nanomolar range, while the corresponding monovalent mannosides require millimolar concentrations. manno‐GNPs containing the disaccharide Manα1‐2Manα are the best inhibitors, showing more than 20 000‐fold increased activity (100 % inhibition at 115 nM ) compared to the corresponding monomeric disaccharide (100 % inhibition at 2.2 mM ). Furthermore, increasing the density of dimannoside on the gold platform from 50 to 100 % does not improve the level of inhibition.     

Differentiation of lisinopril and its RSS diastereomer by liquid chromatography combined with collision‐induced dissociation mass spectrometry

A simple and sensitive liquid chromatography tandem multiple‐stage mass spectrometry (HPLC/MS/MS) method suitable for bulk lisinopril analysis was developed, by which lisinopril and its RSS isomer were separated and differentiated. In the collision‐induced dissociation (CID) mass spectra of the [M + H]⁺ ions, the abundance of the fragment ion of m/z 246 for lisinopril was about two times higher than the ion of m/z 245; however, the former fragment ion was noted to be a little lower than the latter for RSS isomer at all collision energies. In the CID mass spectra of the [M + Li]⁺ ion, the abundance of the rearrangement ion of m/z 315 for the RSS isomer was about three times higher than that for lisinopril. Furthermore, the difference was supported by the results of energy‐resolved mass spectrometry (ERMS) in the test range of collision energies. Similar differences were also observed between the CID mass spectra of lisinopril and RSS isomer methylester, which indicated that the RSS isomer could be rapidly characterized by the CID mass spectra of both the protonated and lithium adduct ion. Elemental compositions of all the ions were confirmed by Fourier Transform ion cyclotron resonance ESI mass spectrometry (FT‐ICR‐ESI/MS). In addition, theoretical computations were carried out to support the experimental results. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of N‐glycans from Ebola virus glycoproteins by matrix‐assisted laser desorption/ionisation time‐of‐flight and negative ion electrospray tandem mass spectrometry

The larger fragment of the transmembrane glycoprotein (GP1) and the soluble glycoprotein (sGP) of Ebola virus were expressed in human embryonic kidney cells and the secreted products were purified from the supernatant for carbohydrate analysis. The N‐glycans were released with PNGase F from within sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS‐PAGE) gels. Identification of the glycans was made with normal‐phase high‐performance liquid chromatography (HPLC), matrix‐assisted laser desorption/ionisation mass spectrometry, negative ion electrospray ionisation fragmentation mass spectrometry and exoglycosidase digestion. Most glycans were complex bi‐, tri‐ and tetra‐antennary compounds with reduced amounts of galactose. No bisected compounds were detected. Triantennary glycans were branched on the 6‐antenna; fucose was attached to the core GlcNAc residue. Sialylated glycans were present on sGP but were largely absent from GP1, the larger fragment of the transmembrane glycoprotein. Consistent with this was the generally higher level of processing of carbohydrates found on sGP as evidenced by a higher percentage of galactose and lower levels of high‐mannose glycans than were found on GP1. These results confirm and expand previous findings on partial characterisation of the Ebola virus transmembrane glycoprotein. They represent the first detailed data on carbohydrate structures of the Ebola virus sGP. Copyright © 2010 John Wiley & Sons, Ltd.     

Peak width‐mass correlation in CID MS/MS of isomeric oligosaccharides using traveling‐wave ion mobility mass spectrometry

Isomeric oligosaccharides γ‐cyclodextrin (γ‐CD), glucosyl‐βCD (Glc₁‐βCD) and maltosyl‐αCD (Glc₂‐αCD) were analyzed by traveling‐wave ion mobility (twIM) mass spectrometry (MS). Their formation of multicharged multimers differed from each other. The ion mobility‐mass spectrometry was useful in the self‐assembling and complex formation analyses of CD isomers. The drift times of the isomers and their product ions with the same mass were almost the same in collision‐induced dissociation (CID) MS/MS. In contrast, the ion mobility peak widths were sensitive to structural differences of the isomeric product ions. The twIM peak width (ms ‐ µs) of the product ions [M ? Glc_n + H]⁺ (n = 0 ～ 6) of γ‐CD correlated linearly with their masses (Da); the large and/or long chain product ions had wider peak widths, which were much wider than those from the general diffusion effect. This was a novel and useful ‘trend line’ to discriminate between the three isomers. Plots of [M ? Glc_{2 ～ 6} + H]⁺ of Glc₁‐βCD and [M ? Glc_{3 ～ 6} + H]⁺ of Glc₂‐αCD product ions' plots were on the same trend line as γ‐CD. The plots of [M ? Glc₁ + H]⁺ of Glc₁‐βCD and [M ? Glc_{1, 2} + H]⁺ of Glc₂‐αCD strayed from the γ‐CD line; their peak widths were narrower than those of γ‐CD. These results indicated that product ions from the chemical species of Glc₁‐β CD and Glc₂‐αCD retained their CD structure. Analyses of the IM peak widths enable us to elucidate the structures of the product ions. Copyright © 2009 John Wiley & Sons, Ltd.     

Differentiation of Boc‐protected α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptide positional isomers by electrospray ionization tandem mass spectrometry

Two new series of Boc‐N‐α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptides containing repeats of L ‐Ala‐δ⁵‐Caa/δ⁵‐Caa‐L ‐Ala and β³‐Caa‐δ⁵‐Caa/δ⁵‐Caa‐β³‐Caa (L ‐Ala = L ‐alanine, Caa = C‐linked carbo amino acid derived from D ‐xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MSⁿ spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc‐group, and the side chain. The dipeptide positional isomers are differentiated by the collision‐induced dissociation (CID) of the protonated peptides. The loss of 2‐methylprop‐1‐ene is more pronounced for Boc‐NH‐L ‐Ala‐δ‐Caa‐OCH₃ (1), whereas it is totally absent for its positional isomer Boc‐NH‐δ‐Caa‐L ‐Ala‐OCH₃ (7), instead it shows significant loss of t‐butanol. On the other hand, second isomeric pair shows significant loss of t‐butanol and loss of acetone for Boc‐NH‐δ‐Caa‐β‐Caa‐OCH₃ (18), whereas these are insignificant for its positional isomer Boc‐NH‐β‐Caa‐δ‐Caa‐OCH₃ (13). The tetra‐ and hexapeptide positional isomers also show significant differences in MS² and MS³ CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed through five‐membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However, b₁⁺ ion is formed in case of δ,α‐dipeptide that may have a six‐membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di‐, tetra‐, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives using electrospray ionization multi‐stage mass spectrometry

N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro‐inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion‐trap multi‐stage mass spectrometry (MSⁿ). The MS² collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N‐Fmoc/Z‐amino acids, dipeptide and tripeptide acids show the [M + Na‐NH₂CHO]⁺ ion, arising from the loss of formamide, as the base peak. Differently, the MS² CID spectra of [M + Na]⁺ ion of all the N‐Boc derivatives yield the abundant [M + Na‐C₄H₈]⁺ and [M + Na‐Boc + H]⁺ ions because of the loss of isobutylene and CO₂ from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N‐protected dipeptidyl and tripeptidyl‐N′‐formamides is provided by MS² and subsequent MSⁿ experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M‐H]^?, [M + HCOO]^? and [M + Cl]^? ions, the presence of in‐source CID fragment ions deriving from the involvement of the N‐protecting group. Furthermore, MSⁿ spectra of [M + Cl]^? ion of N‐protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C‐terminal gem‐diamino residue. The present paper represents an initial attempt to study the ESI‐MS behavior of these important intermediates and lays the groundwork for structural‐based studies on more complex partially modified retro‐inverso peptides. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of an exception to the ‘even‐electron rule’ upon low‐energy collision induced decomposition in negative ion electrospray tandem mass spectrometry

Electrospray‐generated precursor ions usually follow the ‘even‐electron rule’ and yield ‘closed shell’ fragment ions. We characterize an exception to the ‘even‐electron rule.’ In negative ion electrospray mass spectrometry (ES‐MS), 2‐(ethoxymethoxy)‐3‐hydroxyphenol (2‐hydroxyl protected pyrogallol) easily formed a deprotonated molecular ion (M‐H)^? at m/z 183. Upon low‐energy collision induced decomposition (CID), the m/z 183 precursor yielded a radical ion at m/z 124 as the base peak. The radical anion at m/z 124 was still the major fragment at all tested collision energies between 0 and 50 eV (E_lab). Supported by computational studies, the appearance of the radical anion at m/z 124 as the major product ion can be attributed to the combination of a low reverse activation barrier and resonance stabilization of the product ions. Furthermore, our data lead to the proposal of a novel alternative radical formation pathway in the protection group removal of pyrogallol. Copyright © 2009 John Wiley & Sons, Ltd.     

Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision‐induced dissociation

Migration of sulfate groups between hydroxyl groups was identified after collision‐induced dissociation (CID) of sulfated oligosaccharides in an ion trap mass spectrometer in negative ion mode. Analysis of various sulfated oligosaccharides showed that this was a common phenomenon and was particularly prominent in sulfated oligosaccharides also containing sialic acid. It was also shown that the level of migration was increased when the sulfate was positioned on the flexible areas of the oligosaccharides not involved in the pyranose ring, such as the extra‐cyclic C‐6 carbon of hexoses or N‐acetylhexosamines, or on reduced oligosaccharide. This suggested that migration is dependent on the spatial availability of the sulfate in the ion trap during collision. It is proposed that the migration is initiated when the negatively charged ‐SO₃^– residue attached to the oligosaccharide precursor becomes protonated by a CID‐induced proton transfer. This is supported by the CID fragmentation of precursor ions depleted of acidic protons such as doubly charged [M – 2H]^2– ions or the sodiated [M + Na – 2H]^– ions of oligosaccharides containing one sulfate and one sialic acid in the same molecule. Compared to the CID fragmentation of their monocharged [M – H]^– ions, no migration was observed in CID of proton depleted precursors. Alternative fragmentation parameters to suppress migration of sulfated oligosaccharides also showed that it was not present when sulfated oligosaccharides were fragmented by HCD (High‐Energy C‐trap Dissociation) in an Orbitrap mass spectrometer. Copyright © 2011 John Wiley & Sons, Ltd.     

Polyol‐Metall‐Komplexe.47 Kristalline D‐Mannose‐Kupfer(II)‐Komplexe aus Fehlingscher Lösung

Polyol Metal Complexes.47¹⁾ Crystalline D ‐Mannose‐Copper Complexes from Fehling Solutions Blue, unstable crystals of K₃[Cu₅(β‐D ‐Manp)₄H_—13] · α‐D ‐Manp · 16.5 H₂O ( 1 ), which contain a pentanuclear cupric complex of the reducing sugar D ‐mannose in its β‐pyranose form (β‐D ‐Manp), have been obtained from ice‐cold aqueous alkaline solutions. The homoleptic pentacuprate contains bridging mannopyranose ligands, which are charged 4— and 2.5—. Addition of ethylenediamine (en) to such Fehling solutions yields N, N′‐Bis(β‐D ‐mannopyranosyl)‐ethylenediamine (L) as a condensation product of the diamine and mannopyranose. Crystals of [(en)₂Cu₇(β‐D ‐Manp1, 2, 3, 4H_—4)₂(L2, 3, 4H_—3)₂] · 26.6 H₂O ( 2 ) could be isolated. The heptanuclear cupric complex is a structural derivative of the homoleptic mannose complex.     

Viscosalines B1,2 and E1,2: Challenging New 3‐Alkyl Pyridinium Alkaloids from the Marine Sponge Haliclona viscosa

Four new 3‐alkyl pyridinium alkaloids, the viscosalines B₁ ( 1 a ), B₂ ( 1 b ), E₁ ( 2 a ), and E₂ ( 2 b ), were isolated from the Arctic sponge Haliclona viscosa. The structure elucidation of these isomeric compounds was challenging due to ambiguous fragments that derive during “standard” mass spectrometric fragmentation experiments. The final structure elucidation relied on the use of a combination of synthesis, liquid chromatography, and mass spectrometry. Three different mass spectrometers were used to differentiate between the synthetic structural isomers: a time‐of‐flight (TOF) mass spectrometer and two ion‐trap mass spectrometers with different ion‐transfer technologies (i.e., skimmer versus funnel optics). Although at first none of the spectrometers returned spectra that permitted structure elucidation, all three mass spectrometers provided analysis that successfully differentiated between the isomers after thorough method optimization. The use of in‐source collision‐induced dissociation (CID) with the ion trap and TOF instrument returned the most interesting results. The mode of fragmentation of the viscosalines under different experimental conditions is described herein. After successful optimization of the mass spectrometric method applied, the chromatographic method was improved to distinguish the previously inseparable isomers. Finally, both the liquid chromatography and mass spectrometric methods were applied to the natural products and the results compared to those from the synthetic compounds.     

CID of singly charged antioxidants applied in lubricants by means of a 3D ion trap and a linear ion trap-Orbitrap mass spectrometer

The aim of this study was to investigate the fragmentation behavior induced by low‐energy collision‐induced dissociation (LE‐CID) of four selected antioxidants applied in lubricants, by two different types of ion trap mass spectrometers: a three‐dimensional ion trap (3D‐IT) and a linear IT (LIT) Orbitrap MS. Two sterically hindered phenols and two aromatic amines were selected as model compounds representing different antioxidant classes and were characterized by positive‐ion electrospray ionization (ESI) and LE‐CID. Various types of molecular ions (e.g. [M]^+?, [M + H]⁺, [M + NH₄]⁺ or [M + Na]⁺) were used as precursor ions generating a significant number of structurally relevant product ions. Furthermore, the phenolic compounds were analyzed by negative‐ion ESI. For both IT types applied for fragmentation, the antioxidants exhibited the same unusual LE‐CID behavior: (1) they formed stable radical product ions and (2) C? C bond cleavages of aliphatic substituents were observed and their respective cleavage sites depended on the precursor ion selected. This fragmentation provided information on the type of structural isomer usually not obtainable for branched aliphatic substituents utilizing LE‐CID. Comparing the two instruments, the main benefit of applying the LIT‐Orbitrap was direct access to elemental composition of product ions enabling unambiguous interpretation of fragmentation trees not obtainable by the 3D‐IT device (e.g. loss of isobaric neutrals). It should be emphasized that the types of product ions formed do not depend on the type of IT analyzer applied. For characterizing degradation products of antioxidants, the LIT‐Orbitrap hybrid system, allowing the determination of accurate m/z values for product ions, is the method of choice. Copyright © 2011 John Wiley & Sons, Ltd.     

Isomer differentiation via collision‐induced dissociation: The case of protonated α‐, β2‐ and β3‐phenylalanines and their derivatives

A combination of electrospray ionisation (ESI), multistage and high‐resolution mass spectrometry experiments is used to examine the gas‐phase fragmentation reactions of the three isomeric phenylalanine derivatives, α‐phenylalanine, β²‐phenylalanine and β³‐phenylalanine. Under collision‐induced dissociation (CID) conditions, each of the protonated phenylalanine isomers fragmented differently, allowing for differentiation. For example, protonated β³‐phenylalanine fragments almost exclusively via the loss of NH₃, only β²‐phenylalanine via the loss of H₂O, while α‐ and β²‐phenylalanine fragment mainly via the combined losses of H₂O + CO. Density functional theory (DFT) calculations were performed to examine the competition between NH₃ loss and the combined losses of H₂O and CO for each of the protonated phenylalanine isomers. Three potential NH₃ loss pathways were studied: (i) an aryl‐assisted neighbouring group; (ii) 1,2 hydride migration; and (iii) neighbouring group participation by the carboxyl group. Finally, we have shown that isomer differentiation is also possible when CID is performed on the protonated methyl ester and methyl amide derivatives of α‐, β²‐ and β³‐phenylalanines. Copyright © 2010 John Wiley & Sons, Ltd.