Strategies for generating peptide radical cations via ion/ion reactions

Several approaches for the generation of peptide radical cations using ion/ion reactions coupled with either collision induced dissociation (CID) or ultraviolet photo dissociation (UVPD) are described here. Ion/ion reactions are used to generate electrostatic or covalent complexes comprised of a peptide and a radical reagent. The radical site of the reagent can be generated multiple ways. Reagents containing a carbon–iodine (C―I) bond are subjected to UVPD with 266‐nm photons, which selectively cleaves the C―I bond homolytically. Alternatively, reagents containing azo functionalities are collisionally activated to yield radical sites on either side of the azo group. Both of these methods generate an initial radical site on the reagent, which then abstracts a hydrogen from the peptide while the peptide and reagent are held together by either electrostatic interactions or a covalent linkage. These methods are demonstrated via ion/ion reactions between the model peptide RARARAA (doubly protonated) and various distonic anionic radical reagents. The radical site abstracts a hydrogen atom from the peptide, while the charge site abstracts a proton. The net result is the conversion of a doubly protonated peptide to a peptide radical cation. The peptide radical cations have been fragmented via CID and the resulting product ion mass spectra are compared to the control CID spectrum of the singly protonated, even‐electron species. This work is then extended to bradykinin, a more broadly studied peptide, for comparison with other radical peptide generation methods. The work presented here provides novel methods for generating peptide radical cations in the gas phase through ion/ion reaction complexes that do not require modification of the peptide in solution or generation of non‐covalent complexes in the electrospray process. Copyright © 2015 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.     

Ethylenediamine as a liquid chemical reagent to probe hydrogen bonding and host-guest interactions with crown ethers in an ion trap tandem mass spectrometer

Ethylenediamine (EDA) was used as a novel liquid chemical reagent to probe hydrogen bonding and host-guest interactions with crown ether derivatives in an ion trap mass spectrometer (ITMS). Selective ion/molecule reaction product ions were generated by reactions of EDA with oxygenated and aza-crown ethers. For the oxygenated crown ethers, glycols and dimethylglycols, ion/molecule reactions led to the formation of the protonated molecules ([M+H](+)) and adduct ions including [M+30](+), [M+44](+) and [M+61](+). The aza-crown ethers produced [M+H](+), [M+13](+) and [M+27](+) ions. Collisionally activated dissociation (CAD) experiments were applied to probe the binding strength of these ion/molecule reaction products. CAD results indicated that all these hydrogen-bonding complexes are weakly bound except for the [M+44](+) ion of 18-crown-6, since all the complexes dissociate to the protonated polyether and/or protonated EDA. Fragmentation of the [M+H](+) ions under CAD conditions indicates the extensive covalent bond cleavage of the protonated crown ether skeleton.     

Dissociation of disulfide‐intact somatostatin ions: the roles of ion type and dissociation method

The dissociation chemistry of somatostatin‐14 was examined using various tandem mass spectrometry techniques including low‐energy beam‐type and ion trap collision‐induced dissociation (CID) of protonated and deprotonated forms of the peptide, CID of peptide‐gold complexes, and electron transfer dissociation (ETD) of cations. Most of the sequence of somatostatin‐14 is present within a loop defined by the disulfide linkage between Cys‐3 and Cys‐14. The generation of readily interpretable sequence‐related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH₂? S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H]²⁺ species. The products were generated by a combination of S? S bond cleavage and amide bond cleavage. ETD of the [M+3H]³⁺ ion generated rich sequence information, as did CID of the electron transfer products that did not fragment directly upon electron transfer. The electron transfer results suggest that both the S? S bond and an N? C_α bond can be cleaved following a single electron transfer reaction. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Using collision-induced dissociation with corrections for the ion number of degrees of freedom for quick comparisons of relative bonding strength

The number of degrees of freedom-dependent stability of ions and ion-neutral non-covalent complexes under collision-induced dissociation (CID) conditions was studied in a quadrupole ion trap mass spectrometer. It was found that the stability of ions as probed by energy-variable CID has a linear dependence on the total number of degrees of freedom for the ions (or ion-neutral complexes) with the same (or nearly the same) bonding energy. The slope of such a stability vs number of degrees of freedom dependence correlates with the binding energy. Proton-bound amine dimers display the lowest slope as they have weak bonds. Breaking covalent bonds will result in much greater slopes. In addition to the binding energy, the vibrational frequencies of the ion also affect the stability vs number of degrees of freedom behavior. Studying such a dependence of the CID stability in a system paves the way for direct relative binding energy comparisons. The application of this approach is demonstrated by testing the relative heme affinities of anti-malaria drugs and related compounds.     

Stereospecific control of peptide gas‐phase ion chemistry with cis and trans cyclo ornithine residues

We report non‐chiral amino acid residues cis‐ and trans‐1,4‐diaminocyclohexane‐1‐carboxylic acid (cyclo‐ornithine, cO) that exhibit unprecedented stereospecific control of backbone dissociations of singly charged peptide cations and hydrogen‐rich cation radicals produced by electron‐transfer dissociation. Upon collision‐induced dissociation (CID) in the slow heating regime, peptide cations containing trans‐cO residues undergo facile backbone cleavages of amide bonds C‐terminal to trans‐cO. By contrast, peptides with cis‐cO residues undergo dissociations at several amide bonds along the peptide ion backbone. Diastereoisomeric cO‐containing peptides thus provide remarkably distinct tandem mass spectra. The stereospecific effect in CID of the trans‐cO residue is explained by syn‐facially directed proton transfer from the 4‐ammonium group at cO to the C‐terminal amide followed by neighboring group participation in the cleavage of the CO―NH bond, analogous to the aspartic acid and ornithine effects. Backbone dissociations of diastereoisomeric cO‐containing peptide ions generate distinct [b_n]⁺‐type fragment ions that were characterized by CID‐MS³ spectra. Stereospecific control is also reported for electron‐transfer dissociation of cis‐ and trans‐cO containing doubly charged peptide ions. The stereospecific effect upon electron transfer is related to the different conformations of doubly charged peptide ions that affect the electron attachment sites and ensuing N―C_α bond dissociations.     

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.     

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. Copyright © 2012 John Wiley & Sons, Ltd.     

Investigation of collision‐induced dissociations involving odd‐electron ion formation under positive electrospray ionization conditions using accurate mass

Collision induced dissociation (CID) has been extensively used for structure elucidation. CID in the electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) modes has been found to generate mostly even‐electron fragment ions while it has been occasionally reported to form odd‐electron free radical ions. However, the structural requirements and the fragmentation mechanisms for free‐radical CIDs have not been well characterized in the literature. For this purpose, we studied a series of aromatic and non‐aromatic compounds such as sulfonamides, N‐aryl amides, tert‐butyl‐substituted aromatic compounds, aryl alkyl ethers, and O‐alkyl aryl oximes using the LTQ? and LTQ Orbitrap? linear ion trap mass spectrometers. The accurate measurement of the fragment ion masses established the unambiguous assignment of the fragment structures resulting from the test compounds. Our results showed that free radical fragmentation is structure dependent and is to a large extent correlated with the neighboring groups in the structures that stabilize the newly formed free radical ions. Copyright © 2010 John Wiley & Sons, Ltd.     

Electrospray Ionization Tandem Mass Spectrometry of Ammonium Cationized Polyethers

Quaternary ammonium salts (Quats) and amines are known to facilitate the MS analysis of high molar mass polyethers by forming low charge state adduct ions. The formation, stability, and behavior upon collision-induced dissociation (CID) of adduct ions of polyethers with a variety of Quats and amines were studied by electrospray ionization quadrupole time-of-flight, quadrupole ion trap, and linear ion trap tandem mass spectrometry (MS/MS). The linear ion trap instrument was part of an Orbitrap hybrid mass spectrometer that allowed accurate mass MS/MS measurements. The Quats and amines studied were of different degree of substitution, structure, and size. The stability of the adduct ions was related to the structure of the cation, especially the amine’s degree of substitution. CID of singly/doubly charged primary and tertiary ammonium cationized polymers resulted in the neutral loss of the amine followed by fragmentation of the protonated product ions. The latter reveals information about the monomer unit, polymer sequence, and endgroup structure. In addition, the detection of product ions retaining the ammonium ion was observed. The predominant process in the CID of singly charged quaternary ammonium cationized polymers was cation detachment, whereas their doubly charged adduct ions provided the same information as the primary and tertiary ammonium cationized adduct ions. This study shows the potential of specific amines as tools for the structural elucidation of high molar mass polyethers.     

Increased sequence coverage of thymine‐rich oligodeoxynucleotides by infrared multiphoton dissociation compared to collision‐induced dissociation

Infrared multiphoton dissociation (IRMPD) of thymine‐rich oligodeoxynucleotides in a linear ion‐trap mass spectrometer affords far more extensive fragmentation than conventional collision‐induced dissociation (CID). For oligodeoxynucleotides containing one non‐thymine base, CID results primarily in cleavage on the 3′ side of the non‐thymine nucleobase, whereas IRMPD results in cleavages between all the nucleobases and thus provides complete sequence coverage. Furthermore, for oligodeoxynucleotides containing a single non‐thymine base, it is shown that the full series of diagnostic sequence ions observed in the IRMPD mass spectra arise from secondary dissociation of the two primary products formed from the initial cleavage site located next to the non‐thymine base. Copyright © 2010 John Wiley & Sons, Ltd.     

Ion/molecule reactions in a miniature RIT mass spectrometer

Ion/molecule reactions were explored in a newly developed miniature mass spectrometer fitted with a rectilinear ion trap (RIT) mass analyzer. The tandem mass spectrometry performance of this instrument is demonstrated using collision induced dissociation (CID) and ion/molecule reactions. The latter includes Eberlin transacetalization reactions and electrophilic additions. Selective detection of the chemical warfare simulant dimethyl methyl phosphonate (DMMP) was achieved through selective Eberlin reactions of its characteristic phosphonium fragment ion CH3OP(+)(O)CH3 (m/z 93), with 1,4-dioxane or 1,3-dioxolane. Efficient adduct formation as a result of electrophilic attack by the phosphonium ion on various nucleophilic reagents, including 1,1,3,3-tetramethyl urea, methanesulfonic acid methyl ester, dimethyl sulfoxide and methyl salicylate, was also observed using the RIT device. The product ions of these reactions were analyzed using CID and the characteristic fragmentation patterns of the ionic addition products were recorded using multiple-stage experiments in the miniature RIT instrument. This study clearly demonstrates that a small, home-built, miniature RIT mass spectrometer can be used to perform analytically useful ion/molecule reactions and also that instruments like this have the potential to provide a portable platform for in situ detection of organophosphorus esters and related compounds with high specificity using tandem mass spectrometry.     

Tautomerization in gas-phase ion chemistry of isomeric C-8 deoxyguanosine adducts from phenol-induced DNA damage

Collision-induced dissociation (CID) of 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine was investigated using sequential tandem mass spectrometry. These adducts represent biomarkers of DNA damage linked to phenolic radicals and were investigated to gain insight into the effects of chemical structure of a C-8 modification on fragmentation pathways of modified 2'-deoxyguanosine (dG). CID in MS(2) of the deprotonated molecules of both the isomers generated the same product ion having the same m/z values. CID in MS(3) of the product ion at m/z 242 and CID in MS(4) experiments carried out on the selected product ions at m/z 225 and m/z 218 afford distinct fragmentation patterns. The conformational properties of isomeric product ions from CID showed that the ortho-isomers possess the unique ability to tautomerize through an intramolecular proton transfer between the phenolic OH group and the imine nitrogen (N7). Tautomerization of ortho-isomers to their keto-tautomers led to differences in their system of conjugated double bonds compared with either their enol-tautomer or the para-isomer. The charge redistribution through the N-7 site on the imidazole ring is a critical step in guanosine adduct fragmentation which is disrupted by the formation of the keto-tautomer. For this reason, different reaction pathways are observed for 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine. We present herein the dissociation and the gas-phase ion-molecule reactions for highly conjugated ions involved in the CID ion chemistry of the investigated adducts. These will be useful for those using tandem mass spectrometry for structural elucidation of C-8 modified dG adducts. This study demonstrates that the modification at the C-8 site of dG has the potential to significantly alter the reactivity of adducts. We also show the ability of tandem mass spectrometry to completely differentiate between the isomeric dG adducts investigated.     

Energetics and efficiencies of collision‐induced dissociation achieved during the mass acquisition scan in a quadrupole ion trap

Using n‐butylbenzene as a test molecule, evidence is provided that fast, efficient or highly energetic collision‐induced dissociation (CID) can be achieved during the mass acquisition ramp of a commercially available quadrupole ion trap (QIT) mass spectrometer. The method of excitation is very similar to axial modulation for mass range extension except that lower amplitude waveforms are used to excite the precursor ions within the trap instead of ejecting them from the trap. ITSIM simulations verify that fast kinetic excitation followed by kinetic‐to‐internal energy transfer occurs on the rapid time‐scale required for the recapture and mass analysis of product ions during the mass acquisition ramp. CID efficiencies larger than 50% can be obtained using this new approach and ratios of Th 91/92 of n‐butylbenzene fragment ions as large as 9 are possible, albeit at significantly reduced efficiencies. These very large ratios indicate an internal energy above 7 eV for the precursor ions indicating that fragmentation of larger ions could also be possible using this new approach. The main benefits of the new method are that no extra time is required for fragmentation or cooling and that on‐resonance conditions are guaranteed because the ions' secular frequencies are swept through the fixed frequency of excitation. Also presented are the effects of experimental variables such as excitation frequency, excitation amplitude and scan rate on the CID efficiencies and energetics. Copyright © 2005 John Wiley & Sons, Ltd.     

Electrophilic aromatic substitution and single-electron transfer (SET) by the phenylium ion in the gas phase: characterization of a long-lived SET intermediate

Gas-phase mass spectrometric studies and calculations were performed for the reaction of naked phenylium ion with several benzene halides. From these reactions, the molecular ion for biphenyl as the predominant product was obtained only from the reaction of phenylium ions with iodobenzene and bromobenzene. Furthermore, through the collision-induced dissociation (CID) of the ion at m/z 281, the only dissociation observed is the loss of a phenyl radical, which indicates that a single-electron transfer (SET) mechanism might have occurred within the reaction. Additionally, according to the comparison between the CID experiments of those isomeric compounds of the sigma-complexes and the CID experiment of the ion at m/z 281 captured in the ion trap, we have also defined the captured ion at m/z 281 as an SET-intimate ion pair rather than those of sigma-complexes or the diphenyliodonium.     

Gas phase ion–molecule reactions in a modified ion trap: H/D exchange of non-covalent complexes and coordinatively unsaturated platinum complexes

A commercially available electrospray ionization ion trap mass spectrometer has been modified to carry out gas phase ion–molecule reactions. The ability to study gas phase ion–molecule reactions in conjunction with collision induced dissociation (CID) based methods and the multistage trapping capabilities of the ion trap have been exploited in two ways: (i) gas phase H/D exchange reactions inside the ion trap, coupled with CID tandem mass spectrometry have been used to provide insights into the reactivity of non covalent complexes of amino acids and simple peptides, and (ii) CID prior to performing ion–molecule reactions has been used to synthesize and examine the reactivity of coordinatively unsaturated platinum complexes. © 1998 John Wiley & Sons, Ltd.     

High amplitude short time excitation: A method to form and detect low mass product ions in a quadrupole ion trap mass spectrometer

Collision induced dissociation (CID) in a quadrupole ion trap mass spectrometer using the conventional 30 ms activation time is compared with high amplitude short time excitation (HASTE) CID using 2 ms and 1 ms activation times. As a result of the shorter activation times, dissociation of the parent ions using the HASTE CID technique requires resonance excitation voltages greater than conventional CID. After activation, the rf trapping voltage is lowered to allow product ions below the low mass cut-off to be trapped. The HASTE CID spectra are notably different from those obtained using conventional CID and can include product ions below the low mass cut-off for the parent ions of interest. The MS/MS efficiencies of HASTE CID are not significantly different when compared with the conventional 30 ms CID. Similar results were obtained with a two-dimensional (linear) ion trap and a three-dimensional ion trap.     

Infrared ion spectroscopy inside a mass‐selective cryogenic 2D linear ion trap

We demonstrate operation of the first cryogenic 2D linear ion trap (LIT) with mass‐selective capabilities. This trap presents a number of advantages for infrared ion “action” spectroscopy studies, particularly those employing the “tagging/messenger” spectroscopy approach. The high trapping efficiencies, trapping capacities, and low detection limits make 2D LITs a highly suitable choice for low‐concentration analytes from scarce biological samples. In our trap, ions can be cooled down to cryogenic temperatures to achieve higher‐resolution infrared spectra, and individual ions can be mass selected prior to irradiation for a background‐free photodissociation scheme. Conveniently, multiple tagged analyte ions can be mass isolated and efficiently irradiated in the same experiment, allowing their infrared spectra to be recorded in parallel. This multiplexed approach is critical in terms of increasing the duty cycle of infrared ion spectroscopy, which is currently a key weakness of the technique. The compact design of this instrument, coupled with powerful mass selection capabilities, set the stage for making cryogenic infrared ion spectroscopy viable as a bioanalytical tool in small molecule identification.     

L‐Valine assisted distinction between the stereo‐isomers of D‐hexoses by positive ion ESI tandem mass spectrometry

The binary mixtures of 7 hexoses and 20 amino acids were investigated by electrospray ionization ion trap mass spectrometry (ESI‐ITMS). The adduct ions of the amino acid and the hexose were detected for 12 amino acids but not for the other 8 amino acids which are basic acidic amino acids and amides. The ions of amino acid–hexose complexes were further investigated by tandem mass spectrometry (MS/MS), and some of them just split easily into two parts whereas the others gave rich fragmentation, such as the complex ions of isoleucine, phenylalanie, tyrosine, and valine. We found that hexoses could be complexed by two molecules of valine but only by one molecule of the other amino acids. Among seven kinds of valine–hexose complexes coordinated by potassium ion, the MS² spectra of the ion at m/z 453 yielded unambiguous differentiation. And the fragmentation ions are sensitive to the stereochemical differences at the carbon‐4 of hexoses in the complexes, as proved by the MS². Copyright © 2010 John Wiley & Sons, Ltd.