Ionization and collision-induced fragmentation of N-linked and related carbohydrates using divalent cations

Maltoheptaose and several N-linked glycans were ionized by electrospray as adducts with the divalent cations Mg2+, Ca2+, Mn2+, Co2+ and Cu2+. [M + metal]2+ ions were the major species in all cases with calcium giving the highest sensitivity. In addition, copper gave [M + Cu]+ ions. Other cations gave singly charged ions only by elimination of a protonated monosaccharide. Fragmentation of the [M + metal]2+ ions produced both singly and doubly charged ions with the relative abundance of doubly charged ions decreasing in the order Ca > Mg > Mn > Co > Cu. Singly charged ions were formed by elimination of a protonated monosaccharide residue followed, either by successive monosaccharide residue losses, or by a 2,4A cross-ring cleavage of the reducing-terminal monosaccharide. Formation of doubly charged fragments from [M + metal]2+ ions involved successive monosaccharide-residue losses either with or without O,2A or 2,4A cross-ring cleavages of the reducing-terminal monosaccharide. Abundant diagnostic doubly charged ions formed by loss of the 3-antenna from the O,2A cross-ring product were specific to [M + Ca]2+ ions. Fragmentation of [M + Cu]+ ions was similar to that of the corresponding [M + H]+ ions in that most cross-ring fragments were absent.     

Speciation of cyclo(Pro-Gly)<Subscript>3</Subscript> and its divalent metal-ion complexes by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to study the binding of selected group II and divalent transition-metal ions by cyclo(Pro-Gly)3 (CPG3), a model ion carrier peptide. Metal salts (CatXn) were combined with the peptide (M) at a molar ratio of 1:10 M/Cat in aqueous solvents containing 50% vol/vol acetonitrile or methanol and 1 or 10 mM ammonium acetate (NH4Ac). Species detected include [M+H]+, [M+Cat-H]+, [M2+Cat]2+, [M+Cat+Ac]+, and [M+Cat+X]+. The relative stabilities of complexes formed with different cations (Mg2+, Ca2+, Sr2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+) were determined from the abundance of 1:1 and 2:1 M/Cat species relative to that of the unbound peptide. The largest metal ions (Ca2+, Sr2+, and Mn2+) formed the most stable complexes. Reducing the buffer concentration increased the overall extent of metal binding. Results show that the binding specificity of CPG3 depends upon the size of the metal ion and its propensity for electrostatic interaction with oxygen atoms. Product ion tandem mass spectrometry of [M+H]+ and [M+Cu-H]+ confirmed the cyclic structure of the peptide, although the initial site(s) of metal attachment could not be determined.     

An application of electrospray ionization tandem mass spectrometry to probe the interaction of Ca2+/Mg2+/Zn2+ and Cl- with gramicidin A

The peptide, gramicidin A (GrA), has been demonstrated to interact with divalent salts (CaCl2, MgCl2, and ZnCl2) using electrospray ionization mass spectrometry (ESI-MS). The ESI-MS analysis revealed different complexes formed due to the interaction of Val-GrA and Ile-GrA with divalent salts: [Val or Ile-GrA-H+M]+, [Val or Ile-GrA+MCl]+ and [Val or Ile-GrA+M]2+, where M is Ca or Mg or Zn. All these complexes have been subjected to collisionally activated dissociation (CAD). CAD of singly and doubly charged GrA and metal complexes exhibited the losses of water molecules, indicating the ligand preference of GrA. MS/MS and MS3 of [Val or Ile-GrA+MCl]+ resulted in the elimination of chloride ion and water, respectively. The tandem mass spectrometry data of the complex [Val-GrA+MCl]+ suggest that chloride interaction is stronger in the presence of Ca than of Mg and Zn. This study reveals that GrA could interact with Ca, Mg, and Zn in metal ion form as well as in ion pair (MCl) form. The interactions of GrA with Ca support the proposal of a physical basis for the messenger role of Ca (Urry et al., J. Biol. Chem. 1982, 257: 6659-6661).     

Comparison of intensities between doubly charged ions [M + 2H]2+ and singly charged ions [M + H]+ of gramicidin S by electrospray mass spectrometry.

The reason why the intensity of doubly charged ions [M + 2H]2+ of gramicidin S is higher than that of singly charged ions [M + H]+ in electrospray is investigated by ion evaporation theory. As a result of comparison between the total free energies of extracting [M + 2H]2+ and [M + H]+ from a charged droplet to infinity, it is found that the total free energy of [M + 2H]2+ is estimated to be lower than that of [M + H]+. This clearly supports the experimental result. In addition, the importance of the electrostatic contribution in electrospray is demonstrated by showing the result that the total free energy of [M + 2H]2+ without electrostatic contribution is higher than that of [M + H]+.     

Generation and collision-induced dissociation of ammonium tetrafluoroborate cluster ions

Singly and doubly charged cluster ions of ammonium tetrafluoroborate (NH4BF4) with general formula [(NH4BF4)nNH4]+ and [(NH4BF4)m(NH4)2]2+, respectively, were generated by electrospray ionization (ESI) and their fragmentation examined using collision-induced dissociation (CID) and ion-trap tandem mass spectrometry. CID of [(NH4BF4)nNH4]+ caused the loss of one or more neutral NH4BF4 units. The n = 2 cluster, [(NH4BF4)2NH4]+, was unique in that it also exhibited a dissociation pathway in which HBF4 was eliminated to create [(NH4BF4)(NH3)NH4]+. Dissociation of [(NH4BF4)m(NH4)2]2+ occurred through two general pathways: (a) 'fission' to produce singly charged cluster ions and (b) elimination of one or more neutral NH4BF4 units to leave doubly charged product ions. CID profiles, and measurements of changing precursor and product ion signal intensity as a function of applied collision voltage, were collected for [(NH4BF4)nNH4]+ and compared with those for analogous [(NaBF4)nNa]+ and [(KBF4)nK]+ ions to determine the influence of the cation on the relative stability of cluster ions. In general, the [(NH4BF4)nNH4]+ clusters were found to be easier to dissociate than both the sodium and potassium clusters of comparable size, with [(KBF4)nK]+ ions the most difficult to dissociate.     

Characterization of peptides by liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a post-column complexant

Two model peptides, des-Arg1-bradykinin (DAB) and bradykinin (B), were cationized by Ag+ after their separation by reversed-phase liquid chromatography (RPLC) prior to mass spectrometry (MS). Silver nitrate solution was used as a post-column reagent. The RPLC and MS experimental conditions were optimized using flow injection in order to obtain sufficiently abundant silver adducts to permit MS/MS experiments. The use of water-methanol with 0.1% formic acid as mobile phase allowed a good chromatographic separation of the two peptides with a polymeric stationary phase and sufficiently abundant silver-containing adducts, [M + Ag + H]2+ and [M + 2Ag]2+. The gas-phase dissociation of [DAB + Ag + H]2+ and [DAB + 2Ag]2+ led to interpretable mass spectra during the on-line cationization experiment. Most of the ions obtained by dissociating [DAB + Ag + H]2+ and [DAB + 2Ag]2+ species are silver-containing ions but the ions produced depend on the parent. The ions coming from the dissociation of the doubly charged silver adducts [DAB + Ag + H]2+ or [DAB + 2Ag]2+ are of interest compared with those coming from the singly charged silver species or doubly charged protonated species. The fragmentation of the doubly charged silver adducts provides ions over the entire mass range. Although the presence of several prolines in des-Arg1-bradykinin prevents the formation of some expected ions, the observation of triplets [an-H + Ag]+, [bn-H + Ag]+ and [bn + OH + Ag]+ produced by the dissociation of on-line Ag(+)-cationized peptides could contribute to greater success of automatic sequencing of peptides.     

Mobile phase variations in thermospray liquid chromatography-mass spectrometry of pesticides

The effect of four different mobile phase compositions with reversed-phase methanol-water (50:50) + 0.05 M ammonium acetate, methanol-water (50:50) + 0.05 M ammonium formate, acetonitrile-water (50:50) + 0.05 M ammonium acetate and acetonitrile-water (50:50) + 0.05 M ammonium formate were compared in filament-on thermospray liquid chromatography-mass spectrometry for the determination of carbamate and chlorotriazine pesticides. In the positive-ion mode, [M + H]+ and [M + NH4]+ were generally the base peaks for the chlorotriazines and the carbamates, respectively. Depending on the mobile phase used, other adduct ions obtained corresponded to [M + CH3CN + H]+, [M + CH3OH + NH4]+, [M + CH3COONH4 + NH4 - 2H2O]+, [M + CH3CN + NH4]+, [M + CH3COONH4 + H - H2O]+ and the dimer [2M + H]+. In the negative-ion mode, [M - H]- and adducts with the ionizing additive [M + CH3COO]- or [M + HCOO]- were obtained. Other ions for the carbamates carbaryl and oxamyl corresponded to [M - CONHCH3 + CH3COOH]- and [M - CON(CH3)2 + HCOO]-, respectively. The variation of mobile phase composition provides additional structural information in thermospray liquid chromatography-mass spectrometry with no appreciable loss of sensitivity. Applications are reported for the determination of carbamate and chlorotriazine pesticides at the ng/g level in spiked and real soil samples, respectively.     

Tandem mass spectra of transition-metal ion adducts of glycosyl dithioacetals; distinction among stereoisomers

Electrospray ionization mass spectra of some glycosyl dithioacetals recorded in the presence of transition-metal chlorides, XCl2 (where X = Co, Mn and Zn), give abundant adduct ions such as [M+XCl]+ and [2M-H+X]+ and minor ions such as [M-H+X]+ and [2M+XCl]+. The tandem mass spectra of these adducts show characteristic elimination of neutral molecules such as H2O, HCl, EtSH, CH2O, C2H4O2/C2H4O. [M+XCl]+ ions fragment readily and the fragmentation appears to be stereochemically controlled as the relative abundances of the fragments are different for three stereoisomers. The added metal is lost as neutral molecules in the form of XCl(OH) and XCl(SEt). This is a predominant pathway in the ZnCl+ adducts. [2M+XCl]+ ions fragment preferentially by elimination of HCl, indicating strong metal interactions in the resulting dimeric [2M-H+X]+ ion. As there are several electron-rich centers in the molecule, the dimeric complex [2M-H+X]+ can have several structures and the observed fragmentations may reflect the sum of those of all these structures. The dimeric complexes fragment by elimination of neutral molecules leaving the dimeric interactions intact. The extent of fragmentation varies for the stereoisomers, leading to stereochemical differentiation.     

Electrospray ionization studies of transition-metal complexes of 2-acetylbenzimidazolethiosemicarbazone using collision-induced dissociation and ion-molecule reactions

The complexes of transition-metal ions (M2+, where M = Fe, Co, Ni, Cu, Zn, Cd, and Hg) with 2-acetylbenzimidazolethiosemicarbazone (L) are studied under electrospray ionization (ESI) conditions. The ESI mass spectra of Fe and Co complexes showed the complex ions corresponding to [M+2L-2H]+, and those of Ni and Zn complexes showed [M+2L-H]+ ions, wherein the metal/ligand ratio is 1:2 and the oxidation state of the central metal ion is +3 in the case of Fe and Co and +2 in the case of Ni and Zn. The Cd and Cu complexes showed preferentially 1:1 complex ions, i.e., [M+L-H]+ or [M+L+Cl]+, whereas Hg formed both 1:1 and 1:2 complex ions. During formation of the above complex ions one or two ligands are deprotonated after keto-enol tautomerism, depending on the nature and oxidation state of central metal ion. The structures and coordination numbers of the metal ions in the complex ions were studied by their collision-induced dissociation spectra and ion-molecule reactions with acetonitrile or propylamine in the collision cell. Based on these results it is concluded that Fe, Co, Ni and Zn form stable octahedral complexes, whereas tetrahedral or square planar complexes are formed preferentially for other metals. In addition, the Cu complex showed a [2L+2Cu-3H]+ ion with a Cu-Cu bond.     

2-rhodaoxetanes: their formation of oxidation of [RhI(ethene)]+ and their reactivity upon protonation

New cationic, pentacoordinate complexes [(TPA)Rh1(ethene)]+, [1a]+, and [(MeTPA)Rh1(ethene)]+, [1b]+, have been prepared (TPA = N,N,N-tri(2-pyridylmethyl)amine, MeTPA = N-[(6-methyl-2-pyridyl)-methyl]-N,N-di(2-pyridylmethyl)amine). Complex [1a]+ is selectively converted by aqueous HCl to [(TPA)RhIII-(ethyl)Cl]+, [2a]+. The same reaction with [1b]+ results in the [(MeTPA)RhIII-(ethyl)Cl]+ isomers [2b]+ and [2c]+. Treatment of [1a]+ and [1b]+ with aqueous H2O2 results in a selective oxygenation to the unsubstituted 2-rho-da(III)oxetanes (1-oxa-2-rhoda(III)cyclo-butanes) [(TPA)RhIII(kappa2-C,O-2-oxyethyl)]+, [3a]+, and [(MeTPA)RhIII(kappa2-C,O-2-oxyethyl)]+, [3b]+. The reactivity of 2-rhodaoxetanes [3a]+ and [3b]+ is dominated by the nucleophilic character of their 2-oxyethyl oxygen. Reaction of [3a]+ and [3b]+ with the non-coordinating acid HBAr(f)4 results in the dicationic protonated 2-rhodaoxetanes [(TPA)RhIII(kappa2-2-hydroxyethyl)]2+, [4a]2+, and [(MeTPA)RhIII(kappa2-2-hydroxyethyl)]2+, [4b]2+. These eliminate acetaldehyde at room temperature, probably via a coordinatively unsaturated kappa1-2-hydroxyethyl complex. In acetonitrile, complex [4a]2+ is stabilised as [(TPA)-RhIII(kappa1-2-hydroxyethyl)(MeCN)]2+, [5a]2+, whereas the MeTPA analogue [4b]2+ continues to eliminate acetaldehyde. Reaction of [3a]+ with NH4Cl and Mel results in the coordinatively saturated complexes [(TPA)RhIII(kappa1-2-hydroxyethyl)(Cl)]+, [6a]+, and [(TPA)-RhIII(kappa1-2-methoxyethyl)(I)+, [7a]+, respectively. Reaction of [3a]+ with NH4+ in MeCN results in formation of the dicationic metallacyclic amide [(TPA)-RhIII [kappa2-O,C-2-(acetylamino)ethyl]]2+, [9]2+, via the intermediates [4a]2+, [5a]2+ and the metallacyclic iminoester [(TPA)RhIII[kappa2-N,C-2-(acetimidoyloxy)ethyl]]2+, [8]2+. The observed overall conversion of the [Rh(I)(ethene)] complex [1a]+ to the metallacyclic amide [9]2+ via 2-rhodaoxetane [3a]+, provides a new route for the amidation of a [RhI(ethene)] fragment.     

Gas-phase formation of radical cations of monomers and dimers of guanosine by collision-induced dissociation of Cu(II)-guanosine complexes

An electrosprayed water/methanol solution of guanosine and Cu(NO3)2 was observed to give rise to gas-phase copper complexed ions of [CuLn]*2+, [CuL(MeOH)n]*2+, and [CuG n(NO3)]*+, as well as the ions [L]*+, [L+H]+, [G]*+, and [G+H]+ (L=guanosine, G=guanine). The Collision-Induced Dissociation (CID) of [CuL3]*2+ and [CuL(MeOH)n]*2+ (n=2, 3) generates guanosine radical cations [L]*+, while dimeric guanosine radical cations [L2]*+ are generated in the dissociation of [CuL4]*2+. Protonated guanosine [L+H]+ is one of the main products in the primary dissociation of [CuL2]*2+, while the dissociation of the higher-order [CuG2]*2+ produces the [G]*+ radical cation. The guanosine dimer radical cation, [L2]*+ presumably arises from the interaction of two guanosine molecules via proton and hydrogen bonding and is observed to dissociate into [L+H]+ and [L-H]* at low energies. We propose that the first two ligands bind strongly with Cu(II) through N7 and O6 to form a [CuL2]*2+ complex with a four-coordinated planar structure and that a third ligand binds loosely with copper to form [CuL3]*2+. Additional ligation observed in the formation of [CuLn]*2+ (n相似文献   

Mass spectral signature for insect adipokinetic hormones

Insect adipokinetic hormones (AKHs) are structurally similar. They consist of 8 to 10 amino acid residues, and are post-translationally modified at the N-terminus (pyroglutamic acid) and at the C-terminus (carboxyamide). They contain aromatic amino acids at position 4 (mostly Phe, in a few cases Tyr) and at position 8 (Trp). Position 9 is always Gly which is used in the octapeptides for the amidation, and the majority of the peptides have no charge. AKHs exhibit a characteristic ion signature both in matrix-assisted laser desorption/ionization (ion pair [M+Na](+)/[M+K]+) and in electrospray mass spectrometry ([M+H+K]2+, [M-17+H]+, [M+H]+, [M+Na]+, [M+K]+). Their high affinity for Na+ and K+ alkali cations is observed even after reversed-phase purification. AKHs rarely form doubly charged ions with protons or sodium while the [M+H+K]2+ ion is often abundant suggesting a special conformation of the larger metal ion complex possibly related to its size. Here, we present analyses of several AKHs of different insect species and discuss their ionization behavior with respect to their sequence. The mass spectral signature observed is useful for AKH detection from mixtures and so an unassigned 990.7 Da molecule was found in dragonfly which is currently under investigation.     

Establishment of mass spectrometric fingerprints of novel synthetic cholesteryl neoglycolipids: The presence of a unique <Emphasis Type="Italic">C</Emphasis>-glycoside species during electrospray ionization and during collision-induced dissociation tandem mass spectrometry

In this study we evaluated the fragmentation pattern of 16 novel amphiphilic neoglycolipid cholesteryl derivatives that can be efficiently used to increase cationic liposomal stability and to enhance gene transfer ability. These neoglycolipids bear different sugar moieties, such as D-glucosamine, N-acetyl-D-glucosamine, N-trideuterioacetyl-D-glucosamine, N-acetyllactosamine, L-fucose, N-allyloxycarbonyl-D-glucosamine, and some of their per-O-acetylated derivatives. Regardless of the structure of the tested neoglycolipid, QqToF-MS analysis using electrospray ionization (ESI) source showed abundant protonated [M+H]+ species. We also identified by both QqToF-MS and low-energy collision tandem mass spectrometry (CID-MS/MS) of the [M+H]+ ion, the presence of specific common fingerprint fragment ions: [Cholestene]+, sugar [oxonium]+, [(Sugar-spacer-OH)+H]+, [oxonium-H2O]+, and [(Cholesterol-spacer-OH)+H]+. In addition, we observed a unique ion that could not be rationally explained by the expected fragmentation of these amphiphilic molecules. The structure of this ion was tentatively proposed with that of a C-glycoside species formed by a chemical reaction between the sugar portion and the cholesterol. MS/MS analysis of this unique [C-glycoside]+ confirmed the validity of the proposed structure of this ion. The presence of an amino group at position C-2 and free hydroxyl groups of the sugar motif is crucial for the formation of a "reactive" sugar oxonium ion that can form the [C-glycoside]+ species. In summary, we precisely established the fragmentation patterns of the tested series of neoglycolipid cholesteryl derivatives and authenticated their structure as well; moreover, we speculated on the formation of a C-glycoside with the ESI source under atmospheric pressure and in the collision cell during MS/MS analysis.     

Comparative studies of 193-nm photodissociation and TOF-TOFMS analysis of bradykinin analogues: The effects of charge site(s) and fragmentation timescales

The dissociation reactions of [M + H]+, [M + Na]+, and [M + Cu]+ ions of bradykinin (amino acid sequence RPPGFSPFR) and three bradykinin analogues (RPPGF, RPPGFSPF, PPGFSPFR) are examined by using 193-nm photodissociation and post-source decay (PSD) TOF-TOF-MS techniques. The photodissociation apparatus is equipped with a biased activation cell, which allows us to detect fragment ions that are formed by dissociation of short-lived (<1 mus) photo-excited ions. In our previously reported photodissociation studies, the fragment ions were formed from ions dissociating with lifetimes that exceeded 10 mus; thus these earlier photofragment ion spectra and post-source decay (PSD) spectra [composite of both metastable ion (MI) and collision-induced dissociation (CID)] were quite similar. On the other hand, short-lived photo-excited ions dissociate by simple bond cleavage reactions and other high-energy dissociation channels. We also show that product ion types and abundances vary with the location of the charge on the peptide ion. For example, H+ and Na+ cations can bind to multiple polar functional groups (basic amino acid side chains) of the peptide, whereas Cu+ ions preferentially bind to the guanidino group of the arginine side-chain and the N-terminal amine group. Furthermore, when Cu+ is the charge carrier, the abundances of non-sequence informative ions, especially loss of small neutral molecules (H2O and NH3) is decreased for both photofragment ion and PSD spectra relative to that observed for [M + H]+ and [M + Na]+ peptide ions.     

Structural determination of saponins extracted from starfish by fast atom bombardment collision-induced dissociation mass spectrometry.

Three saponins were extracted and isolated from starfish by reversed-phase high performance liquid chromatography (HPLC), and analyzed by fast atom bombardment mass spectrometry (FAB-MS). Their molecular weight information could be obtained by the presence of abundant [M+Na]+ ions and weak [M+H]+ ions in FAB-MS spectra. Moreover, high resolution mass measurements of their [M+Na]+ ions were performed at the resolution of 10000 to elucidate the element composition of extracted saponins. The collision-induced dissociation (CID) of sodium-adducted molecules [M+Na]+ yielded diverse product ions via dissociated processes. In the collision-induced dissociation (CID)-MS/MS analysis of [M+Na]+ ion, the sulfate-containing saponins produced characteristic ions such as SO4Na+, [NaHSO4+Na]+, [M+Na-sugar]+ and [M+Na-2sugar]+ ions, whereas the sulfate-free compound showed characteristic ions produced by cleavage of sugar moiety and side chain of aglycone. The fragmentation patterns could provide information on the linkage position of sugar groups in aglycone and sulfate groups.     

刺五加寡糖的电喷雾多级串联质谱研究

采用小柱层析法从刺五加中分离得到刺五加寡糖类系列化合物(刺五加二糖刺五加六糖).实验结果表明,在正离子模式下的ESI-MS谱中,此类化合物呈现出特征的加合离子峰簇[M+Na]⁺/[M+K]⁺或[M+H₂O+Na]⁺/[M+H₂O+K]⁺,可以确定其分子量;在负离子模式下的ESI-MS谱中,刺五加寡糖易形成[M-H]^-/[M+nH₂O-H]^-(n<3).还利用电喷雾多级串联质谱(ESI-MSⁿ)对刺五加三糖进行了系统的研究,推断出刺五加三糖的组成与结构.     

Tandem electrospray mass spectrometric studies of proton and sodium ion adducts of neutral peptides with modified N- and C-termini: synthetic model peptides and microheterogeneous peptaibol antibiotics

The fragmentations of [M+H]+ and [M+Na]+ adducts of neutral peptides with blocked N- and C-termini have been investigated using electrospray ion trap mass spectrometry. The N-termini of these synthetically designed peptides are blocked with a tertiarybutyloxycarbonyl (Boc) group, and the C-termini are esterified. These peptides do not possess side chains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage patterns of the protonated peptides are strikingly different from those of sodium ion adducts. While the loss of the N-terminal blocking group occurs quite readily in the case of MS/MS of [M+Na]+, the cleavage of the C-terminal methoxy group seems to be a facile process in the case of MS/MS of [M+H]+ * Fragmentation of the protonated adducts yields only bn ions, while yn and a(n) ions are predominantly formed from the fragmentation of sodium ion adducts. The a(n) ions arising from the fragmentation of [M+Na](+) lack the N-terminal Boc group (and are here termed a(n)* ions). MS/MS of [M+Na]+ species also yields b(n) ions of substantially lower intensities that lack the N-terminal Boc group (b(n)*). A similar distinction between the fragmentation patterns of proton and sodium ion adducts is observed in the case of peptides possessing an N-terminal acetyl group. An example of the fragmentation of the H+ and Na+ adducts of a naturally occurring peptaibol from a Trichoderma species confirms that fragmentation of these two ionized species yields complementary information, useful in sequencing natural peptides. Inspection of the isotopic pattern of b(n) ions derived from [M+H]+ adducts of peptaibols provided insights into the sequences of microheterogeneous samples. This study reveals that the combined use of protonated and sodium ion adducts should prove useful in de novo sequencing of peptides, particularly of naturally occurring neutral peptides with modified N- and C-termini, for example, peptaibols.     

Analysis of neutral oligosaccharides for structural characterization by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

We have acquired multi-stage mass spectra (MSn) of four branched N-glycans derived from human serum IgG by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF-MS) in order to demonstrate high sensitivity structural analysis. [M+H]+ and [M+Na]+ ions were detected in the positive mode. The detection limit of [M+Na]+ in MS/MS and MS3 measurements for structural analysis was found to be 100 fmol, better than that for [M+H]+. The [M+H]+ ions subsequently fragmented to produce predominantly a Y series of fragments, whereas [M+Na]+ ions fragmented to give a complex mixture of B and Y ions together with some cross-ring fragments. Three features of MALDI-QIT-CID fragmentation of [M+Na]+ were cleared by the analysis of MS/MS, MS3 and MS4 spectra: (1) the fragment ions resulting from the breaking of a bond are more easily generated than that from multi-bond dissociation; (2) the trimannosyl-chitobiose core is either hardly dissociated, easily ionized or it is easy to break a bond between N-acetylglucosamine and mannose; (3) the fragmentation by loss of only galactose from the non-reducing terminus is not observed. We could determine the existence ratios of candidates for each fragment ion in the MS/MS spectrum of [M+Na]+ by considering these features. These results indicate that MSn analysis of [M+Na]+ ions is more useful for the analysis of complicated oligosaccharide structures than MS/MS analysis of [M+H]+, owing to the higher sensitivity and enhanced structural information. Furthermore, two kinds of glycans, with differing branch structures, could be distinguished by comparing the relative fragment ion abundances in the MS3 spectrum of [M+Na]+. These analyses demonstrate that the MSn technology incorporated in MALDI-QIT-TOF-MS can facilitate the elucidation of structure of complex branched oligosaccharides.     

Electrospray mass spectrometry and fragmentation of N-linked carbohydrates derivatized at the reducing terminus

Derivatives were prepared from N-linked glycans by reductive amination from 2-aminobenzamide, 2-aminopyridine, 3-aminoquinoline, 2-aminoacridone, 4-amino-N-(2-diethylaminoethyl)benzamide, and the methyl, ethyl, and butyl esters of 4-aminobenzoic acid. Their electrospray and collision-induced dissociation (CID) fragmentation spectra were examined with a Q-TOF mass spectrometer. The strongest signals were obtained from the [M + Na]+ ions for all derivatives except sugars derivatized with 4-amino-N-(2-diethylaminoethyl)benzamide which gave very strong doubly charged [M + H + Na]2+ ions. The strongest [M + Na]+ ion signals were obtained from the butyl ester of 4-aminobenzoic acid and the weakest from 2-aminopyridine. The most informative spectra were recorded from the [M + Li]+ or [M + Na]+ ions. These spectra were dominated by ions produced by sequence-revealing glycosidic cleavages and "internal" fragments. Linkage-revealing cross-ring cleavage ions were reasonably abundant, particularly from high-mannose glycans. Although the nature of the derivative was found to have little effect upon the fragmentation pattern, 3-aminoquinoline derivatives gave marginally more abundant cross-ring fragments than the other derivatives. [M + H]+ ions formed only glycosidic fragments with few, if any, cross-ring cleavage ions. Doubly charged molecular ions gave less informative spectra; singly charged fragments were weak, and molecular ions containing hydrogen ([M + 2H]2+ and [M + H + Na]2+) fragmented as the [M + H]+ singly charged ions with no significant cross-ring cleavages.     

Improved protonation, collision-induced decomposition efficiency and structural assessment for 'red tide' brevetoxins employing nanoelectrospray mass spectrometry

Brevetoxins are a group of natural neurotoxins found in blooms of red tide algae. Previous electrospray mass spectrometry (ES-MS) studies show that all brevetoxins have high affinities for sodium ions, and they form abundant sodium adduct ions, [M + Na]+, in ES-MS, even when trace contamination is the only source of sodium ions. Attempts to obtain informative product ions from the collision-induced decomposition (CID) of [M + Na]+ brevetoxin precursor ions resulted only in uninformative sodium ion signals, even under elevated collision energies. In this study, a nano-ES-MS approach was developed wherein ammonium fluoride was used to form cationic [M + NH4]+ adducts of brevetoxin-2 and brevetoxin-3; a significant increase in the abundance of protonated brevetoxin molecules [M + H]+ also resulted, whereas the abundance of sodium adducts of brevetoxins [M + Na]+ was observed to decrease. Under CID, both [M + NH4]+ and [M + H]+ gave similar, abundant product ions and thus underwent the same types of fragmentation. This indicated that ammonium ions initially attached to brevetoxins forming [M + NH4]+ easily lose neutral ammonia in a first step in the gas phase, leaving protonated brevetoxin [M + H]+ to readily undergo further fragmentation under CID.