Glycosidic Cleavage Research Articles

Matrix-assisted laser desorption/ionization tandem mass spectrometry of N-glycans derivatized with isonicotinic hydrazide and its biotinylated form.

Successful structural characterization of glycans often requires derivatization prior to mass spectrometric analysis. Here we report on a new derivatization reagent for glycans, biotinylated isonicotinic hydrazide, allowing glycan analysis by both mass spectrometry (MS) and biochemically. Fragmentation behavior in MS and its use in structural elucidation were investigated and compared with other labels. Glycans, released from ribonuclease B and ovalbumin, were derivatized with hydrazine labels (isoniazid (INH), biotinylated isonicotinic hydrazide (BINH) and biotinamidocaproylhydrazide (BACH)). In addition, native counterparts and 2-aminobenzamide (2-AB) derivatives were prepared. Comparative matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI TOF/TOF) experiments were carried out to investigate the fragmentation pattern of the derivatives. Finally, the capability of BINH derivatives to bind lectins was explored. Generally, derivatization provided beneficial enhancement in the mass spectrometric signal intensity as compared to native counterparts. The mass spectrometric fragmentation varied with the kind of label used. The most significant structure-revealing ions (cross-ring cleavages) were observed in the spectra of BINH derivatives, whereas mainly glycosidic cleavages were found with native form of glycans and 2-AB derivatives. Hydrazine derivatization provided the means to obtain structurally informative fragment ions. Due to BINH derivatization, specific fragments of the isomers allowed the identification of diverse glycans. The derivatization reaction can be carried out without the need for purification. The biotin residue of BINH enabled for biochemical studies, i.e. protein-glycan interactions.

ChemInform Abstract: 5‐Nitrosalicylic Acid as a Novel Matrix for In‐source Decay in Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry

AbstractReview: 55 refs.

Evaluation of quantitative variation of secondary metabolites in Bergenia ciliata (Haw.) using high performance thin layer chromatography.

Evaluation of quantitative variation of secondary metabolites in Bergenia ciliata (Haw.) using high performance thin layer chromatography.

Top-Down Strategies for the Structural Elucidation of Intact Gram-negative Bacterial Endotoxins.

Re-modelling of lipopolysaccharides, which are the primary constituent of the outer cell membrane of Gram-negative bacteria, modulates pathogenesis and resistance to microbials. Reported herein is the characterization of intact Gram-negative bacterial lipooligosaccharides (LOS) via a new strategy utilizing online liquid chromatography (LC) coupled with ultraviolet photodissociation (UVPD) mass spectrometry. Compared to collision-based MS/MS methods, UVPD and UVPD/HCD promoted a greater array of cleavages within both the glycan and lipid moieties, including C-C, C-N, C-O cleavages in the acyl chains as well as glycosidic and cross-ring cleavages, thus providing the most far-reaching structural characterization of LOS. This LC-MS/MS strategy affords a robust analytical method to structurally characterize complex mixtures of bacterial endotoxins that maintains the integrity of the core oligosaccharide and lipid A domains of LOS, providing direct feedback about the cell envelope architectures and LOS modification strategies involved in resistance host innate immune defense.

Unusual Enzymatic Glycoside Cleavage Mechanisms

Over the sixty years since Koshland initially formulated the classical mechanisms for retaining and inverting glycosidases, researchers have assembled a large body of supporting evidence and have documented variations of these mechanisms. Recently, however, researchers have uncovered a number of completely distinct mechanisms for enzymatic cleavage of glycosides involving elimination and/or hydration steps. In family GH4 and GH109 glycosidases, the reaction proceeds via transient NAD(+)-mediated oxidation at C3, thereby acidifying the proton at C2 and allowing for elimination across the C1-C2 bond. Subsequent Michael-type addition of water followed by reduction at C3 generates the hydrolyzed product. Enzymes employing this mechanism can hydrolyze thioglycosides as well as both anomers of activated substrates. Sialidases employ a conventional retaining mechanism in which a tyrosine functions as the nucleophile, but in some cases researchers have observed off-path elimination end products. These reactions occur via the normal covalent intermediate, but instead of an attack by water on the anomeric center, the catalytic acid/base residue abstracts an adjacent proton. These enzymes can also catalyze hydration of the enol ether via the reverse pathway. Reactions of α-(1,4)-glucan lyases also proceed through a covalent intermediate with subsequent abstraction of an adjacent proton to give elimination. However, in this case, the departing carboxylate "nucleophile" serves as the base in a concerted but asynchronous syn-elimination process. These enzymes perform only elimination reactions. Polysaccharide lyases, which act on uronic acid-containing substrates, also catalyze only elimination reactions. Substrate binding neutralizes the charge on the carboxylate, which allows for abstraction of the proton on C5 and leads to an elimination reaction via an E1cb mechanism. These enzymes can also cleave thioglycosides, albeit slowly. The unsaturated product of polysaccharide lyases can then serve as a substrate for a hydration reaction carried out by unsaturated glucuronyl hydrolases. This hydration is initiated by protonation at C4 and proceeds in a Markovnikov fashion rather than undergoing a Michael-type addition, giving a hemiketal at C5. This hemiketal then undergoes a rearrangement that results in cleavage of the anomeric bond. These enzymes can also hydrolyze thioglycosides efficiently and slowly turn over substrates with inverted anomeric configuration. The mechanisms discussed in this Account proceed through transition states that involve either positive or negative charges, unlike the exclusively cationic transition states of the classical Koshland retaining and inverting glycosidases. In addition, the distribution of this charge throughout the substrate can vary substantially. The nature of these mechanisms and their transition states means that any inhibitors or inactivators of these unusual enzymes probably differ from those presently used for Koshland retaining or inverting glycosidases.

Enzymatic production of modified 2‐dodecyl‐sophorosides (biosurfactants) and their characterization

AbstractAlkyl sophorosides, a special type of sophorolipids, can be produced using Candida bombicola cultures with glucose as main carbon source and 2‐dodecanol as co‐substrate. The dominating component, 2‐dodecyl‐sophoroside SL‐E2‐12, was purified via medium pressure liquid chromatography (MPLC) and used as substrate for further enzymatic modifications. Using β‐glucuronidase in aqueous media the first modification gave a glycosidic cleavage of the acetyl glucose unit leading to 2‐dodecyl‐glucoside GL‐A2‐12. In a subsequent lipase‐catalyzed acylation with sebacic acid in toluene this compound was functionalized regioselectively at the primary C‐6′ position of glucose. Similar lipase‐catalyzed reactions in toluene, but now using SL‐E2‐12 as acyl acceptor and unusual hydroxyl fatty acids – 3‐hydroxy decanoic acid and 17‐hydroxy stearic acid – as acyl donors, resulted in mono‐ and diacylations of primary hydroxyl positions of the sophorose unit (C‐6′ and C‐6′′) as shown by NMR and MS studies. In physicochemical characterization experiments the new glycoconjugates lowered the surface tension of water from 72 to 27–32 mN/m. Moreover it was observed that the new products inhibit the growth of particular Gram‐positive bacteria. Additionally they indicate potential for anti‐tumor promoting activity.Practical applications: According to literature data 2‐alkyl‐sophorosides can be produced using Candida bombicola cultures with yields of more than 20 g/L. Based on this starting material, 2‐dodecyl‐sophoroside, several regioselective enzymatic modifications were performed. Initially, β‐glucuronidase catalysis in aqueous buffer solution gave 2‐dodecyl‐glucoside which was then successfully acylated at the C‐6 position of glucose by lipase catalysis using a common dicarboxylic acid, sebacic acid. The resulting free carboxylic function of the acidic derivative opens up the possibility of achieving additional chemo‐enzymatic reactions at this position for conversion, for instance, into polyesters. In addition lipase catalysis in organic solvents of 2‐dodecyl‐sophoroside allows acylation with unusual hydroxyl fatty acids of microbial origin. Physicochemical studies showed that these new glycoconjugates may be of interest for the cosmetic industry due to moisture conservation of the skin. For practical applications the conditions of the above biocatalytic reactions have been improved and scaled up.

The Mutational Effect of Ile58 at Subsite C in Hen Egg-White Lysozyme on Substrate Binding, Enzymatic Activity, and Protein Stability

Ile58 of hen egg-white lysozyme (HEL) is buried in the interior of the molecule and is considered to participate in sugar residue binding at subsite C through hydrophobic interaction. The contribution of Ile58 to lysozyme function and stability was investigated by replacement of Ile58 with less hydrophobic residues, Val (I58V) and Ala (I58A). Replacement of Ile58 with Ala decreased substrate binding ability to an N-acetylglucosamine trisaccharide, (GlcNAc)3, and a GlcNAc polymer, chitin, whereas replacement with Val had little effect. Similar results were obtained as to enzymatic activity toward both the bacterial cell substrate and glycol chitin. Kinetic analysis by substrate (GlcNAc)5 revealed that replacement of the Ile residue reduced the sugar residue affinity at subsite C and the rate constant of glycosidic bond cleavage. The rate constant of glycosidic cleavage for mutant I58A was about one-third of that for the wild-type. Guanidine hydrochloride unfolding experiments showed that mutants I58V and I58A were less stable than the wild-type, by 1.88 and 2.88 kcal/mol respectively. Moreover, the stability of the protein inserted at this position decreased linearly with decreasing hydrophobicity of the inserted residue. It appears that the hydrophobicity of Ile58 is an important factor in the efficient substrate binding, enzymatic reaction, and structural stability of HEL.

5-nitrosalicylic Acid as a novel matrix for in-source decay in matrix-assisted laser desorption/ionization mass spectrometry.

The matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) of peptides and glycans was studied using an oxidizing chemical, 5-nitrosalicylic acid (5-NSA) as the matrix. The use of 5-NSA for the MALDI-ISD of peptides and glycans promoted fragmentation pathways involving "hydrogen-deficient" radical precursors. Hydrogen abstraction from peptides resulted in the production of a "hydrogen-deficient" peptide radical that contained a radical site on the amide nitrogen in the peptide backbone with subsequent radical-induced cleavage at the Cα-C bonds. Cleavage at the Cα-C bond leads to the production of an a (•)/x fragment pair and the radical a (•) ions then undergo further hydrogen abstraction to form a ions after Cα-C bond cleavage. Since the Pro residue does not contain a nitrogen-centered radical site, Cα-C bond cleavage does not occur at this site. Alternatively, the specific cleavage of CO-N bonds leads to a b (•)/y fragment pair at Xxx-Pro which occurs via hydrogen abstraction from the Cα-H in the Pro residue. In contrast, "hydrogen-deficient" glycan radicals were generated by hydrogen abstraction from hydroxyl groups in glycans. Both glycosidic and cross-ring cleavages occurred as the result of the degradation of "hydrogen-deficient" glycan radicals. Cross-ring cleavage ions are potentially useful in linkage analysis, one of the most critical steps in the characterization of glycans. Moreover, isobaric glycans could be distinguished by structure specific ISD ions, and the molar ratio of glycan isomers in a mixture can be estimated from their fragment ions abundance ratios. MALDI-ISD with 5-NSA could be a useful method for the sequencing of peptides including the location of post-translational modifications, identification and semi-quantitative analysis of mixtures of glycan isomers.

Fragmentation of chromophore labelled oligosaccharides induced by photodissociation with visible light

Photodissociation with visible light (Vis-PD) has been applied to chromophore labelled oligosaccharide ions produced by electrospray in a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer. As chromophores four aromatic dyes, such as Rhodamine 110, have been attached to maltopentaose, lacto-N-fucopentaose I (LNFP I) and lacto-N-fucopentaose II (LNFP II). While in the positive ion mode the Vis-PD mass spectra show predominantly glycosidic cleavages and fragments from the reducing end of the carbohydrate, the negative ion spectra result in cross-ring cleavages and also glycosidic fragments mainly from the non-reducing end of the carbohydrate. It will be shown that Vis-PD mass spectrometry leads to sequence specific fragmentation.

Electron Detachment Dissociation of Underivatized Chloride-Adducted Oligosaccharides

Chloride anion attachment has previously been shown to aid determination of saccharide anomeric configuration and generation of linkage information in negative ion post-source decay MALDI tandem mass spectrometry. Here, we employ electron detachment dissociation (EDD) and collision activated dissociation (CAD) for the structural characterization of underivatized oligosaccharides bearing a chloride ion adduct. Both neutral and sialylated oligosaccharides are examined, including maltoheptaose, an asialo biantennary glycan (NA2), disialylacto-N-tetraose (DSLNT), and two LS tetrasaccharides (LSTa and LSTb). Gas-phase chloride-adducted species are generated by negative ion mode electrospray ionization. EDD and CAD spectra of chloride-adducted oligosaccharides are compared to the corresponding spectra for doubly deprotonated species not containing a chloride anion to assess the role of chloride adduction in the stimulation of alternative fragmentation pathways and altered charge locations allowing detection of additional product ions. In all cases, EDD of singly chloridated and singly deprotonated species resulted in an increase in observed cross-ring cleavages, which are essential to providing saccharide linkage information. Glycosidic cleavages also increased in EDD of chloride-adducted oligosaccharides to reveal complementary structural information compared to traditional (non-chloride-assisted) EDD and CAD. Results indicate that chloride adduction is of interest in alternative anion activation methods such as EDD for oligosaccharide structural characterization.

Identification and Relative-Quantification of Glycans by Matrix-Assisted Laser Desorption/Ionization In-Source Decay with Hydrogen Abstraction

The use of specific matrixes allows enhancing the scope of in-source decay (ISD) applications in matrix-assisted laser desorption/ionization (MALDI) thanks to the specificity of analyte-matrix chemistry. The use of an oxidizing matrix, 5-nitrosalicylic acid (5-NSA), for MALDI-ISD of glycans is shown to promote fragmentation pathways involving radical precursors. Both glycosidic and cross-ring cleavages are promoted by hydrogen abstraction from hydroxyl group of glycans by 5-NSA molecules. Cross-ring cleavage ions are potentially useful in linkage analysis, one of the most critical steps of glycan characterization. Moreover, we show here that isobaric glycans could be distinguished by structure specific ISD ions and that the molar ratio of glycan isomers in the mixture can be estimated from their fragment ions abundance. The use of 5-NSA also opens the possibility to perform pseudo-MS(3) analysis of glycans. Therefore, MALDI-ISD with 5-NSA is a useful method for identification of glycans and semiquantitative analysis of mixture of glycan isomers.

Hexuronic Acid Stereochemistry Determination in Chondroitin Sulfate Glycosaminoglycan Oligosaccharides by Electron Detachment Dissociation

Electron detachment dissociation (EDD) has previously provided stereo-specific product ions that allow for the assignment of the acidic C-5stereochemistry in heparan sulfate glycosaminoglycans (GAGs), but application of the same methodology to an epimer pair in the chondroitin sulfate glycoform class does not provide the same result. A series of experiments have been conducted in which glycosaminoglycan precursor ions are independently activated by electron detachment dissociation (EDD), electron induced dissociation (EID), and negative electron transfer dissociation (NETD) to assign the stereochemistry in chondroitin sulfate (CS) epimers and investigate the mechanisms for product ion formation during EDD in CS glycoforms. This approach allows for the assignment of electronic excitation products formed by EID and detachment products to radical pathways in NETD, both of which occur simultaneously during EDD. The uronic acid stereochemistry in electron detachment spectra produces intensity differences when assigned glycosidic and cross-ring cleavages are compared. The variations in the intensities of the doubly deprotonated (0,2)X(3) and Y(3) ions have been shown to be indicative of CS-A/DS composition during the CID of binary mixtures. These ions can provide insight into the uronic acid composition of binary mixtures in EDD, but the relative abundances, although reproducible, are low compared with those in a CID spectrum acquired on an ion trap. The application of principal component analysis (PCA) presents a multivariate approach to determining the uronic acid stereochemistry spectra of these GAGs by taking advantage of the reproducible peak distributions produced by electron detachment.

Sequence analysis of the sulfated rhamno-oligosaccharides derived from a sulfated rhamnan

Three sulfated rhamno-oligosaccharides, designated O1, O2 and O3, were obtained by mild acid hydrolysis of the sulfated rhamnan and purified by gel-permeation chromatography. On the basis of one- and two-dimensional nuclear magnetic resonance (1D, 2D NMR) spectroscopic analyses, the oligosaccharide O1 was characterized to be α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap. The fragmentation pattern of the homogeneous disaccharide in the product ion spectra was recognized by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation (ES-CID MS/MS). With the principles established, the sequences of the oligosaccharides O2 and O3 were deduced to be α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap, and α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap (2SO4), respectively. The investigation demonstrated that the sulfated rhamnan-derived oligosaccharides were novel sulfated oligosaccharides different from those of other polysaccharides-degraded from algae, and it could be possible to determine the sequence of the sulfated rhamno-oligosaccharides directly from the glycosidic cleavage fragmentation in the product ion spectra.

Use of procaine and procainamide as derivatizing co-matrices for the analysis of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Analysis of oligosaccharides by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry often yields only alkali metal cation adducts, which results in lower fragmentation yields and difficulty to retrieve sequence information. Derivatization by reductive amination may be used to promote Y-type glycosidic cleavages. However, this involves time-consuming preparations and purifications with sample loss. Here, procaine and procainamide were used directly as co-matrices with 2,5-dihydroxybenzoic acid (DHB). Acidified 10 g/L procaine hydrochloride or procainamide hydrochloride solutions in water/acetonitrile were added to the oligosaccharide solution one minute before preparing our MALDI targets using DHB with the dried-droplet method. This simple protocol resulted in deposits of very fine homogeneous crystals. Positive ion mass spectra, easily acquired in an automated mode, presented a high percentage of oligosaccharides derivatized as Schiff base or glycosylamine notably detected as protonated molecules [M + H](+). The high abundance of procaine or procainamide on the target did not impede the ionization process, improved the signal-to-noise ratio and eliminated the need to search for 'sweet spots'. Fragmentation of the protonated precursor ions of the derivatives largely favored Y-type glycosidic cleavages. This easy and fast sample preparation, involving low toxicity and easily accessible chemicals, allowed the selection of protonated molecules as precursor ions for post-source decay analyses. This opened the possibility of simplifying sequence retrieval in routine oligosaccharide analyses.

Production and fragmentation of negative ions from neutral N‐linked carbohydrates ionized by matrix‐assisted laser desorption/ionization

Although negative ion fragmentation mass spectra of neutral N-linked carbohydrates (those attached to Asn in glycoproteins) provide much more structural information than spectra recorded in positive ion mode, neutral carbohydrates are reluctant to form negative ions by matrix-assisted laser desorption/ionization (MALDI) unless ionized from specific matrices such as nor-harmane or adducted with anions such as chloride. This paper reports the results of experiments to optimize negative ion formation from adducts of N-linked glycans with respect to ion abundance and fragment ion production. The best results were obtained with 2,4,6-trihydroxyacetophenone (THAP) as the matrix with added ammonium nitrate as the salt providing the anion. This approach is demonstrated to be applicable for a wide range of N-linked glycan structures. Phosphate adducts, analogous to those that are usually encountered in electrospray spectra from N-glycans released by protein N-glycosidase F, were produced by addition of ammonium phosphate to the matrix but in relatively low yield allowing competitive ionization of endogenous anionic compounds leading to complex spectra. Fragmentation of the nitrate adducts, which were formed in higher yield, generally paralleled that seen by collision-induced dissociation following ionization by electrospray, with the first stage of the dissociation being the elimination of the nitrate with a proton from one of the hydroxyl groups of the sugar. The spectra of the resulting [M-H](-) species displayed very specific fragment ions, mainly cross-ring and C-type glycosidic cleavage products, that revealed more structural (linkage and branching) information of the compounds than the mainly glycosidic cleavage products that dominated the positive ion spectra.

Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared with infrared multiphoton dissociation. Neutral losses and satellite ions such as C-2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA-labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared with 2-AA-labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag.

Glycoside Cleavage by a New Mechanism in Unsaturated Glucuronyl Hydrolases

Unsaturated glucuronyl hydrolases (UGLs) from GH family 88 of the CAZy classification system cleave a terminal unsaturated sugar from the oligosaccharide products released by extracellular bacterial polysaccharide lyases. This pathway, which is involved in extracellular bacterial infection, has no equivalent in mammals. A novel mechanism for UGL has previously been proposed in which the enzyme catalyzes hydration of a vinyl ether group in the substrate, with subsequent rearrangements resulting in glycosidic bond cleavage. However, clear evidence for this mechanism has been lacking. In this study, analysis of the products of UGL-catalyzed reactions in water, deuterium oxide, and dilute methanol in water, in conjunction with the demonstration that UGL rapidly cleaves thioglycosides and glycosides of inverted anomeric configuration (substrates that are resistant to hydrolysis by classical glycosidases), provides strong support for this new mechanism. A hydration-initiated process is further supported by the observed UGL-catalyzed hydration of a C-glycoside substrate analogue. Finally, the observation of a small β-secondary kinetic isotope effect suggests a transition state with oxocarbenium ion character, in which the hydrogen at carbon 4 adopts an axial geometry. Taken together, these observations validate the novel vinyl ether hydration mechanism and are inconsistent with either inverting or retaining direct hydrolase mechanisms at carbon 1.

193 nm Ultraviolet Photodissociation of Deprotonated Sialylated Oligosaccharides

The fragmentation patterns of deprotonated sialylated oligosaccharides and glycans from fetuin obtained upon collisionally induced dissociation (CID) and 193 nm ultraviolet photodissociation (UVPD) in a linear ion trap are presented. UVPD produced a more extensive series of cross-ring cleavage ions, such as A- and X-type ions, and dual-cleavage internal ions, including A/Y and X/B fragment ions. In addition, UVPD generated unique fragment ions which arise from site-specific cleavage of the triol substituent of the sialic acid residue. In contrast, CID produced more conventional glycosidic cleavages and relatively few A-type ions. UVPD of doubly deprotonated sialylated oligosaccharides produced mostly singly deprotonated fragment ions, whereas the product ions in the CID spectra were overwhelmingly doubly charged ions, an outcome attributed to the more extensive cleavages of sialic acid residues upon UVPD and products from electron photodetachment. The larger array of product ions, including those arising from extensive cross-ring cleavages and dual-cleavage ions, generated by 193 nm UVPD relative to CID gives greater confidence for identification of glycans. Several key site-specific cleavages by UVPD, such as ones involving the sialic acid moieties, provide evidence of glycan composition.

Electron detachment dissociation and infrared multiphoton dissociation of heparin tetrasaccharides

Heparin glycosaminoglycans (GAGs) present the most difficult glycoform for analytical characterization due to high levels of sulfation and structural heterogeneity. Recent contamination of the clinical heparin supply and subsequent fatalities has highlighted the need for sensitive methodologies of analysis. In the last decade, tandem mass spectrometry has been increasingly applied for the analysis of GAGs, but developments in the characterization of highly sulfated compounds have been minimal due to the low number of cross-ring cleavages generated by threshold ion activation by collisional induced dissociation (CID). In the current work, electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) are applied to a series of heparin tetrasaccharides. With both activation methods, abundant glycosidic and cross-ring cleavages are observed. The concept of Ionized Sulfate Criteria (ISC) is presented as a succinct method for describing the charge state, degree of ionization and sodium/proton exchange in the precursor ion. These factors contribute to the propensity for useful fragmentation during MS/MS measurements. Precursors with ISC values of 0 are studied here, and shown to yield adequate structural information from ion activation by EDD or IRMPD.

Virion glycosylation governs integrity and infectivity of infectious pancreatic necrosis virus.

The possible importance of the O‐linked glycosylation in virion stability and infectivity of infectious pancreatic necrosis virus (IPNV) was analysed. Enzymatic treatment with O‐glycosidase of radiolabelled virions under different ionic conditions, to allow for possible alternative exposure of glycosidic enzyme cleavage sites, did not alter the specific infectivity of virions re‐isolated after rate‐zonal centrifugation in glycerol gradients. As an alternative method to assess the significance of carbohydrates in IPNV integrity, periodate oxidation in the presence of an aldehyde quencher was chosen. Following re‐isolation of viruses, a 3–5 10log‐unit reduction in specific infectivity was revealed and, at higher concentrations, a total disruption or virion aggregation was observed. The loss of infectivity of intact virions was not because of a lack of attachment to cells. Additionally, re‐evaluation of reading values from UV‐spectra of purified IPNV yielded a specific infectivity of 3 × 1011 TCID50‐units mg−1 of protein and a ratio of 40 virions per TCID50‐unit in the CHSE‐214 cell system.