Determination of sulfation pattern in brain glycosaminoglycans by chip-based electrospray ionization ion trap mass spectrometry

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans display variability of sulfation in their constituent disaccharide repeats during chain elongation. Since a large proportion of the extracellular matrix of the central nervous system (CNS) is composed of proteoglycans, CS/DS disaccharide degree and profile of sulfation play important roles in the functional diversity of neurons, brain development, and some of its pathological states. To investigate the sulfation pattern of CS/DS structures expressed in CNS, we introduced here a novel method based on an advanced system encompassing fully automated chip nanoelectrospray ionization (nanoESI) in the negative ion mode and high capacity ion trap multistage mass spectrometry (MS²–MS³) by collision-induced dissociation (CID). This method, introduced here for the first time in glycomics of brain glycosaminoglycans, was particularly applied to structural investigation of disaccharides obtained by β-elimination and digestion with chondroitin B and AC I lyase of hybrid CS/DS chains from wild-type mouse brain. Screening in the chip-MS mode of DS disaccharide fraction resulting after depolymerization with chondroitin B lyase revealed molecular ions assigned to monosulfated disaccharide species having a composition of 4,5-Δ-[IdoA-GalNAc]. By optimized CID MS²–MS³, fragment ions supporting the localization of sulfate ester group at C4 within GalNAc were produced. Chip ESI MS profiling of CS disaccharide fraction obtained by depolymerization of the same CS/DS chain using chondroitin AC I lyase indicated the occurrence of mono- and bisulfated 4,5-Δ-[GlcA-GalNAc]. The site of oversulfation was determined by MS²–MS³, which provided sequence patterns consistent with a rare GlcA-3-sulfate–GalNAc-6-sulfate structural motif.      

LC-MS<Superscript><Emphasis Type="BoldItalic">n</Emphasis></Superscript> Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS ⁿ . The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS ⁿ fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS ⁿ experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS ⁿ methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.     

Complexation of magnesium and calcium ions with heparin

The acid-base properties of unfractionated heparin (H₄L) and the complexation of biometal ions (Mg²⁺ and Ca²⁺) with heparin in aqueous solutions have been studied by pH titration, using mathematical modeling methods in data processing. The heparin concentration is taken to be equal to the concentration of heparin disaccharide units. The formation constants of the protonated heparin species H_iL (i = 1, 2) have been estimated. The most abundant Mg²⁺-heparin and Ca²⁺-heparin complex species have been identified, and their formation constants have been estimated.     

Comparative analysis of complex formation of magnesium and calcium ions with low-molecular-weight and unfractionated heparin

The acid-base and complexing properties of Logiparin (a low-molecular-weight variety (LMH) of heparin (H₄L) with magnesium and calcium ions were studied using mathematical modeling of chemical equilibria in aqueous solutions of Logiparin and its solutions with magnesium and calcium ions and pH titration. The heparin concentration was set equal to the concentration of its disaccharide units. The protonated heparin form HL^3? and the most significant heparin complexes of Mg²⁺ and Ca²⁺ were identified; relevant formation constants were estimated. Comparative analysis of the results is carried out against available data on ion complexing with unfractionated (high-molecular-weight) heparin (HMH). The Logiparin complexes of calcium and magnesium ions are inferior to the HMH complexes in both their formation constants in solution and their effect on the decrease in the equilibrium concentration of free calcium ions, a participant of all blood coagulation processes. This distinction creates prerequisites for the lower blood-coagulating activity of Logiparin compared to that of HMH, which is for the first time confirmed in biological experiments with the in vitro administration of the same concentration of HMH or LMH into the blood plasma of laboratory rats.     

NMR methods to monitor the enzymatic depolymerization of heparin

Heparin and the related glycosaminoglycan, heparan sulfate, are polydisperse linear polysaccharides that mediate numerous biological processes due to their interaction with proteins. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, digestion to produce smaller oligosaccharides is commonly performed prior to separation and analysis. Current techniques used to monitor the extent of heparin depolymerization include UV absorption to follow product formation and size exclusion or strong anion exchange chromatography to monitor the size distribution of the components in the digest solution. In this study, we used ¹H nuclear magnetic resonance (NMR) survey spectra and NMR diffusion experiments in conjunction with UV absorption measurements to monitor heparin depolymerization using the enzyme heparinase I. Diffusion NMR does not require the physical separation of the components in the reaction mixture and instead can be used to monitor the reaction solution directly in the NMR tube. Using diffusion NMR, the enzymatic reaction can be stopped at the desired time point, maximizing the abundance of larger oligosaccharides for protein-binding studies or completion of the reaction if the goal of the study is exhaustive digestion for characterization of the disaccharide composition. In this study, porcine intestinal mucosa heparin was depolymerized using the enzyme heparinase I. The unsaturated bond formed by enzymatic cleavage serves as a UV chromophore that can be used to monitor the progress of the depolymerization and for the detection and quantification of oligosaccharides in subsequent separations. The double bond also introduces a unique multiplet with peaks at 5.973, 5.981, 5.990, and 5.998 ppm in the ¹H-NMR spectrum downfield of the anomeric region. This multiplet is produced by the proton of the C-4 double bond of the non-reducing end uronic acid at the cleavage site. Changes in this resonance were used to monitor the progression of the enzymatic digestion and compared to the profile obtained from UV absorbance measurements. In addition, in situ NMR diffusion measurements were explored for their ability to profile the different-sized components generated over the course of the digestion.     

Linkage Determination of Linear Oligosaccharides by MSn (n > 2) Collision-Induced Dissociation of Z1 Ions in the Negative Ion Mode

Obtaining unambiguous linkage information between sugars in oligosaccharides is an important step in their detailed structural analysis. An approach is described that provides greater confidence in linkage determination for linear oligosaccharides based on multiple-stage tandem mass spectrometry (MSⁿ, n >2) and collision-induced dissociation (CID) of Z₁ ions in the negative ion mode. Under low energy CID conditions, disaccharides ¹⁸O-labeled on the reducing carbonyl group gave rise to Z₁ product ions (m/z 163) derived from the reducing sugar, which could be mass-discriminated from other possible structural isomers having m/z 161. MS³ CID of these m/z 163 ions showed distinct fragmentation fingerprints corresponding to the linkage types and largely unaffected by sugar unit identities or their anomeric configurations. This unique property allowed standard CID spectra of Z₁ ions to be generated from a small set of disaccharide samples that were representative of many other possible isomeric structures. With the use of MSⁿ CID (n = 3 – 5), model linear oligosaccharides were dissociated into overlapping disaccharide structures, which were subsequently fragmented to form their corresponding Z₁ ions. CID data of these Z₁ ions were collected and compared with the standard database of Z₁ ion CID using spectra similarity scores for linkage determination. As the proof-of-principle tests demonstrated, we achieved correct determination of individual linkage types along with their locations within two trisaccharides and a pentasaccharide.

Ion mobility-mass spectrometry analysis of isomeric carbohydrate precursor ions

The rapid separation of isomeric precursor ions of oligosaccharides prior to their analysis by mass spectrometry to the nth power (MS ⁿ ) was demonstrated using an ambient pressure ion mobility spectrometer (IMS) interfaced with a quadrupole ion trap. Separations were not limited to specific types of isomers; representative isomers differing solely in the stereochemistry of sugars, in their anomeric configurations, and in their overall branching patterns and linkage positions could be resolved in the millisecond time frame. Physical separation of precursor ions permitted independent mass spectra of individual oligosaccharide isomers to be acquired to at least MS³, the number of stages of dissociation limited only practically by the abundance of specific product ions. IMS–MS ⁿ analysis was particularly valuable in the evaluation of isomeric oligosaccharides that yielded identical sets of product ions in tandem mass spectrometry experiments, revealing pairs of isomers that would otherwise not be known to be present in a mixture if evaluated solely by MS dissociation methods alone. A practical example of IMS–MSⁿ analysis of a set of isomers included within a single high-performance liquid chromatography fraction of oligosaccharides released from bovine submaxillary mucin is described.     

Tandem Mass Spectrometry Imaging Enables High Definition for Mapping Lipids in Tissues

Mass spectrometry imaging (MSI) of lipids in biological tissues is useful for correlating molecular distribution with pathological results, which could provide useful information for both biological research and disease diagnosis. It is well understood that the lipidome could not be clearly deciphered without tandem mass spectrometry analysis, but this is challenging to achieve in MSI due to the limitation in sample amount at each image spot. Here we develop a multiplexed MS² imaging (MS²I) method that can provide MS² images for 10 lipid species or more for each sampling spot, providing spatial structural lipidomic information. Coupling with on-tissue photochemical derivatization, imaging of 20 phospholipid C=C location isomers is also realized, showing enhanced molecular images with high definition in structure for mouse brain and human liver cancer tissue sections. Spatially mapped t-distributed stochastic neighbor embedding has also been adopted to visualize the tumor margin with enhancement by structural lipidomic information.     

Isolation and characterization of an exopolygalacturonase from Fusarium oxysporum f.sp. cubense race 1 and race 4

Background

Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [→4IdoA(2S)β-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units.

Results

Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfate)β-1→3GalNAcβ-1→] modulate heparin cofactor II activity of dermatan sulfate polymers. Thus, high and low heparin cofactor II stimulating activity is observed in 2,4-sulfated dermatan sulfates and 2,6-sulfated dermatan sulfates, respectively, confirming the clear correlation between the anticoagulant activities of dermatan sulfates and the presence of 2,4-sulfated units.

Conclusions

Our results indicate that in ascidian dermatan sulfates the position of sulfation on the GalNAc in the disaccharide [→4IdoA(2S)β-1→3GalNAcβ-1→] is directly related to the taxon and that the 6-O sulfation is a novelty apparently restricted to the Phlebobranchia. We also show that the increased content of [→4IdoA(2S)β-1→3GalNAc(4S)β-1→] disaccharide units in dermatan sulfates from Stolidobranchia accounts for the increased heparin cofactor II stimulating activity.     

Engineered bio-active polysaccharides from heparin

[Image: see text] Heparin, the well-known anticoagulant polysaccharide, is also active in many other biological systems owing to its structural similarity to HS, but usually lacks selectivity because it is more highly sulfated. A series of straightforward chemical reactions (de-O-sulfation, de-N-sulfation and re-N-acetylation), carried out to partial or complete extent, were combined, resulting in a number of modified heparin polysaccharide derivatives with altered properties. These exhibited a range of abilities to promote cell signalling through the FGF/FGFR tyrosine kinase signalling system, in an in vitro cell assay with combinations of FGF-1, -2, -3 and FGFR 1 and 3. One polysaccharide (N-acetylated, 6-O- and 2-O-sulfated heparin), with only a fraction (<10(-3)) of the anticoagulant activity of heparin (200 U . mg(-1)), promoted FGF-2-mediated angiogenesis (10-fold) and therefore had an improved ratio of pro-angiogenic activity to anticoagulant activity in excess of 10(4) compared to heparin. These results demonstrate that heparin-derived polysaccharides can be engineered for selected activities and have potential in a wide range of medical, biotechnological and tissue-engineering applications. Effect of selected engineered heparin polysaccharides on angiogenesis.     

Modular synthesis of heparin oligosaccharides

A general, modular strategy for the first completely stereoselective synthesis of defined heparin oligosaccharides is described. Six monosaccharide building blocks (four differentially protected glucosamines, one glucuronic and one iduronic acid) were utilized to prepare di- and trisaccharide modules in a fully selective fashion. Installation of the alpha-glucosamine linkage was controlled by placing a conformational constraint on the uronic acid glycosyl acceptors thereby establishing a new concept for stereochemical control. Combination of disaccharide modules to form trans-uronic acid linkages was completely selective by virtue of C2 participating groups. Coupling reactions between disaccharide modules exhibited sequence dependence. While the union of many glucosamine uronic acid disaccharide modules did not meet any problems, certain sequences proved not accessible. Elaboration of glucosamine uronic acid disaccharide building blocks to trisaccharide modules by addition of either one additional glucosamine or uronic acid allowed for stereoselective access to oligosaccharides as demonstrated on the example of a hexasaccharide resembling the ATIII-binding sequence. Final deprotection and sulfation yielded the fully synthetic heparin oligosaccharides.     

Differentiating Isobaric Steroid Hormone Metabolites Using Multi-Stage Tandem Mass Spectrometry

Steroid hormones and their metabolites are currently undergoing clinical trials as potential therapeutics for traumatic brain injury (TBI). To support this work, it is necessary to develop improved procedures for differentiating isobaric species in this compound class. Equilin sulfate (E-S), estrone sulfate (E1-S), 17α-dihydroequilin sulfate (ADHE-S), and 17β-dihydroequilin sulfate (BDHE-S) are primary constituents in hormone replacement therapies, such as Premarin, which are among pharmaceuticals being investigated for TBI treatment. The latter three compounds are isomers and can be difficult to differentiate in trace analytical determinations. In this work, a systematic study of the fragmentation of ADHE-S, BDHE-S, E1-S, and E-S under different stages of higher order tandem mass spectrometry (MSⁿ) and variation of collision energy, allowed optimization of conditions for distinguishing the isomeric structures. For epimeric variants (e.g., ADHE-S versus BDHE-S; α- versus β-stereoisomerization in the C-17 position), differentiation was achieved at MS⁴ and fragmentation was demonstrated through MS⁵. Computational analysis was performed to further explore differences in the fragmentation pathways due to changes in stereochemistry.      

Identification of triacylglycerol using automated annotation of high resolution multistage mass spectral trees

High complexity of identification for non-target triacylglycerols (TAGs) is a major challenge in lipidomics analysis. To identify non-target TAGs, a powerful tool named accurate MSⁿ spectrometry generating so-called ion trees is used. In this paper, we presented a technique for efficient structural elucidation of TAGs on MSⁿ spectral trees produced by LTQ Orbitrap MSⁿ, which was implemented as an open source software package, or TIT. The TIT software was used to support automatic annotation of non-target TAGs on MSⁿ ion trees from a self-built fragment ion database. This database includes 19108 simulate TAG molecules from a random combination of fatty acids and corresponding 500582 self-built multistage fragment ions (MS ≤ 3). Our software can identify TAGs using a “stage-by-stage elimination” strategy. By utilizing the MS¹ accurate mass and referenced RKMD, the TIT software can discriminate unique elemental composition candidates. The regiospecific isomers of fatty acyl chains will be distinguished using MS² and MS³ fragment spectra. We applied the algorithm to the selection of 45 TAG standards and demonstrated that the molecular ions could be 100% correctly assigned. Therefore, the TIT software could be applied to TAG identification in complex biological samples such as mouse plasma extracts.     

Utilization of MS spectra for the multicomponent quantification of diastereomeric N-acetylhexosamines

A rapid and accurate means of quantifying mixtures of diastereomeric N-acetylhexosamine monosaccharides using MS³ product ions is introduced. The method involves derivatizing the monosaccharides with [Co(DAP)₂Cl₂]Cl (where DAP is diaminopropane), and subjecting the derivatized products to collision-induced dissociation (CID) in a quadrupole ion trap mass spectrometer. Each diastereomer provides unique MS³ product ion abundances. The abundances for the pure monosaccharide standards are used in a system of equations in order to quantify mixtures of these diastereomers. Using the system of equations is quite advantageous, as it is the only mass spectrometric method that has been shown to successfully quantify mixtures of more than two isomers. The utility of the method is demonstrated by successfully quantifying various two and three component mixtures of the diastereomeric monosaccharides. Furthermore, the method is used to quantify the recovery of a single diastereomeric monosaccharide from an acidic resin. Although the multicomponent quantification method described herein is used to quantify mixtures of N-acetylhexosamine diastereomers, it could be applied to any group of isomers, provided distinguishing CID spectra are obtained. This is the first known report of utilizing MS³ product ions for quantification of structural isomeric mixtures.     

ESI-MS differential fragmentation of positional isomers of sulfated oligosaccharides derived from carrageenans and agarans

We have prepared a number of isomeric red seaweed galactan-derivative sulfated oligosaccharides to determine whether there were diagnostic differences among the isomeric mass spectra obtained using ESI CID MS/MS (triple quadrupole instrument). Fragmentation of the single or multicharged molecular ions from di-, tetra-, and hexasaccharides indicated that the relative positioning of the sulfate groups and type of monosaccharide unit affect the rate of cleavage of the glycosidic bonds. We also performed a comparative [M-Na]⁻ fragmentation study of positional isomers of sulfated disaccharides that present all four monosulfation possibilities on the galactopyranosidic ring. In this case, negative-ion ESI CID MS/MS approach gave diagnostic product ions from cross-ring cleavages along with the same main B₁ ion (from sulfated Gal_p), at m/z 241, for all isomers. The isomeric disaccharides were also submitted to increased spray energy conditions inducing in-source fragmentation; preformed B₁ ions were then fragmented to give similar product ions as those found in [M-Na]⁻ analysis. Evaluation of the relative abundances mainly for cross-ring fragment ions at m/z 138, 139, 151, 153 allowed clear distinction among the members of the disaccharide series. The different ratios for m/z 151/153 ions were consistent with the predominance of m/z 153 being related to the cases when the bond involved in the cleavage process links a sulfated carbon. A quadrupole ion trap instrument (MSⁿ analysis) was also utilized to compare the results obtained with the triple quadrupole instrument.     

Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity

O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [³⁵S] 3′-phosphoadenosine-5′-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.

Electrospray Mass Spectrometry with Consecutive Fragmentation Steps (ESI‐MSn) as a Tool for Rapid and Sensitive Analysis of Ginsenosides and Their Galactosyl Derivatives

The ginsenosides Rb₁ ( 3 ) and Rg₁ ( 4 ) isolated from Panax ginseng were enzymatically modified with galactosyltransferase to furnish new derivatives carrying galactose units in one or both sugar chains at position C(20) and/or C(3) or C(6) of the protopanaxadiol and protopanaxatriol aglycones 1 and 2 , respectively. To determine the linkage position(s) of the introduced galactose unit(s), an electrospray‐ionization MS analysis with consecutive fragmentation steps (ESI‐MSⁿ) was carried out using an ion‐trap mass spectrometer (Figs. 2 and 3). It was shown that both sugar moieties, located at different positions of the protopanaxadiol and protopanaxatriol aglycone, can be easily differentiated and analyzed in the subsequent fragmentation steps. Collision‐induced dissociation (CID) of the Na⁺‐ionized molecule (MS²) leads to cleavage of the most labile O−C(20) glycosidic bond, liberating the C(20) oligosaccharide fragment ion that can be analyzed in a subsequent fragmentation step (MS³). MS³ of the C(20) monodeglycosylated ginsenoside leads to cleavage of the second sugar moiety, allowing structure analysis of this fragment ion (MS⁴). By this method, the linkages of the monosaccharides and branching positions can be rapidly determined using only a few μl of a 10⁻⁵ M sample solution.     

Mixed-ligand complexation of calcium and magnesium ions with heparin and glycine

Chemical equilibria in dilute aqueous solutions containing high-molecular-weight heparin (Na₄hep) and Glycine (HGly), as well as in solutions of the MCl₂-Na₄hep-HGly-H₂O-NaCl system (M = Ca²⁺, Mg²⁺) against the background of 0.15 M NaCl at 37°C, have been studied by mathematical modeling of chemical equilibria on the basis of pH-metric titration data. The model of equilibria of the Na₄hep-HGly-H₂O-NaCl system for the range 2.30 ≤ pH ≤ 10.50 at different ratios of initial heparin and glycine concentrations showed that, in the pH range of blood plasma stability (pH 6.80–7.40), the protonated H H₃hepGly₃⁴⁻ species prevailed. This was supported by UV absorption spectra of heparin and glycine solutions in the presence of 0.15 M NaCl and absorbance dynamics for solutions containing heparin and glycine. The results of modeling equilibria in the five-component MCl₂-Na₄hep-HGly-H₂O-NaCl systems (M = Ca²⁺, Mg²⁺) showed that the Ca²⁺ and Mg²⁺ ions form with heparin and glycine stable protonated mixed-ligand complexes M H₃hepGly₃²⁻. The formation constants of these species are one order of magnitude higher than the formation constants of the homoligand calcium and magnesium with heparin. In the pH range 6.80–7.40, the calcium content decreases depending on the ratio of the initial concentrations of Na₄hep, HGly, and CaCl₂: at the 1 : 3 : 1 ratio, it decreases by a factor of 5.7 owing to the formation of the predominant species CaH₃hepGly₃²⁻, and at equimolar amounts of the reagents (1 : 1 : 1), the calcium content decreases by a factor of 3.5 (the CaH₃hepGly₃²⁻ concentration is three time as low as the NaCahep⁻ concentration).     

二肽衍生物的电喷雾质谱研究

基于HIV整合酶核心结构域,合成了以HIV整合酶为靶标的二肽衍生物,采用多级质谱技术（二级、三级）研究二肽衍生物在质谱条件下的化学键断裂途径,发现主要的断裂方式为：氨基与羰基间的NH-CO键的断裂以及N-(苯并噻唑-2-基)甲酰氨基与亚甲基间的CO-C间的断裂。     

Structural Definition of Trehalose 6-Monomycolates and Trehalose 6,6'-Dimycolates from the Pathogen <Emphasis Type="BoldItalic">Rhodococcus equi</Emphasis> by Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization

The cell wall of the pathogenic bacterium Rhodococcus equi (R. equi) contains abundant trehalose monomycolate (TMM) and trehalose dimycolate (TDM), the glycolipids bearing mycolic acids. Here, we describe multiple-stage (MS ⁿ ) linear ion-trap (LIT) mass spectrometric approaches toward structural characterization of TMM and TDM desorbed as [M + Alk]⁺ (Alk = Na, Li) and as [M + X]^– (X = CH₃CO₂, HCO₂) ions by electrospray ionization (ESI). Upon MS ⁿ (n = 2, 3, 4) on the [M + Alk]⁺ or the [M + X]^– adduct ions of TMM and TDM, abundant structurally informative fragment ions are readily available, permitting fast assignment of the length of the meromycolate chain and of the α-branch on the mycolyl residues. In this way, structures of TMM and TDM isolated from pathogenic R. equi strain 103 can be determined. Our results indicate that the major TMM and TDM molecules possess 6, and/or 6'-mycolyl groups that consist of mainly C14 and C16 α-branches with meromycolate branches ranging from C18 to C28, similar to the structures of the unbound mycolic acids found in the cell envelope. Up to 60 isobaric isomers varying in chain length of the α-branch and of the meromycolate backbone were observed for some of the TDM species in the mixture. This mass spectrometric approach provides a direct method that affords identification of various TMM and TDM isomers in a mixture of which the complexity of this lipid class has not been previously reported using other analytical methods.