On-target derivatization of keratan sulfate oligosaccharides with pyrenebutyric acid hydrazide for MALDI-TOF/TOF-MS

In the present work, a rapid and novel method of on-target plate derivatization of keratan sulfate (KS) oligosaccharides for subsequent analysis by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is described. MALDI-(time-of-flight)-TOF spectra of labeled KS oligosaccharides revealed that significantly improved ionization can be accomplished through derivatization with pyrenebutyric acid hydrazide (PBH), and the most abundant peak in each spectrum corresponds to the singly charged molecular ion [M - H]- or [M + (n - 1)Na - nH]-, where n = the number of sulfates (n = 1, 2, 3...). The high-energy collision-induced dissociation (heCID) spectra of labeled KS oligosaccharides displayed fragments of compounds similar to those observed with laser-induced dissociation (LID) analysis, suggesting that both heCID and LID fragmentations can be used to analyze KS oligosaccharides. Moreover, fragmentation analysis of all labeled KS oligosaccharides was performed by MALDI-TOF/TOF-MS. With LID mode, sodium adducts showed fragmentation of glycosidic linkages with mainly Y/B/C ions, as well as various cross-ring cleavages providing exact information for the positions of sulfate groups along the KS oligosaccharide chains. This one-step on-target derivatization method makes MALDI-TOF/TOF-MS identification of KS fast, simple and highly throughput for trace amounts of biological samples.     

Simultaneous analysis of monosaccharides and oligosaccharides by high-performance liquid chromatography with postcolumn fluorescence derivatization

To develop a fluorimetric HPLC technique for the simultaneous microanalysis of reducing mono- and oligosaccharides, the technique of linear gradient elution was introduced into the postcolumn fluorimetric detemination system of reducing saccharides with benzamidine. Fluorescence measurement was performed at 288 nm for excitation and 470 nm for emission and an optimization study for this postcolumn fluorescence derivatization carried out. Under optimum conditions, the detection limits of D-glucose and maltohexaose were 1.78 and 2.59 pmol, respectively. The present method was successfully applied to saccharide analysis and should prove useful for automated simultaneous microanalysis of reducing mono- and oligosaccharides in foods.     

Highly sensitive capillary electrophoresis analysis of N-linked oligosaccharides in glycoproteins following fluorescence derivatization with rhodamine 110 and laser-induced fluorescence detection

We describe a highly sensitive CE with laser-induced fluorescence (LIF) detection for the analysis of N-linked oligosaccharides in glycoproteins using rhodamine 110 as a fluorescence derivatization reagent. One CE separation is performed using a fused-silica capillary and neutral pH buffer conditions and allows for the separation of sialo-oligosaccharides according to the number of sialic acids. An alternate separation is performed using the same capillary and acidic pH buffer conditions, enabling the separation of asialo-oligosaccharides according to their sizes. The derivatization and separation conditions for the analysis of sialo- and asialo-oligosaccharides were optimized. Furthermore, we applied the proposed method for the analyses of N-linked sialo- and asialo-oligosaccharides in glycoproteins (ribonuclease B, fetuin, and recombinant human erythropoietin).     

电喷雾质谱在寡糖序列分析中的应用

选取具有不同结构特征的N-糖链、硫酸软骨素寡糖、人乳寡糖以及海洋来源的壳寡糖、褐藻胶寡糖、卡拉胶寡糖和硫酸岩藻寡糖等,对电喷雾质谱在寡糖的主链序列、分支位点、硫酸基取代位置确定、单糖组成和聚合度分析等方面的应用技术及碎片离子的断裂规律进行了总结.根据相邻同类碎片离子之间的质荷比差值可初步判断寡糖的单糖组成类型;通过与色谱分离技术联用或衍生化方法可提高寡糖的分辨率和离子化效率,并测得寡糖的分子量及聚合度;借助串联质谱及对寡糖还原端的特异性标记,可获得寡糖的还原端残基和部分序列信息;根据寡糖产生的特征碎片离子及其丰度大小可判断残基的特定位置和类型.另外,寡糖的分支通常作为一个整体发生糖苷键断裂或产生D离子,据此可判断分支点的位置;根据硫酸寡糖产生的特异性跨环断裂碎片,可以确定硫酸基的连接位置.这些规律和方法的总结为未知寡糖的结构和序列的分析提供了启发和指导.     

3-氨基-9-乙基咔唑衍生化寡糖混合物的高效液相色谱分离及激光解吸电离飞行时间质谱分析

建立了以3-氨基-9-乙基咔唑(AEC)为衍生化试剂对寡糖的标记方法。寡糖的还原端与AEC的伯氨基反应生成烯胺,再被NaBH3CN还原为二级胺,使得寡糖被AEC标记。衍生物通过反相高效液相色谱分离纯化,采用的色谱柱为Waters Symmetry C18柱(3.9 mm×150 mm,5 μm),乙腈和乙酸铵水溶液(pH 4.5)为流动相,梯度洗脱,在254 nm波长处检测,并以基质辅助激光解吸电离飞行时间质谱进行分析。在此衍生化条件和色谱条件下,葡寡糖衍生物分离良好,并且AEC衍生可显著提高葡寡糖的质谱检测灵敏度。该方法适用于寡糖的分离纯化和结构分析,并与生物质谱具有良好的兼容性,表明该方法在微量寡糖链分析方面有广阔的应用前景。     

寡糖衍生化及基质辅助激光解吸电离飞行时间质谱分析方法研究

为提高中性寡糖在基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)中的检测灵敏度,建立了以1-(4-氰基苯基)-4-哌啶碳酰肼(CPH)为衍生化试剂对寡糖的标记方法。寡糖的还原端与CPH的酰肼基团反应生成腙,使得寡糖被CPH标记,衍生物以MALDI-TOF质谱进行分析。结果表明:在反应温度95℃,醋酸浓度为0.125%(V/V),CPH过量100倍的条件下,衍生产率可达最大,并且CPH衍生可使中性寡糖在MALDI-TOF质谱中的检测灵敏度提高10倍。本方法简便快速,灵敏度高,适合微量寡糖链的质谱分析。     

A low-cost efficient online derivatization system for the determination of saccharides by high-performance liquid chromatograph-ultraviolet detector

In this study, a low-cost efficient online derivatization system was developed which allows for the detection of various types of mono- and oligo-saccharides only utilizing high-performance liquid chromatography (HPLC)-ultraviolet detector (UV) system. In the proposed method, phenylhydrazine was used as the derivatization reagent and directly spiked in the mobile phase, allowing for the separation and detection of mono- and oligosaccharides in an accessible instrument system (HPLC-UV). And the online derivatization design of the proposed method has significantly reduced the potential harm of derivatization reagents to the analysts. Furthermore, critical chromatographic parameters were optimized via the Box-Behnken design strategy, culminating in the ideal response for saccharides. Finally, the methodology validation of the proposed method was conducted. The proposed method showed satisfactory linear ranges with acceptable correlation coefficients (R² > 0.99), outstanding accuracy (Recovery: 95.3%–105.6%), high intra-day precision (relative standard deviation [RSD]: 1.4%–7.1%) and inter-day precision (RSD: 2.0%–7.4%). The robustness and ruggedness of the proposed method were proved as the recovery values in the range of 95.0%–104.6% and 95.1%–104.8% for robustness and ruggedness, respectively. These satisfactory validation results confirm the applicability and reliability of the proposed method for the analysis of saccharides in various complex real-world samples.     

Malononitrile as a new derivatizing reagent for high-sensitivity analysis of oligosaccharides by electrospray ionization mass spectrometry

A new method for the high-sensitivity analysis of oligosaccharides by negative ion electrospray ionization mass spectrometry was developed through a chemical derivatization of oligosaccharides. Oligosaccharides were derivatized to dinitrile compounds from the reaction with malononitrile under mildly basic conditions. The derivative of maltoheptaose was detected mainly as the [M-2H]2- ion in negative ion mode with 20 fmol sensitivity, even in unpurified samples. In this malononitrile derivatization method, no inorganic reagent, other than sodium hydroxide as a base catalyst, is used. Also, because excess ligand (malononitrile) is volatile, high sensitivity detection is realized without any solvent extraction or chromatographic purification. The detection limit can also be decreased by simple on-line cartridge filtration to 200 attomol which is 10(5) times better than that of free maltoheptaose. Structural information for oligosaccharide derivatives was obtained by collision induced dissociation. This malononitrile derivatization method is convenient and efficient for the sensitive analysis of oligosaccharides.     

Mass spectrometric analysis of carbohydrates labeled with a biotinylated tag

A derivatization method for mass spectrometric analysis of oligosaccharides is presented. Small saccharides, complex, high‐mannose‐type oligosaccharides and oligosaccharides released from hen ovalbumin were converted into their biotin derivatives by incubating them with biotinamidocaproyl hydrazide (BACH). Improved sensitivity of mass spectrometric analysis of labeled glycans in comparison with their natural counterparts was achieved after derivatization. The labeling reagent contains a biotin handle at one end and a hydrazide group at the other. Hence, the key feature of biotinylated sugars is that in addition to their usefulness in functional studies (e.g. analysis of the interaction between lectins and biotin‐derivatized oligosaccharides) they might be utilized also for structural analysis of oligosaccharides. Mass spectrometric studies were performed by matrix‐assisted laser desorption/ionization time‐of‐flight and electrospray ionization mass spectrometry. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural characterisation of underivatised olive pulp xylo-oligosaccharides by mass spectrometry using matrix-assisted laser desorption/ionisation and electrospray ionisation

Purified olive pulp glucuronoxylans, with a Xyl/GlcA ratio of 7:1, were subjected to mild acid hydrolysis and the mixture of oligosaccharides obtained was fractionated by size exclusion chromatography. One elution fraction representative of low molecular weight oligosaccharides was analysed by mass spectrometry using matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) as ionisation methods, in the positive mode. Both types of spectra showed cationised molecules [M + Na](+) of xylo-oligosaccharides in a range below m/z 1,000. The xylo-oligosaccharide structures identified were series of neutral oligosaccharides of xylose (Xyl(n), n = 3-7), of acidic oligosaccharides substituted by one glucuronic acid (Xyl(n)GlcA, n = 3-5) and by two glucuronic acid residues (Xyl(n)GlcA(2), n = 2 and 3), and also of acidic oligosaccharides substituted with one 4-O-methylglucuronic acid residue (Xyl(n)meGlcA, n = 2-4). The proposed structures were confirmed by tandem mass (MS/MS) spectra obtained using collision induced dissociation of the molecular ions. Fragmentation of cationised adducts of neutral Xyl(n) yielded C- and A-type fragments, while ammonium adducts mainly yielded B-type fragments. The fragmentation of the sodium adducts of acidic oligosaccharides (Xyl(n)meGlcA, Xyl(n)GlcA) resulted in the loss of the substituting residue (GlcA or meGlcA) as the predominant fragment, while the corresponding ammonium adducts yielded B-type fragments.     

Determination of phenols and chlorophenols as trimethylsilyl derivatives using gas chromatography-mass spectrometry

A rapid and simple method is described for the simultaneous determination of 6 phenols (phenol, o-, m-, p-cresol, catechol and resorcinol) and 19 chlorophenols (all mono-, di-, tri-, and tetrachlorophenol isomers and pentachlorophenol) present in aqueous samples. The method is based on derivatization with trimethylsilyl-N,N-dimethylcarbamate (TMSDMC). In contrast to other derivatization agents, TMSDMC instantaneously reacts with the phenolic compounds at room temperature and no further sample processing is necessary prior to instrumental analysis. The determination of the derivatives was performed by capillary gas chromatography-mass spectrometry (GC-MS). The stability of the most instable trimethylsilyl derivative (pentachlorophenol) was studied using different excess levels of the derivatization reagent. The derivatization method was tested on spiked water samples preconcentrated by solid phase extraction on Isolute ENV+ cartridge. The overall method gave detection limits of 0.01-0.25 microg/L for all compounds and < 0.05 microg/L for 17 of them.     

Mass spectrometric study of N‐glycans from serum of woodchucks with liver cancer

Woodchucks have been a preferred lab animal model of chronic hepatitis B viral infection. The model recapitulates the disease progression of HBV infection to hepatocellular carcinoma (HCC) and has documented similarities in protein glycosylation with human HCC. This study examined N‐glycans in serum of animals with(out) HCC. Oligosaccharides were released enzymatically using PNGaseF from total serum or from serum partially fractionated by extraction. Two different extraction procedures – reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and solid‐phase extraction (SPE) on a cation‐exchange/reversed‐phase STRATA‐XC cartridge – were used with the purpose of confirming glycosylation profiles. Oligosaccharides were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) after derivatization with phenylhydrazine and/or permethylation. Characteristic fragment ions produced under MS/MS conditions allowed discrimination between isomeric structures of oligosaccharides, including those sialylated with two types of acidic residues. The complementary methods allowed structural characterization of oligosaccharides from various N‐glycan classes. Furthermore, to validate results, glycosylation profiles of woodchuck sera were compared to glycans obtained from mouse serum on the same conditions. In summary, we have identified 40 N‐glycan structures in the serum of woodchucks and some types of oligosaccharide structures appeared to increase in HCC samples following protease digest. The study provides improved tools for the characterization of N‐glycans from total serum in the progression of liver disease. Copyright © 2009 John Wiley & Sons, Ltd.     

Introducing porous graphitized carbon liquid chromatography with evaporative light scattering and mass spectrometry detection into cell wall oligosaccharide analysis

Separation and characterization of complex mixtures of oligosaccharides is quite difficult and, depending on elution conditions, structural information is often lost. Therefore, the use of a porous-graphitized-carbon (PGC)-HPLC-ELSD-MSⁿ-method as analytical tool for the analysis of oligosaccharides derived from plant cell wall polysaccharides has been investigated. It is demonstrated that PGC-HPLC can be widely used for neutral and acidic oligosaccharides derived from cell wall polysaccharides. Furthermore, it is a non-modifying technique that enables the characterization of cell wall oligosaccharides carrying, e.g. acetyl groups and methylesters. Neutral oligosaccharides are separated based on their size as well as on their type of linkage and resulting 3D-structure. Series of the planar β-(1,4)-xylo- and β-(1,4)-gluco-oligosaccharides are retained much more by the PGC material than the series of β-(1,4)-galacto-, β-(1,4)-manno- and α-(1,4)-gluco-oligosaccharides. Charged oligomers such as α-(1,4)-galacturonic acid oligosaccharides are strongly retained and are eluted only after addition of trifluoroacetic acid depending on their net charge. Online-MS-coupling using a 1:1 splitter enables quantitative detection of ELSD as well as simple identification of many oligosaccharides, even when separation of oligosaccharides within a complex mixture is not complete. Consequently, PGC-HPLC-separation in combination with MS-detection gives a powerful tool to identify a wide range of neutral and acidic oligosaccharides derived from various cell wall polysaccharides.     

Towards a Non-Biased Formaldehyde Quantification in Leather: New Derivatization Conditions before HPLC Analysis of 2,4-Dinitrophenylhydrazine Derivatives

In leathers, formaldehyde is currently analyzed according to EN ISO 17226-1 standard, by reversed phase liquid chromatography after off-line precolumn derivatization with 2,4 dinitrophenylhydrazine (DNPH) in strong acidic conditions. We first demonstrate that this standard is not adapted to leather retanned with resins likely to release formaldehyde by hydrolysis. Indeed, formaldehyde content may be largely overestimated due to concomitant resin hydrolysis (in harsh acidic conditions) that releases formaldehyde during the derivatization step and during the waiting time on autosampler before analysis. Therefore, we thoroughly studied the derivatization step in order to propose new derivatization conditions. Replacing orthophosphoric acid by less acidic buffer solutions is not enough to avoid hydrolysis. A derivatization without adding acid is realized by solubilizing DNPH in acetonitrile instead of orthophosphoric acid. These conditions lead to a complete derivatization of formaldehyde in 3 h at 50 °C (in a water bath) while avoiding the hydrolysis of co-extracted dicyandiamide and melamine resins. The as-obtained leather extracts are stable over time. Formaldehyde contents found with this method agree with the formaldehyde content measured immediately at the end of derivatization reaction in standard conditions or with formaldehyde content measured by a home-designed flow injection analysis with acetylacetone online derivatization and UV detection.     

Sequencing of oligosaccharides derivatized with benzylamine using electrospray ionization-quadrupole time of flight-tandem mass spectrometry

Oligosaccharides were derivatized by reductive amination using benzylamine and analyzed by nanoelectrospray ionization-quadrupole time of flight-tandem mass spectrometry (nanoESI-QTOF-MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+H]+ ions for all benzylamine-derivatized oligosaccharides. To obtain structural information from these derivatized oligosaccharides, MS/MS was applied. Protonated molecular ions underwent extensive fragmentation, even under low-energy collision-induced dissociation. MS/MS spectra of [M+H]+ ions are characterized by simple fragmentation patterns which result from cleavage of the glycosidic bonds and thus allow a straightforward interpretation. Fragmentation of the [M+H]+ ions gave predominantly B- and Y-type glycosidic fragments. A systematic study of various oligosaccharides showed that information on sugar sequence and branching could easily be obtained. Predictable and reproducible fragmentation patterns could be obtained in all cases. This derivatization procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from 10 microg of glycoproteins separated by gel electrophoresis. Moreover, the derivatives retain their sensitivity to exoglycosidases. Thus a series of sequential on-target exoglycosidase treatments combined with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was found to be useful for the determination of structural features of the glycans released from proteins separated by gel electrophoresis such as the monosaccharide sequence, branching pattern, and anomeric configurations of the corresponding glycosidic linkages. Our strategy can be used successfully to assign the major glycans released from proteins separated by gel electrophoresis.     

Direct methylation at the surface of Carbopack B Part II: Determination of phenoxyalkanoic acid herbicides

The determination of phenoxyalkanoic acids in different types of water within the limits of the European drinking water guideline is described. Most of these acids are used world-wide as herbicides and growth regulators. Compounds having an acidic hydrogen in their structure are very strongly adsorbed on Carbopack B, a special graphitized carbon black. This effect was used for the transformation of analytes to methyl esters directly on the surface of the adsorbent, using trimethylsulfonium hydroxide (TMSH) as derivatization reagent. After elution with ethyl acetate the derivatives were identified and quantified by gas chromatography/mass spectrometry (GC/MS). The interaction between analyte and adsorbent and the yields of the derivatization step are influenced by the length of the alkanoic chain. Received: 21 June 1999 / Revised: 23 August 1999 / Accepted: 24 August 1999     

Simultaneous high-performance liquid chromatographic determination of catecholamine-related compounds by post-column derivatization involving coulometric oxidation followed by fluorescence reaction

A highly selective and sensitive high-performance liquid chromatographic method for the determination of catecholamines (norepinephrine, epinephrine and dopamine) and related compounds (L-DOPA, normetanephrine, metanephrine, 3-methoxytyramine, 3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid, 3,4-dihydroxyphenylethylene glycol, 4-hydroxy-3-methoxyphenylethylene glycol and 4-hydroxy-3-methoxyphenylethanol) with a post-column technique involving coulometric oxidation followed by fluorescence derivatization is described. These compounds, 3,4-dihydroxybenzylamine and ferulic acid are separated within 35 min by ion-pair reversed-phase chromatography using acidic buffers (pH 3.1) with methanol-acetonitrile (3:2, v/v) gradient elution, and then oxidized by a commercial coulometric detector to the corresponding o-quinones, which are converted into fluorescent derivatives by reaction with 1,2-diphenylethylenediamine. The detection limits (signal-to-noise ratio = 3) on-column are 1.5-4 pmol for the two mandelic acids, 600 fmol for L-DOPA and 20-70 fmol for the others.     

Oligosaccharide analysis using anion attachment in negative mode electrospray mass spectrometry

Eleven different anionic species were able to form adducts with neutral oligosaccharides at low cone voltage in negative ion mode electrospray mass spectrometry. Among them, fluoride and acetate have the ability to significantly enhance the absolute abundance of [M - H](-) for neutral oliogosaccharides, which otherwise have low tendencies to deprotonate due to the lack of a highly acidic group. Evidence shows that the source of high abundances of [M - H](-) for neutral oligosaccharides arises from the decomposition of [M + F](-) and [M + Ac](-) with neutral losses of HF and HAc, respectively. The chloride adducts have the best stability among all the adduct species investigated, and chloride adducts consistently appeared in higher abundances relative to [M - H](-). In tandem mass spectrometry (ES-MS/MS) experiments, upon collision induced dissociation (CID), F(-) and Ac(-) adducts gave purely analyte-related product ions, i.e., no detection of the attaching anion and no incorporation of these anions into decomposition products. Cl(-) adducts produced both Cl(-) and analyte-related product ions. For the above three anions, CID of adduct species may be used for structural determination of neutral oligosaccharides because, in each case, structurally-informative fragment ions were produced. In the presence of F(-) and Ac(-), simultaneous detection of acidic and neutral oligosaccharides was achieved, because the problem of the presence of an acidic group that can impede the deprotonation of a neutral oligosaccharide was minimized. The ratio of Cl(-):non-Cl-containing product ions obtained in CID spectra of chloride adducts of disaccharides was used to differentiate anomeric configurations of disaccharides. Density functional theory (DFT) was employed to evaluate the optimized structures of chloride adducts of disaccharides, and it was found that chloride anions favor close contact with the hydrogen from the anomeric hydroxyl group. Multiple hydrogen bonding further stabilizes the chloride adduct.     

Miniaturization in carbohydrate analysis

Recent progress of microchip electrophoresis (ME) of carbohydrates is overviewed. Carbohydrate analysis by ME encounters difficulties such as lack of electric charge and deficiency of a chromophore/fluorophore in analyte molecules, however, it benefits from the accumulated knowledge of capillary electrophoresis (CE) and rapid separation of simple sugars also by ME, with high column efficiency comparable to CE, has become possible. Analysis at high pH, with electrochemical detection, is a promising approach because carbohydrates can be ionized by weak dissociation of the hydroxyl groups and the in situ formed ionic species can be effectively separated by the zone electrophoresis mode. The separated species can be sensitively monitored by electrochemical detection on a gold or copper electrode. Ionization as borate complexes and refractometric detection is also possible, though sensitivity is lower. Introduction of UV-absorbing or fluorescent tags is potentially useful but the time-consuming derivatization processes sacrifice the rapidity of ME. Examples of ME of carbohydrates as 1-phenyl-3-methyl-5-pyrazolone (PMP; for simple mono- and oligosaccharides with UV detection), 8-aminopyrene-1,3,6-trisulfonate (APTS; for oligosaccharides ladders with LIF detection), and 4-nitro-2,1,3-benzoxadiazole (NBD-F; for amino sugars and aminoalditols with LIF detection) derivatives are presented, with details of the analytical conditions. Since ME in a short separation channel enables rapid analysis within 1 min, it presents an ideal tool for clinical analysis, as shown in a few papers reporting protocols for specific blood glucose assay. Finally, the usefulness of microfluidic reactors and microarrays for enzyme-assisted carbohydrate analysis as well as glycan profiling is pointed out.     

Reductive amination of carbohydrates using NaBH(OAc)<Subscript>3</Subscript>

An improved protocol for reductive amination of carbohydrates is developed. This derivatization facilitates the detection of oligosaccharides in HPLC-UV and mass spectrometric applications by enhancing the signal of the carbohydrates. In this study, reductive amination was achieved using NaBH(OAc)₃.This reducing agent is an attractive alternative to the toxic, but extensively used reducing agent, NaBH₃CN. Several types of carbohydrates were successfully derivatized using NaBH(OAc)₃, and the results obtained from this protocol were compared with those obtained with NaBH₃CN. Both reducing agents were equally effective in side-by-side analysis. Two purification strategies (purification by zip-tip and HPLC) were implemented and the instrumental limit of detection of each method was compared. The detection limit was ~1,000 times lower when the purification was done using HPLC, compared to using the zip-tip. Since the derivatization by-products in this protocol are not toxic, MS analysis also could also be performed directly, without purification. The MS/MS data of derivatized and underivatized oligosaccharides were acquired as well. The derivatized oligosaccharides produce more easily interpretable product ions than underivatized oligosaccharides.