Matrix optimization for matrix-assisted laser desorption/ionization mass spectrometry of oligosaccharides from human milk

Neutral and acidic oligosaccharides from human milk were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). These experiments require suitable matrices; their selection and particularly their preparation protocols must be optimized. Important criteria are sensitivity, reproducibility, tolerance against impurities and resolution over a wide mass range. For analytical investigations of these oligosaccharides, containing labile fucosylated and sialylated components, another property of a matrix becomes a significant factor, namely the influence on ion stability and the extent of (metastable) fragmentation. The experience gained with the MALDI/MS of neutral and acidic oligosaccharides is summarized taking into account different intentions of measurement and typical problems, such as impurities after enzymatic treatment. For a rapid screening of an oligosaccharide sample, superior results were obtained with a new preparation technique using 5-chloro-2-mercaptobenzothiazole (CMBT) as the first layer for 2,5-dihydroxybenzoic acid. For structural analysis by post-source decay, CMBT as the first layer for 3-aminoquinoline is a favoured preparation protocol, because extensive fragmentation is achieved. For acidic oligosaccharides, a special preparation protocol makes it possible to determine the number of sialic acids by inducing highly effective cationization. Matrix-assisted laser desorption/ionization mass spectrometry; matrices; oligosaccharides; post-source decay.     

Capillary electrophoresis of acidic oligosaccharides from human milk

Interest in defining the array of oligosaccharides of human milk has been increasing. Pathogens that bind glycans on their host mucosal surfaces may be inhibited by human milk oligosaccharides. It has been postulated that acidic oligosaccharides in human milk may inhibit binding by pathogens that bind acidic glycans in the gut, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides of human milk have been quantified by HPLC and CE. A recent CE technique uses the MEKC mode with direct detection at 205 nm to resolve and quantify, in the native form, the 12 most dominant sialyloligosaccharides of human milk in a single 35-min run. The method gives a linear response from 39 to 2500 microg/mL with a coefficient of variation between 2 to 9% and accuracy from 93 to 109%. This was used to detect variation in expression of specific sialyloligosaccharides in milk. Individual sialyloligosaccharide concentrations in milk differ among individual donors and between less and more mature milk. Thus, CE can be used to measure variation in sialyloligosaccharide expression in milk, and thereby test the relationship of this variation-to-variation in risk of specific diseases in breastfed infants.     

Separation of lactose from human milk oligosaccharides with simulated moving bed chromatography

The successful separation of the disaccharide lactose from a complex mixture of human milk oligosaccharides (HMOS) with the continuous chromatography of simulated moving bed (SMB) technique is described. Since lactose is the main carbohydrate in human milk with well-known functions for the infant, it is necessary to separate it from the rest of the oligosaccharides to divide them into less complex fractions and analyse their partial unknown functions. For separation of lactose from HMOS two different stationary phases (size-exclusion gel as well as ion-exchange gel) were used. As the main result, it is shown that a size-exclusion gel with the particle size of 50-100 microm and porosity of 50 A was the preferred stationary phase for our separation process with almost complete lactose separation and stable conditions.     

Liquid chromatography-electrospray mass spectrometry as a tool for the analysis of sulfated oligosaccharides from mucin glycoproteins.

An approach for analyzing sulfated oligosaccharide alditol mixtures by liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) is described. Two columns, an amino-bonded column and a porous graphitized carbon column (PGC) were used. Oligosaccharides were eluted with linear gradients of acetonitrile and water, with 5 mM ammonium hydrogencarbonate or formate buffers at a basic pH. The methods were evaluated on a mixture of sulfated oligosaccharide alditols prepared from mucin glycoproteins from pig stomach. Results from LC-ESI-MS of the mixture were compared with the structural information obtained by high energy collision fragmentation using fast atom bombardment tandem mass spectrometry (FAB-MS-MS). The separation ability of the two columns was also tested using a more complex mixture of sulfated oligosaccharides from pig colon, where several isomers were detected. The potential use of in-source collision-induced dissociation (CID) to gain sequence information of sulfated oligosaccharides was also evaluated. The major fragment ions obtained by in-source CID of the trisaccharide Hex-3HexNAcol6-HexNAc6-SO3 were sufficient for assigning the oligosaccharide sequence and the position of the sulfate group within the monosaccharide moiety. The LC-ESI-MS approach should be a valuable tool for characterization of mucin glycosylation and alterations during pathological conditions.     

Trace analysis of aminoglycoside antibiotics in bovine milk by MEKC with LIF detection

This work describes a straightforward and sensitive method for the multi-residue analysis of aminoglycoside antibiotics (kanamycin B, amikacin, neomycin B and paromomycin I) in bovine milk samples. The method involves the pre-capillary derivatization of antibiotics with sulfoindocyanine succinimidyl ester (Cy5) and their separation and determination by MEKC with LIF detection. The optimum procedure includes a derivatization step of the antibiotics at 25 degrees C for 30 min and direct injection for MEKC analysis, which is performed in about 20 min by using borate buffer (35 mM; pH 9.2) with 55 mM SDS as an anionic surfactant and 20% ACN as the organic modifier. Under these conditions, dynamic ranges of 10-500 microg/L and RSDs (within-day precision) from 3.8 to 5.3% were obtained. These results indicate that the proposed MEKC-LIF method is useful as a selective and sensitive tool for the determination of these antibiotics and surpasses other reported electrophoretic alternatives. Finally, the method was successfully applied to bovine milk samples after a simple solid-phase extraction clean-up and preconcentration procedure. The aminoglycosides were readily detected at 0.5-1.5 microg/kg levels with average recoveries ranging from 89.4 to 93.3%.     

Structural analysis of the N-linked oligosaccharides from human urinary trypsin inhibitor.

The N-linked oligosaccharides from human urinary trypsin inhibitor were purified and their structures were investigated by compositional analysis, the two-dimensional sugar map method and 500 MHz 1H-NMR. The results revealed that they were composed of disialosyl, monosialosyl and asialosyl oligosaccharides, which have the common biantennary core structure; Gal1-4GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)M an1-4GlcNAc1-4GlcNAc.     

Critical parameters for the analysis of anionic oligosaccharides by desorption electrospray ionization mass spectrometry

Sulfated oligosaccharides derived from glycosaminoglycans (GAGs) are fragile compounds, highly polar and anionic. We report here on the rare but successful application of desorption electrospray ionization (DESI) — LTQ‐Orbitrap mass spectrometry (MS) to the high‐resolution analysis of anionic and sulfated oligosaccharides derived from the GAGs hyaluronic acid and heparin. For that purpose, key parameters affecting DESI performance, comprising the geometric parameters of the DESI source, the probed surface and the spraying conditions, applied spray voltage, flow rates and solvent composition were investigated. Under suitable conditions, the DESI technique allows the preservation of the structural integrity of such fragile compounds. DESI enabled the sensitive detection of anionic hyaluronic acid and heparin oligosaccharides with a limit of detection (LOD) down to 5 fmol (≈10 pg) for the hyaluronic acid decasaccharide. Detection of hyaluronic acid oligosaccharides in urine sample was also successfully achieved with LOD values inferior to the ng range. Multistage tandem mass spectrometry (MSⁿ) through the combination of the DESI source with a hybrid linear ion trap‐orbitrap mass spectrometer allowed the discrimination of isomeric sulfated oligosaccharides and the sequence determination of a hyaluronic acid decasaccharide. These results open promising ways in glycomic and glycobiology fields where structure–activity relationships of bioactive carbohydrates are currently questioned. Copyright © 2012 John Wiley & Sons, Ltd.     

High-throughput mass finger printing and Lewis blood group assignment of human milk oligosaccharides

The structural diversity of human milk oligosaccharides (HMOs) strongly depends on the Lewis (Le) blood group status of the donor which allows a classification of these glycans into three different groups. Starting from 50 μL of human milk, a new high-throughput, standardized, and widely automated mass spectrometric approach has been established which can be used for correlation of HMO structures with the respective Lewis blood groups on the basis of mass profiles of the entire mixture of glycans together with selected fragment ion spectra. For this purpose, the relative abundance of diagnostically relevant compositional species, such as Hex₂Fuc₂ and Hex₃HexNAc₁Fuc₂, as well as the relative intensities of characteristic fragment ions obtained thereof are of key importance. For each Lewis blood group, i.e., Le(a − b+), Le(a + b−), and Le(a − b−), specific mass profile and fragment ion patterns could be thus verified. The described statistically proven classification of the derived glycan patterns may be a valuable tool for analysis and comparison of large sets of milk samples in metabolic studies. Furthermore, the outlined protocol may be used for rapid screening in clinical studies and quality control of milk samples donated to milk banks.     

On-line sheathless capillary electrophoresis/nanoelectrospray ionization-tandem mass spectrometry for the analysis of glycosaminoglycan oligosaccharides

A novel approach in glycosaminoglycomics, based on sheathless on-line capillary electrophoresis/nanoelectrospray ionization-quadrupole time of flight-mass spectrometry (CE/nanoESI-QTOF-MS) and tandem MS of extended chondroitin sulfate/dermatan (CS/DS) oligosaccharide chains is described. The methodology required the construction of a new sheathless CE/nanoESI-QTOF-MS configuration, its implementation and optimization for the high sensitivity analysis of CS/DS oligosaccharide mixtures from conditioned culture medium of decorin transfected human embryonic kidney (HEK) 293 cells. Under newly established sheathless on-line CE/(-)nanoESI conditions for glycosaminoglycan (GAG) ionization and MS detection, single CS/DS oligosaccharide components of extended chain length and increased sulfation degree were identified. Molecular ions corresponding to species carrying 5 and 6 negative charges could be generated for large GAG oligosaccharide species in the negative ion nanoESI-MS. The optimized on-line conditions enabled the detection of molecular ions assigned to oversulfated tetradeca-, octadeca-, and eicosasaccharide CS/DS molecules, which represent the category of largest sulfated GAG-derived oligosaccharides evidenced by CE/ESI-MS. By on-line CE/ESI tandem MS in data-dependent acquisition mode the oversulfated eicosasaccharide species could be sequenced and the localization of the additional sulfate group along the chain could be determined.     

Direct analysis of fatty acid profile from milk by thermochemolysis-gas chromatography-mass spectrometry

The fatty acid composition of milk is of considerable interest due to their nutritional and functional properties. Although rapid milk fat separation and transesterification procedures have been developed, the overall procedure remains time consuming, specially, for the analysis of a large number of samples. In this work, a fast and simple method for direct profiling of fatty acids from milk using thermochemolysis has been developed. This method has the capability of directly analyse fatty acids from one drop of milk without fat extraction or cleanup. Our approach for thermochemolysis is based on thermal desorption integrated with a cold trap inlet. The optimized method does not present isomerisation/degradation of polyunsaturated fatty acid and shows milk fatty acid profiles comparable to the conventional method based on fat extraction and alkaline transesterification. Overall, this method has demonstrated significant potential for high throughput analysis of fatty acids in milk.     

Tannins analysis from different medicinal plants extracts using MALDI-TOF and MEKC

The matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and micellar electrokinetic chromatography (MEKC) methods were used to identify and quantify five tannins, (+)-catechin, (?)-epigallocatechin, (?)-epigallocatechin gallate, (?)-epicatechin gallate and (?)-epicatechin, from aqueous, ethanolic and acetonic extracts of Calendula officinalis, Hy-pericum, perforatum, Galium verum and Origanum vulgare. The MALDI-TOF technique was used for screening tannins monomers and oligomers in plant extracts. The sandwich method and matrix 2,5-dihydroxybenzoic acid with a concentration of 10 mg mL^?1 in acetonitrile/ultrapure water/trifluoroacetic acid (20: 80: 0.1, vol.) were used. The electrophoretic method developed for the separation and quantification of 5 catechins in 15 min exhibited good efficiency and precision, low limits of detection (0.0032–0.0153 μ.g mL-¹) and quantification (0.0096–0.0466 μ.g mL^?1). The correlation coefficients (R²) exceeded 0.9986 and the recovery values ranged between 94.25 % and 102.50 %. The present work provides new information on some of the less studied compounds present in plants frequently used in traditional medicine.     

Sequence analysis of alginate-derived oligosaccharides by negative-ion electrospray tandem mass spectrometry

Negative-ion electrospray tandem mass spectrometry (ES-MS/MS) with collision-induced dissociation (CID) is attempted for sequence determination of alginate oligosaccharides, derived from polyanionic alginic acid, polymannuronate, and polyguluronate by partial depolymerization using either alginate lyase or mild acid hydrolysis. Sixteen homo- and hetero-oligomeric fragments were obtained after fractionation by gel-filtration and strong anion exchange high performance liquid chromatography. The product-ion spectra of these alginate oligosaccharides were dominated by intense B-, C-, Y-, and Z-type ions together with (0,2)A- and (2,5)A-ions of lower intensities. Internal mannuronate residues (M) produce weak but specific decarboxylated Z(int)-ions (Z(int) - 44 Da; int: denotes internal), which can be used for distinction of M and a guluronate residue (G) at an internal position. A reducing terminal M or G, although neither gives rise to a specific ion, can be identified by differences in the intensity ratio of fragment ions of the reducing terminal residue [(2,5)A(red)]/[(0,4)A(red)] (red: denotes reducing terminal).     

On-line liquid chromatography/thermospray mass spectrometry in the analysis of oligosaccharides.

The analysis of intact neutral oligosaccharides by on-line liquid chromatography/thermospray mass spectrometry is described. Molecular-weight information on oligomers up to a degree of polymerization of 10 is obtained using an aqueous mobile phase containing 10(-4) mol/L sodium acetate, which was found to be compatible with thermospray interfacing and ionization. Ions due to sodiated and disodiated oligosaccharides are observed under these conditions without fragmentation. The aqueous 10(-4) mol/L sodium acetate mobile phase is demonstrated to be applicable in the separation of mixtures of oligosaccharides on a reversed-phase octadecyl-modified silica column.     

Enzymatic release of oligosaccharides from glycoproteins for chromatographic and electrophoretic analysis

For most separations-based analyses of glycoprotein oligosaccharides, the first step is release of the oligosaccharides from the polypeptide. Historically, O-linked and N-linked oligosaccharides have been released from glycoproteins using chemical means, such as alkaline degradation (β-elimination) or hydrazinolysis. In the last two decades, a growing repertoire of enzymes, including endoglycosidases and glycoamidases, able to release glycoprotein oligosaccharides under mild conditions, have become available. This review traces the discovery characterization and use of these glycoprotein oligosaccharide releasing enzymes. Emphasis is placed on providing information of practical value for the researcher wishing to incorporate enzymatic oligosaccharide release into their study of glycoprotein oligosaccharide structure and function.     

Identification of oligosaccharides from histopathological sections by MALDI imaging mass spectrometry

Direct tissue analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) provides the means for in situ molecular analysis of a wide variety of biomolecules. This technology—known as imaging mass spectrometry (IMS)—allows the measurement of biomolecules in their native biological environments without the need for target-specific reagents such as antibodies. In this study, we applied the IMS technique to formalin-fixed paraffin-embedded samples to identify a substance(s) responsible for the intestinal obstruction caused by an unidentified foreign body. In advance of IMS analysis, some pretreatments were applied. After the deparaffinization of sections, samples were subjected to enzyme digestion. The sections co-crystallized with matrix were desorbed and ionized by a laser pulse with scanning. A combination of α-amylase digestion and the 2,5-dihydroxybenzoic acid matrix gave the best mass spectrum. With the IMS Convolution software which we developed, we could automatically extract meaningful signals from the IMS datasets. The representative peak values were m/z 1,013, 1,175, 1,337, 1,499, 1,661, 1,823, and 1,985. Thus, it was revealed that the material was polymer with a 162-Da unit size, calculated from the even intervals. In comparison with the mass spectra of the histopathological specimen and authentic materials, the main component coincided with amylopectin rather than amylose. Tandem MS analysis proved that the main components were oligosaccharides. Finally, we confirmed the identification of amylopectin by staining with periodic acid-Schiff and iodine. These results for the first time show the advantages of MALDI-IMS in combination with enzyme digestion for the direct analysis of oligosaccharides as a major component of histopathological samples.     

Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis of hyaluronan oligosaccharides

A new method is presented for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronan (HA) with bacterial hyaluronidase (E.C. 4.2.2.1, from Streptomyces hyalurolyticus) using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS). Mixtures containing HA oligosaccharides of tetrasaccharide (4-mer)-34-mer were analyzed using this method. The carboxyl groups of the glucuronate residues in the prepared HA oligomers, were modified as the acidic form (COOH), sodium salts (COONa), organic ammonium salts, or methylesters before MALDI-TOFMS measurement. Among these samples, the methylester form of glucuronate residues in HA oligosaccharides, prepared by methylation using trimethylsilyl diazomethane, afforded high sensitivity for spectra. This simple modification method for carboxyl group methylation of acidic polysaccharides [Hirano et al., Carbohydr. Res., 340, (2005) 2297-2304] provides samples suitable for MALDI-TOF mass spectrometric analysis throughout a significantly enhanced range of masses.     

样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响

人乳内源肽是乳蛋白在乳腺中被降解形成的具有生理功能的肽,是人乳的重要组成部分,研究人乳内源肽对于婴儿健康具有重要的意义.高效液相色谱-串联质谱(LC-MS/MS)联用技术的应用,促使人乳内源肽的研究取得了突破性的进展.人乳中内源肽含量低、干扰组分多,样品制备方法是影响分析结果的关键步骤.为了研究样品制备方法对分析结果的...     

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.     

Detection of milk oligosaccharides in plasma of infants

Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are non-digestible by the human gut, where they are known to play important functions as prebiotics and decoys for binding pathogens. Moreover, it has been proposed that HMO may provide sialic acids to the infant that are important in brain development, however this would require absorption of HMO into the bloodstream. HMO have consistently been found in the urine of humans and other mammals, suggesting systemic absorption. Here, we present a procedure for the profiling of milk oligosaccharides (MO) in plasma samples obtained from 13 term infants hospitalized for surgery for congenital heart disease. The method comprises protein denaturation, oligosaccharide reduction, and porous graphitized carbon solid phase extraction for purification followed by analysis using nHPLC-PGC-chip-TOF-MS. Approximately 15 free MO were typically observed in the plasma of human infants, including LNT, LDFP, LNFT, 3′SL, 6′SL, 3′SLN, and 6′SLN, of which the presence was confirmed using fragmentation studies. A novel third isomer of SLN, not found in human or bovine milk was also consistently detected. Differences in the free MO profiles were observed between infants that were totally formula-fed and infants that received at least some part breast milk. Our results indicate that free MO similar in structure to those found in human milk and urine are present in the blood of infants. The method and results presented here will facilitate further research toward the possible roles of free MO in the development of the infant.