MALDI linear-field reflectron TOF post-source decay analysis of underivatized oligosaccharides: Determination of glycosidic linkages and anomeric configurations using anion attachment

Six different anionic species (fluoride, chloride, bromide, iodide, nitrate, and acetate) are tested for their abilities to form anionic adducts with neutral oligosaccharides that are detectable by MALDI-TOF mass spectrometry. Fluoride and acetate cannot form anionic adducts with the oligosaccharides in significant yields. However, bromide, iodide, and nitrate anionic adducts consistently appear in higher abundances relative to [M - H](-), just like the highly stable chloride adducts. Post-source decay (PSD) decompositions of Br(-), I(-), and NO(3)(-) adducts of oligosaccharides provide no structural information, i.e., they yield the respective anions as the main product ions. However, determination of linkage types is achieved by analysis of structurally-informative diagnostic peaks offered by negative ion PSD spectra of chloride adducts of oligosaccharides, whereas the relative peak intensities of pairs of diagnostic fragment ions allow differentiation of anomeric configurations of glycosidic bonds. Thus, simultaneous identification of the linkage types and anomeric configurations of glycosidic bonds is achieved. Our data indicate that negative ion PSD fragmentation patterns of chloride adducts of oligosaccharides are mainly determined by the linkage types. Correlation may exist between the linkage positions and fragmentation mechanisms and/or steric requirements for both cross-ring and glycosidic bond fragmentations. PSD of the chloride adducts of saccharides containing a terminal Glcalpha1-2Fru linkage also yields chlorine-containing fragment ions which appear to be specifically diagnostic for a fructose linked at the 2-position on the reducing end. This also allows differentiation from saccharides with a 1-1 linked pyranose on the same position.     

Quantitative aspects in electrospray ionization ion trap and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of malto-oligosaccharides

Mass spectrometry is widely applied in carbohydrate analysis, but still quantitative evaluation of data is critical due to different ionization efficiencies of the constituents in a mixture. Different size and chemical structure of the analytes cause their uneven distribution in droplets (electrospray ionization, ESI) or matrix spots (matrix-assisted laser desorption/ionization, MALDI). In addition, instrumental parameters affect final ion yields. In order to study and optimize the latter, an equimolar mixture of malto-oligosaccharides (DP1-6) was analyzed using varying target masses for ESI as well as different matrices and laser power for MALDI. The sodium adducts and derivatives for positive ion mode (hydrazones with Girard's T Reagent, GT) and negative ion mode (reductively aminated with o-aminobenzoic acid, oABA) were studied. Negatively charged oABA-labeled malto-oligosaccharides turned out to be unsuitable for quantification of the malto-oligomeric composition. Best agreement was achieved when applying target masses in the range of the highest homolog in the mixture in electrospray ionization ion trap (ESI-IT) (1-2% deviation with GT label or as Na(+) adducts). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) gave best results when the laser power was adjusted significantly over the desorption/ionization threshold (1% deviation with GT label). Both parameters show significant influence on the determined oligomeric composition. Consequently, estimation and even quantitative determination of amounts of oligosaccharides in a mixture can be achieved when the analytes are labeled and the proper instrumental parameters are used.     

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.     

Structural characterization of 2-aminobenzamide-derivatized oligosaccharides using a matrix-assisted laser desorption/ionization two-stage time-of-flight tandem mass spectrometer

Oligosaccharides were derivatized by reductive amination using 2-aminobenzamide (2-AB) and analyzed by matrix-assisted laser desorption/ionization two-stage time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+Na]+ ions for all 2-AB-derivatized oligosaccharides. A systematic study was conducted on a series of 2-AB-derivatized oligosaccharides to allow rationalization of the fragmentation processes. The MALDI-TOF/TOF-MS/MS spectra of the [M+Na]+ ions of 2-AB-derivatized oligosaccharides were dominated by glycosidic cleavages. These fragments originating both from the reducing and the non-reducing ends of the oligosaccharide yield information on sequence and branching. Moreover, the MALDI-TOF/TOF-MS/MS spectra were also characterized by abundant cross-ring fragments which are very informative on the linkages of the monosaccharide residues constituting these oligosaccharides. MALDI-TOF/TOF-MS/MS analysis of 2-AB-derivatized oligosaccharides, by providing structural information at the low-picomole level, appears to be a powerful tool for carbohydrate structural analysis.     

基质辅助激光解吸电离飞行时间质谱表征双二氮杂萘酮化合物

采用基质辅助激光解吸电离飞行时间质谱(MAIDI-TOF MS)，以2，5-二羟基苯甲酸(DHB)为基体对10种合成的新型双二氮杂萘酮化合物进行了质谱分析，得到了较强的样品准分子离子信号；对校正标样进行了筛选并讨论了标样对测定准确度的影响；研究了样品与金属离子形成加成物的性质。     

A novel strategy for MALDI-TOF MS analysis of small molecules

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) does not work efficiently on small molecules (usually with molecular weight below 500 Da) because of the interference of matrix-related peaks in low m/z region. The previous methods developed for this problem focused on reducing the peaks caused by the traditional matrices. Here, we report a novel strategy to analyze small molecules in a high and interference-free mass range by using metal-phthalocyanines (MPcs) as matrices which should be capable of forming matrix-analyte adducts. The mass of the target analyte was calculated by subtracting the mass of MPc from the mass of the MPc-analyte adduct. MPcs were also detectable and could serve as internal standards. Various MPcs with aromatic or aliphatic groups and different metal centers were then synthesized and explored. Aluminum-phthalocyanines (AlPcs), gallium-phthalocyanines (GaPcs), and indium-phthalocyanines (InPcs) were efficient matrices to form MPc-analyte adducts in either the positive or negative ion mode. The detection limits varied from 17 to 75 fmol, depending on analyte types. The mechanism of adducts formation was also proposed. Collectively, our strategy provides a novel and efficient way to analyze small molecules by MALDI-TOF MS.     

Analysis of 8-aminonaphthalene-1,3,6-trisulfonate-derivatized oligosaccharides by capillary electrophoresis-electrospray ionization quadrupole ion trap mass spectrometry.

Dextran was partially hydrolyzed with 0.1 mol/l HCl and the hydrolysate was derivatized with 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) by reductive amination. The derivatized-oligosaccharide mixture was separated by capillary electrophoresis (CE) in a buffer of 1% HAc-NH4OH, pH 3.4, and the separated components were detected on-line by electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT-MS) in the negative ion mode. A mass accuracy lower than 0.01% could be achieved and as low as 1.6 pmol of detxran octaose could be detected. ANTS-derivatized dextran oligosaccharide with a degree of polymerization (DP) lower than 6 produced both [M-H]- and [M-2H]2- ions, whereas those with a DP of 6 or higher than 6 produced only [M-2H]2- ion. As 1< or =DP< or =6, the percentage of [M-2H]2- ion in the total ions of [M-H]- and [M-2H]2- was found to be a linear function of the logarithmic DP. Molecular mass determination with ESI-QIT-MS strengthens the power of CE analysis of oligosaccharides.     

Quantitative analysis of malto-oligosaccharides by MALDI-TOF mass spectrometry, capillary electrophoresis and anion exchange chromatography

The analysis of malto-oligosaccharides by MALDI-TOF mass spectrometry (MS), capillary electrophoresis (CE) and anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is described. Appropriate methods were developed which enabled the resolution of the oligosaccharides and quantification of the peak areas. It could be shown that each technique provided a different distribution profile of the maltodextrins. Using MALDI-TOF MS signals of higher molecular weight oligomers were enhanced while low molecular weight analogues were discriminated. Thus, the response factor depends on the degree of polymerization (DP) of the carbohydrates. Homologues up to DP-15 could be detected. Analysis of the maltodextrins by CE was accomplished by derivatization of the sugars with 4-aminobenzonitrile (ABN) and 8-aminonaphthalene-1,3,6-trisulfonic acid, respectively. By using the latter reagent oligosaccharides up to DP-13 were detected while derivatization with ABN allowed detection up to DP-9. The molecular weight distribution obtained by both approaches were the same. HPAEC-PAD enabled the determination of oligomers up to DP-9. The distribution obtained by this technique showed somewhat lower signals of the small homologues than those found by CE while the opposite held for higher molecular weight compounds. Hydrolysis of the carbohydrates by the derivatization reaction prior to CE analysis, which increased the proportion of low molecular weight homologues, may account for these findings.     

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.     

Analysis of 8-aminonaphthalene-1,3,6-trisulfonic acid labelled N-glycans by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a fast and efficient analytical method which is now widely used in glycobiology for the separation and quantification of free or glycoprotein-released oligosaccharides. However, since identification by FACE of N-glycan structures is only based on their electrophoretic mobility after labelling with 8-aminonaphthalene-1,3, 6-trisulfonic acid (ANTS), co-migration of derived glycans on gel could occur which may result in erroneous structural assignments. As a consequence, a protocol was developed for the fast and efficient matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of ANTS-labelled N-glycans. N-Glycans were isolated from plant and mammalian glycoproteins, reductively aminated with the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. The ANTS-labelled glycans were eluted from FACE gel slices and then analysed by MALDI-TOF mass spectrometry in negative ion mode. Using 3-aminoquinoline containing 2.5 mM citrate NH(4)(+) as matrix, neutral N-linked N-glycans, as well as labelled sialylated oligosaccharides, were found to be easily detected in the 2-10 picomole range giving rise to ?M - H(-) ions.     

Quantitative analysis of malto-oligosaccharides by MALDI-TOF mass spectrometry, capillary electrophoresis and anion exchange chromatography

The analysis of malto-oligosaccharides by MALDI-TOF mass spectrometry (MS), capillary electrophoresis (CE) and anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is described. Appropriate methods were developed which enabled the resolution of the oligosaccharides and quantification of the peak areas. It could be shown that each technique provided a different distribution profile of the maltodextrins. Using MALDI-TOF MS signals of higher molecular weight oligomers were enhanced while low molecular weight analogues were discriminated. Thus, the response factor depends on the degree of polymerization (DP) of the carbohydrates. Homologues up to DP-15 could be detected. Analysis of the maltodextrins by CE was accomplished by derivatization of the sugars with 4-aminobenzonitrile (ABN) and 8-aminonaphthalene-1,3,6-trisulfonic acid, respectively. By using the latter reagent oligosaccharides up to DP-13 were detected while derivatization with ABN allowed detection up to DP-9. The molecular weight distribution obtained by both approaches were the same. HPAEC-PAD enabled the determination of oligomers up to DP-9. The distribution obtained by this technique showed somewhat lower signals of the small homologues than those found by CE while the opposite held for higher molecular weight compounds. Hydrolysis of the carbohydrates by the derivatization reaction prior to CE analysis, which increased the proportion of low molecular weight homologues, may account for these findings. Receiverd: 28 August 1997 / Revised: 24 November 1997 / Accepted: 25 November 1997     

Selective molecular recognition of arginine by anionic salt bridge formation with bis-phosphate crown ethers: implications for gas phase peptide acidity from adduct dissociation

Arginine forms a stable noncovalent anionic salt bridge complex with DP (a crown ether which contains two endocyclic dialkylhydrogenphosphate esters). Abundant adduct formation with DP is observed for complexes with arginine, YAKR, HPPGFSPFR, AAKRKAA, RR, RPPGFSPFR, RYLGYL, RGDS, and YGGFMRGL in electrospray ionization mass spectrometry (ESI-MS) experiments. DFT calculations predict a hydrogen bonded salt bridge structure with a protonated guanidinium flanked by two deprotonated phosphates to be the lowest energy structure. Dissociation of DP/peptide adducts reveals that, in general, the relative gas phase acidity of a peptide is dependent on peptide length, with longer peptides being more acidic. In particular, peptides that are six residues or more in length can stabilize the deprotonated C-terminus by extensive hydrogen bonding with the peptide backbone. Dissociation of DP/peptide complexes often yields the deprotonated peptide, allowing for the facile formation of anionic peptides that otherwise would be difficult to generate in high abundance. Although DP has a preference for binding to arginine residues in peptides, DP is also observed to form less abundant complexes with peptides containing multiple lysines. Lys-Xxx-Lys and Lys-Lys sequences form low abundance anionic adducts with DP. For example, KKKK exclusively forms a double adduct with one net negative charge on the complex.     

Fragmentation characteristics of permethylated oligosaccharides using a matrix-assisted laser desorption/ionization two-stage time-of-flight (TOF/TOF) tandem mass spectrometer

Matrix-assisted laser desorption/ionization two-stage time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS) was applied to characterize permethylated oligosaccharides. Under these ionization conditions such derivatives yield intense signals corresponding to sodium-cationized molecular species. A systematic study was conducted on a series of neutral and sialylated permethylated oligosaccharides to allow rationalization of the fragmentation processes. The major fragments observed in the MALDI-TOF/TOF-MS/MS spectra result from cleavage of glycosidic bonds, preferentially at N-acetylhexosamine and sialic acid residues. The fragments originating from both the reducing and the non-reducing ends of the glycan yield information on sequence and branching. Cross-ring cleavages, which are very informative of the linkages of the monosaccharide residues constituting these oligosaccharides, and 'internal' cleavage ions which are derived from elimination of substituents from around the pyranose ring, were also observed. This extensive fragmentation was shown to be useful for the structural characterization of oligosaccharides. MALDI-TOF/TOF-MS/MS of permethylated oligosaccharides appears to be a powerful tool for carbohydrate structural analysis.     

Nano-HPLC of Oligosaccharides – Method Development and Optimization

 The separation of homologous maltodextrins using methyl-, ethyl- and n-butyl esters of aminobenzoic acid and n-heptyloxyaniline by means of reversed phase nano-HPLC is presented. Fused silica capillaries packed with four different stationary phases were tested, the separation of the derivatized sugars was optimized and the separation properties of these columns were tested on the basis of the van Deemter plots. UV/VIS detection was used (285 nm for the aminobenzoic esters and 275 nm for the aniline derivatives) in combination with on-line ESI triple quadrupole mass spectrometry and off-line MALDI-TOF mass spectrometry. The choice of the stationary phase had a tremendous influence on the separation. Optimal results were obtained with an encapsulated ODS stationary phase and n-butyl aminebenzoate as label for the oligosaccharides. Malto-oligosaccharides with a degree of polymerization of up to of 25 could be resolved to baseline. On-line coupling with ESI-MS yielded additional information as a result of induced fragmentation and increased sensitivity by monitoring precursor ion. Off-line mass detection with MALDI-TOF was performed on the separated derivatized saccharides collected from nano-HPLC runs. The MALDI-TOF mass spectra confirm the ESI-MS data. Received January 25, 2001. Revision April 28, 2001.     

刺五加寡糖的电喷雾多级串联质谱研究

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

MALDI-MS/MS with Traveling Wave Ion Mobility for the Structural Analysis of N-Linked Glycans

The preference for singly charged ion formation by MALDI makes it a better choice than electrospray ionization for profiling mixtures of N-glycans. For structural analysis, fragmentation of negative ions often yields more informative spectra than fragmentation of positive ones but such ions are more difficult to produce from neutral glycans under MALDI conditions. This work investigates conditions for the formation of both positive and negative ions by MALDI from N-linked glycans released from glycoproteins and their subsequent MS/MS and ion mobility behaviour. 2,4,6-Trihydroxyacetophenone (THAP) doped with ammonium nitrate was found to give optimal ion yields in negative ion mode. Ammonium chloride or phosphate also yielded prominent adducts but anionic carbohydrates such as sulfated N-glycans tended to ionize preferentially. Carbohydrates adducted with all three adducts (phosphate, chloride, and nitrate) produced good negative ion CID spectra but those adducted with iodide and sulfate did not yield fragment ions although they gave stronger signals. Fragmentation paralleled that seen following electrospray ionization providing superior spectra than could be obtained by PSD on MALDI-TOF instruments or with ion traps. In addition, ion mobility drift times of the adducted glycans and the ability of this technique to separate isomers also mirrored those obtained following ESI sample introduction. Ion mobility also allowed profiles to be obtained from samples whose MALDI spectra showed no evidence of such ions allowing the technique to be used in conditions where sample amounts were limiting. The method was applied to N-glycans released from the recombinant human immunodeficiency virus glycoprotein, gp120.     

Differently complex oligosaccharides can be easily identified by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry directly from a standard thin-layer chromatography plate

Thin-layer chromatography (TLC) is a simple, fast and inexpensive separation method. Unambiguous identification of the TLC spots is, however, often a problem. Here we show for the first time that oligosaccharides (derived from dextran, alginate, hyaluronan and chondroitin sulfate) can be characterized by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) directly on a TLC plate. The applied oligosaccharides were either commercially available or obtained from the polysaccharides by HCl-induced hydrolysis. Normal phase TLC was followed by MALDI-TOF MS subsequent to matrix deposition. It will be shown that high quality mass spectra can be obtained that enable unequivocal assignments. It will also be shown that the high content of formic acid in the solvent system does not confer major problems but is responsible for the partial formylation of the analyte and minor N-acetyl loss from hyaluronan and chondroitin sulfate.     

Nano-HPLC-mass spectrometry and MEKC for the analysis of oligosaccharides from human milk

The separation of oligosaccharides derivatized with various esters of aminobenzoic acid by means of reversed-phase nano-HPLC (nHPLC) with on-line ESI mass spectrometry and off-line MALDI-TOF mass spectrometry as well as MEKC is described. For this purpose methyl, ethyl and butyl aminobenzoates and heptyloxyaniline were used as derivatization agents for homologous maltodextrins and oligosaccharides from human milk. Four different C(18) stationary phases were tested for this purpose because the type of stationary phase was shown to have a dramatic effect on the performance of the separation. Optimal results were obtained using n-butyl aminobenzoate as label and an encapsulated ODS stationary phase. The on-line coupling of nHPLC to ESI MS allowed to separate and identify various oligosaccharides from human milk. This technique enabled the exact attribution of the molecular structure to a signal in the chromatogram. In a second approach oligosaccharides were separated by nHPLC and subsequently fractionated. The fractions were analyzed by MALDI-TOF mass spectrometry. The results obtained by this approach confirmed the ESI MS data. An analogous separation profile was obtained by using sodium dodecyl sulfate in MEKC, which proves that the retention mechanisms of both techniques are identical.     

基质辅助激光解吸附飞行时间质谱分析缩合单宁的阳离子化问题

以单价金属离子Cs 和Na 作为离子化试剂,对3种缩合单宁进行基质辅助激光解吸附飞行时间(MALD I-TOF)质谱分析。加入Na 作为阳离子化试剂,能得到较高质量的质谱图。但由于实验通道中几乎不可能完全去除的K 的干扰而会高估棓儿茶酚/表棓儿茶酚单元的组成比例,从而影响对棓儿茶酚/表棓儿茶酚单元存在与否的判断;选择Cs 作为阳离子化试剂可以避免此问题,但在复杂的缩合单宁分析中,同样因为杂质离子Na 和K 的干扰而使得质谱图变得更复杂;未去离子处理直接对缩合单宁进行MALD I-TOF质谱分析与去离子并加入Cs 的处理比去离子并加入Na 能检测到更高聚合度的高聚物,检测到离子峰强度最高的聚合物随离子不同而不同。     

Rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry

A rapid and quantitative method for solid-phase methyl esterification of carboxy groups of various sialylated oligosaccharides has been established. The method employed a triazene derivative, 3-methyl-1-p-tolyltriazene, for facile derivatization of oligosaccharides immobilized onto general solid supports such as Affi-Gel Hz and gold colloidal nanoparticles in a multiwell plate. The workflow protocol was optimized for the solid-phase processing of captured sialylated/unsialylated oligosaccharides separated from crude sample mixtures by chemical ligation. From tryptic and/or PNGase F-digest mixtures of glycoproteins, purification by chemoselective immobilization, esterification and recovery were achieved in the same well of the filter plate within three hours when used in conjunction with "glycoblotting technology" (S.-I. Nishimura, K. Niikura, M. Kurogochi, T. Matsushita, M. Fumoto, H. Hinou, R. Kamitani, H. Nakagawa, K. Deguchi, N. Miura, K. Monde, H. Kondo, High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry: Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 91-96). The recovered materials were directly applicable to subsequent characterization by mass spectrometric techniques such as MALDI-TOF for large-scale glycomics of both neutral and sialylated oligosaccharides. On-bead/on-gold nanoparticle derivatization of glycans containing sialic acids allowed rapid and quantitative glycoform profiling by MALDI-TOF MS with reflector and positive ion mode. In addition to its simplicity and speed, the method eliminates the use of unfavorable halogenated solvents such as chloroform and dichloromethane or volatile solvents such as diethyl ether and hexane, resulting in a practical and green chemical method for automated robotic adaptation.