Size exclusion chromatography/mass spectrometry applied to the analysis of polysaccharides.

A novel method for analysing polysaccharide materials is described which employs size-exclusion chromatography (SEC) followed by detection by on-line electrospray ionisation mass spectrometry (ESI-MS) and off-line matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). It is demonstrated through SEC/ESI ion trap mass spectrometry that the formation of multiply charged oligomer ions, which bind up to five sodium cations, allows the rapid analysis of polysaccharide ions with molecular weights in excess of 9 kDa. MALDI spectra generated from fractionation of the effluent collected from the same SEC separation are shown to be in good agreement with the ESI spectra with respect to molecular weight distributions and types of ions generated. ESI and MALDI mass spectra of samples obtained from sequential graded ethanol precipitation and SEC fractionation of acid and enzymatically digested arabinoxylan polysaccharides show important structural differences between polysaccharide fragments. In addition, a comparison is made between the mass spectra of native and permethylated SEC-separated fragments of acid and enzymatically treated arabinogalactan. Linkage information of the permethylated arabinogalactan oligomers can be rapidly established through the use of on-line SEC/ESI-MS( n) experiments.     

Structural analysis of sulfated glycans by sequential double-permethylation using methyl iodide and deuteromethyl iodide

MALDI mass spectrometric characterization of sulfated glycans is often challenging due to their low ionization response in the positive ion mode. Here we demonstrate a new analytical approach, allowing the measurement of sulfated glycans by substituting the sulfate group with a deuteromethyl group. Sulfated glycan samples are initially permethylated before the methanolytic cleavage of their sulfate groups. Desulfated and permethylated glycans are then subjected to another permethylation step using deuteromethyl iodide to label the hydroxyl groups resulting from methanolysis. The number of attached sulfate groups is subsequently calculated from the mass-shift resulting from the chemical cleavage of these sulfate groups. The position of the sulfate substitution is then determined by collision-induced dissociation (CID) tandem mass spectrometry of permethylated and permethylated plus deuteromethylated samples. The described approach was initially optimized and validated using linear standard glycans, while its effectiveness has also been demonstrated here for the N-glycans derived from bovine thyroid-stimulating hormone (bTSH).     

Characterization of the O-4 phosphorylated and O-5 substituted Kdo reducing end group and sequencing of the core oligosaccharide of Aeromonas salmonicida ssp salmonicida lipopolysaccharide using tandem mass spectrometry

The molecular structure of the wild strain of the lipopolysaccharide core of Aeromonas salmonicida, ssp salmonicida has been sequenced using tandem mass spectrometry. The core oligosaccharide was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group, and its structure is proposed as the follows: [structure: see text] After the core oligosaccharide of LPS was released from the lipid A portion by conventional treatment with 1% acetic acid, we demonstrated the existence of a homogeneous mixture composed mainly of the native core oligosaccharide containing the Kdo with its O-4 phosphate group intact, and a degraded core oligosaccharide mixture, which eliminated the O-4 phosphate group with extreme facility. The precise molecular structure and glycone sequence of the homogeneous mixture of phosphorylated and dephosphorylated core oligosaccharides was determined by electrospray ionization (ESI) mass spectrometry and tandem mass spectrometric analysis. CID-MS/MS of the homogeneous mixture of permethylated core oligosaccharides afforded a series of diagnostic product ions which confirmed the established sequence of the glycones to be determined. Matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry reconfirmed the molecular structure of the dephosphorylated homogeneous permethylated mixture of the core oligosaccharides containing the diastereomeric 4,8- and 4,7-anhydro-alpha-keto acids.     

Stereochemical effects on the mass spectrometric behavior of native and derivatized trisaccharide isomers: comparisons with results from molecular modeling

Differentiation of oligosaccharide isomers by mass spectrometry (MS) is a challenging task. For native, permethylated and peracetylated trisaccharides, matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) MS and liquid secondary ionization (LSI) MS experiments can produce complementary results that are useful for molecular mass and sugar sequence determination and isomer differentiation. Linear MALDI/TOF-MS analysis of native and derivatized oligosaccharides usually produces cationized molecular ions. Characterization by LSI-MS and tandem mass spectrometry (LSI-MS/MS) typically may yield only low-abundance protonated molecular ions but produces dominant B-type ions by elimination of ROH (R = Me, Ac) from the C-1 position at the reducing end and distinctive sequence-related fragments. Results for four milk trisaccharides, two neutral (fucosyllactoses) and two sialylated (sialyllactoses), are presented to demonstrate the utility of microscale permethylation and gas-to-solid phase peracetylation for high sensitivity structural elucidation. For the pairs of carbohydrates investigated in this study by LSI-MS, LSI-MS/MS and linear MALDI/TOF-MS, the fragmentation patterns of the native, permethylated and peracetylated isomer pairs are shown to differ markedly as a consequence of their limited dissimilarities. In addition, the tendency of sialylated carbohydrates to form lactones upon peracetylation has been exploited to take advantage of the variation in the extent of lactonization with orientation of the sialic acid moiety relative to the adjacent sugar rings. Lactone formation is favored for 3'-sialyllactose compared with its 6'-isomer; Hyperchem was employed to indicate the relative stabilities of the molecular and fragment ions and to visualize the molecules in 3D (rather than to obtain absolute conformational energy values). The relative conformational energies of lactonized and non-lactonized ions were calculated using the Hyperchem software; their values support the trends observed by MS.     

A new naphthimidazole derivative for saccharide labeling with enhanced sensitivity in mass spectrometry detection

A series of saccharides, including maltoheptose, blood type B antigen, pullulan and the glucan of Ganoderma lucidum, are easily converted into the naphthimidazole (NAIM) derivatives in high yields by the iodine‐promoted oxidative condensation. The NAIM‐labeled saccharides, without further purification, show enhanced signals in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS). The combined use of NAIM derivatization and MALDI‐TOFMS analysis thus provides a rapid method for identification of saccharides even in less than 1 pmol of saccharide in the sample. Characterization of the biologically active saccharides and complex polysaccharides is also achieved through the NAIM‐derivatization method. This study can be further applied to facilitate the isolation and analysis of novel saccharides. Copyright © 2009 John Wiley & Sons, Ltd.     

基质辅助激光解吸电离飞行时间质谱研究（I）：林芳烃化合物

采用基质辅助激光解吸电离飞行时间质谱（ＭＡＬＤＩ－ＴＯＦ－ＭＳ）对杯芳烃化合物进行了分析。探讨了样品的制备条件、仪器操作参数等因素对测定结果的影响。２５种样品的质谱数据表明,利用ＭＡＬＤＩ－ＴＯＦ－ＭＳ可非常方便地得到灵敏度、分辨率、准确度均较高,且易于识别和解析的质谱图,为此类化合物的质谱表征提供和建立了一种新的高效分析方法。     

Post-source decay mass spectrometry: optimized calibration procedure and structural characterization of permethylated oligosaccharides

Permethylated oligosaccharides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) using a reflectron time-of-flight instrument in the post-source decay (PSD) mode. Under these ionization conditions, such derivatives yield intense signals corresponding to sodium or potassium cationized molecular species. Fragments observed in the PSD spectra result exclusively from cleavage of glycosidic bonds, preferentially at N-acetylhexosamine residues. A systematic study was carried out on a series of permethylated oligosaccharides to allow rationalization of the fragmentation processes. Fragments originating from both the reducing and the non-reducing ends of the oligosaccharide yield information on sequence and branching. Moreover, glycosyl residues linked in position 3 of HexNAc units give rise to a highly specific elimination process, which allows unambiguous assignment of (1-3) interglycosidic linkages. Special attention was paid to the structural analysis of oligosaccharides carrying the commonly encountered fucosyl and sialyl end-caps. In the case of sialylated residues, a targeted methodology involving desialylation and specific CD3-labeling of the nascent free hydroxyl groups was developed to mark the initial location of sialic acid residues along the oligosaccharide backbone. As accurate mass determination of fragment ions is essential for their assignment, a simplified protocol for the calibration in the PSD mode is described. This procedure allows the determination of the correction function parameters required to process the data for an instrument that employs post-acceleration detection. MALDI/PSD-MS of permethylated oligosaccharides, by providing structural information at the low picomole level, appears to be a valuable complement, or an alternative, to the techniques currently in use for carbohydrate structural analysis.     

Matrix-assisted laser desorption/ionization in-source decay combined with tandem time-of-flight mass spectrometry of permethylated oligosaccharides: targeted characterization of specific parts of the glycan structure

Matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) has been introduced in recent years as a valuable tool for the structural characterization of permethylated oligosaccharides. In this report, we describe the combination of MALDI in-source decay (ISD) with the subsequent TOF/TOF-MS analyses of specific fragments, allowing the detailed characterization of the selected part of the oligosaccharide molecule. Part of the second-generation fragment ions were different from those observed in conventional MALDI-TOF/TOF-MS experiments. Other fragments, which had already been observed in conventional MALDI-TOF/TOF-MS and again showed up in second-generation fragment analysis, could be assigned to specific parts of the molecule. Our approach disclosed different structural features of the oligosaccharides: due to permethylation, the glycosidic linkage fragments allowed the distinction between terminal, monosubstituted and disubstituted monosaccharides and indicated the oligosaccharide sequence. Moreover, substitution positions were deduced based on characteristic cross-ring fragmentation by high-energy collision-induced fragmentation. In conclusion, combination of MALDI-ISD with TOF/TOF-MS allows the detailed characterization of specific moieties of permethylated oligosaccharides and is, therefore, a powerful technique for structural glycomics.     

Characterization of Minor N-linked Glycans on Antibodies Using Endo H Release and MALDI–Mass Spectrometry

Abstract

A MALDI mass spectrometry method using Bruker Daltonic's LIFT technology for MS/MS analysis has been developed for profiling and characterizing low abundant N-glycans from recombinant immunoglobulin G (IgG) antibodies. In this method, Endoglycosidase H (Endo H) released N-glycans are derivatized at their reducing end with 2-aminobenzamide (2-AB) and separated by normal phase chromatography. Endo H hydrolyses the bond between the two GlcNAc residues of the trimannosyl core of high mannose and hybrid N-linked glycans, leaving the core GlcNAc attached to the protein. High mannose and hybrid type N-glycans are released from the glycoprotein whereas the more abundant, complex biantennary type oligosaccharide structures are unaffected. Analysis of Endo H treated glycan moieties by MALDI mass spectrometry identified several minor species of high mannose and hybrid type glycans. Subsequent MALDI TOF MS/MS analysis of the resulting products yielded information about structural features of the high mannose and hybrid type glycans. This study involving Endo H treatment followed by MALDI mass spectrometry coupled with LIFT technology for MS/MS analysis offers a specific and sensitive technique for visualizing, and characterizing minor glycan species.     

MALDI imaging directed laser ablation tissue microsampling for data independent acquisition proteomics

A multimodal workflow for mass spectrometry imaging was developed that combines MALDI imaging with protein identification and quantification by liquid chromatography tandem mass spectrometry (LC‐MS/MS). Thin tissue sections were analyzed by MALDI imaging, and the regions of interest (ROI) were identified using a smoothing and edge detection procedure. A midinfrared laser at 3‐μm wavelength was used to remove the ROI from the brain tissue section after MALDI mass spectrometry imaging (MALDI MSI). The captured material was processed using a single‐pot solid‐phase‐enhanced sample preparation (SP3) method and analyzed by LC‐MS/MS using ion mobility (IM) enhanced data independent acquisition (DIA) to identify and quantify proteins; more than 600 proteins were identified. Using a modified database that included isoform and the post‐translational modifications chain, loss of the initial methionine, and acetylation, 14 MALDI MSI peaks were identified. Comparison of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the identified proteins was achieved through an evolutionary relationships classification system.     

基质辅助激光解吸附电离飞行时间质谱分析化学合成酵母转录活化因子

基质辅助激光解吸附电离飞行时间质谱是近年来发展很快的一种新颖的质谱分析仪，尤其适用于生物大分子的分子量测定，本文介绍它在确证化学合成酵母转录活化因子Ａｃ（Ｑ＾２３６，Ｑ＾２４２），ＧＣＮ４（２２６－２５２）－ＧＧＣ－ＮＨ２的一级结构中的重要作用。     

Massenspektrometrie von flavonoid-O-glykosiden—I

Thirteen naturally occuring flavonol-O-monoglycosides and twelve flavonol-bis-O-glycosides were permethylated in micro-scale and subjected to mass spectrometry. Analysis of the spectra yielded a simple and quick method for determining the linkage position of the sugar in monoglycosides and for differentiating between possible isomers in the case of flavonol-3,7-bis-O-glycosides.     

双步激光解析/激光电离质谱法及其应用的新进展

激光光电离技术已广泛应用于质谱领域.基于单束激光的基质辅助激光解析(MALDI)质谱分析方法,已成为质谱分析生物大分子的标准方法之一.本文介绍的是另一种新的激光质谱分析方法:双步激光解析/激光电离质谱法(L2MS),与MALDI相比,该方法不需要加入与样品形成共结晶的基质,同时可通过独立地改变两束激光的光强和波长达到优...     

MALDI-TOF MS analysis of native and permethylated or benzimidazole-derivatized polysaccharides

MALDI-TOF MS provides rapid and sensitive analyses of larger biomolecules. However, MS analyses of polysaccharide have been reported to have lower sensitivity compared to peptides and proteins. Here, we investigated some polysaccharides chemically derivatized by permethylation and ortho-phenylene diamine (OPD) tagging. Methylated glycan is obviously able to improve the sensitivity for mass spectrometry detection. Oxidative condensation by UV-activation tagging to saccharides by OPD and peptide-OPD also improve the sensitivity of MALDI-TOF MS analyses. Polysaccharides including dextran, glucomannan, arabinoxylan, arabinogalactan and beta-1,3-glucan, isolated from nutritional supplements of Ganoderma lucidum and Saccharomyces pastorianus were measured using MALDI-TOF MS with 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix. These glycans were also derivatized to methylated and benzimidazole-tagged glycans by chemical transformation for molecular weight analysis. The derivatized polysaccharides showed excellent MALDI-TOF MS signal enhancement in the molecular weight range from 1 to 5 kDa. Here, we demonstrate an efficient method to give glycan-benzimidazole (glycan-BIM) derivatives for polysaccharide determination in MALDI-TOF MS. Therefore, permethylated or benzimidazole-derivatized polysaccharides provide a new option for polysaccharide analysis using MALDI-TOF MS.     

High-throughput one-bead-one-compound approach to peptide-encoded combinatorial libraries: MALDI-MS analysis of single TentaGel beads

The identification of pharmacologically promising compounds (lead compounds) from combinatorial libraries is frequently limited by the throughput of the analytical technique employed. Fourier transform mass spectrometry (FTMS) offers high sensitivity, mass accuracy (m/Deltam > 500 000), and sequencing capabilities. A rapid and efficient method for high-throughput analysis of single beads from peptide-encoded combinatorial libraries with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is presented. Encoding peptides on single beads are identified and structurally characterized by MALDI time-of-flight (TOF) and ultrahigh-resolution MALDI Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. A strategy of on-probe sample preparation is developed to minimize handling of the beads.     

Identification of volatile basic components in tobacco by headspace liquid-phase microextraction coupled to matrix-assisted laser desorption/ionization with Fourier transform mass spectrometry

A method incorporating headspace liquid-phase microextraction (HS-LPME) coupled to matrix-assisted laser desorption/ionization (MALDI) with Fourier transform mass spectrometry (FTMS) was established to analyze volatile basic components in tobacco. The sample preparation volume for MALDI-MS was compatible with the volume of the solvent microdrop in the HS-LPME procedure. The pH and the polarity of the solvent for HS-LPME were adjusted by choice of the MALDI matrix and matrix additive. Based on the elemental composition and tandem mass spectrometry information, 25 volatile nitrogenous compounds in tobacco were detected and identified. The approach is fast and sensitive, and has the potential for automation for high-throughput analysis. This approach offers an alternative method for analysis of trace volatile organic compounds in complex samples.     

Solid phase microextraction with matrix assisted laser desorption/ionization introduction to mass spectrometry and ion mobility spectrometry

Solid phase microextraction (SPME) with matrix assisted laser desorption/ionization (MALDI) introduction was coupled to mass spectrometry and ion mobility spectrometry. Nicotine and myoglobin in matrix 2,5-dihydroxybenzonic acid (DHB), enkephalin and substance P in alpha-cyano-4-hydroxy cinnaminic acid were investigated as the target compounds. The tip of an optical fiber was silanized for extraction of the analytes of interest from solution. The optical fiber thus served as the sample extraction surface, the support for the sample plus matrix, and the optical pipe to transfer the laser energy from the laser to the sample. The MALDI worked under atmospheric pressure, and both an ion mobility spectrometer and a quadrupole/time-of-flight mass spectrometer were used for the detection of the SPME/MALDI signal. The spectra obtained demonstrate the feasibility of the SPME with MALDI introduction to mass spectrometry instrumentation.     

Structural analysis of permethylated oligosaccharides using electrospray ionization quadrupole time-of-flight tandem mass spectrometry and deutero-reduction

Deutero-reduced permethylated oligosaccharides were analyzed by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) using a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, fitted with a nanoflow ESI source. Under these ionization conditions such derivatives preferentially form sodiated molecular species in addition to protonated molecular species. Under collision-induced dissociation, protonated and sodiated molecular species yield simple and predictable fragment mass spectra. A systematic study was conducted on a series of deutero-reduced permethylated glycans to allow rationalization of the fragmentation processes. MS/MS spectra were characterized by fragments resulting from the cleavage of glycosidic bonds. These fragments originating from both the reducing and the non-reducing ends of the glycan yield information on sequence and branching. Furthermore, the substituent 3-linked to a HexNAc unit was readily eliminated. Special attention was devoted to a systematic study of fucosylated glycans. The fucosylated deutero-reduced permethylated glycans were submitted to an acidic hydrolysis, releasing specifically the fucosyl residues. The nascent free hydroxyl groups were subsequently CD3-labelled in order to determine the positions initially bearing the fucosyl residues along the oligosaccharide backbone. This methodology was finally applied to characterize a glycan pool enzymatically released from glycoproteins. The present data show that structural elucidation can be achieved at the 50 fmol level.     

基质辅助激光解吸电离飞行时间质谱研究2: 四硫 富瓦烯化合物

采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),对四硫富瓦烯化合物进行质谱表征。在所用的实验条件下,样品很容易解吸电离生成单电荷分子离子,得到单同位素分辨的质谱图。26种实际样品的质谱分析结果表明;MALDI-TOF-MS可以比其它质谱方法更有效、更方便地用于此类化合物的质谱分析,解决了此类化合物不易进行质谱鉴定的难题。     

Hyphenation of capillary high-performance liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for nano-scale screening of single-bead combinatorial libraries

This paper focuses on the technical aspects of chemical screening from 384-well plate nano-scale single-bead combinatorial libraries. The analytical technique utilized is a combination of capillary liquid chromatography with ultraviolet detection and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The HPLC/MALDI-MS hyphenation is achieved by means of a micro-fraction collector with a peak detection system that automatically collects the peaks onto the MALDI targets for subsequent characterization. Several experimental parameters such as type of 384-well plate, well-plate sealing foils, and a column-switching procedure were investigated using a small test library of nine components. Additionally, the influence of different MALDI matrices, different MALDI targets and sample-spotting techniques on the MALDI detection sensitivity as well as the ruggedness and sample throughput capacity of this technique were studied. Optimum results for the analytes investigated were obtained with 2,5-dihydroxybenzoic acid using on-line mixing of HPLC effluent and matrix solution. To demonstrate the potential of this capillary HPLC/MALDI-TOFMS method, its application to several single-bead libraries was investigated. The instrumental method allowed for the rapid identification and purity assessment of combinatorial libraries with detection limits down to the higher femtomole level using both UV detection and MALDI mass spectrometry.