Comprehensive Analytical Approach toward Glycomic Characterization and Profiling in Urinary Exosomes.
ABSTRACT: Exosomes are extracellular nanosized vesicles with lipid bilayers encapsulating nucleic acids and proteins, both with and without glycosylation. While exosomal nucleic acids and proteins have previously been explored to identify cancer biomarkers with some promising results, little information has been available concerning their glycoconjugate content. Exosomes were isolated from normal urine samples through multistep differential centrifugation. The isolated exosomes have an average size of 146 nm and a spherical shape, as determined by dynamic light scattering and transmission electron microscopy, respectively. N-Glycans were enzymatically released from the isolated vesicles. After being reduced and permethylated, N-glycans were measured by MALDI mass spectrometry. Paucimannosidic, high-mannose, and complex type glycans were identified and their relative abundances were determined. Some detailed structures of these glycans were revealed through liquid chromatography/tandem mass spectrometry (LC/MS-MS). The reduced N-glycans, without being permethylated, were also separated and analyzed by LC/MS-MS, and their structures were further detailed through isomeric separation on porous graphitized carbon (PGC) packed in long capillaries. Using microfractionation before LC/MS-MS, minor multiantennary N-glycans were preconcentrated as based on hydrophobicity or charge. Preconcentration of the reduced and permethylated glycans on a C18 cartridge revealed numerous large glycans, whereas fractionation of the reduced N-glycans by ion-exchange cartridges facilitated detection of sulfated glycans. After removing N-glycans from the original sample aliquot, O-glycans were chemically released from urinary exosomes and profiled, revealing some unusual structures.
Project description:Liquid chromatography/mass spectrometry (LC/MS) is currently considered to be a conventional glycomics analysis strategy due to the high sensitivity and ability to handle complex biological samples. Interpretation of LC/MS data is a major bottleneck in high-throughput glycomics LC/MS-based analysis. The complexity of LC/MS data associated with biological samples prompts the needs to develop computational tools capable of facilitating automated data annotation and quantitation.An LC/MS-based automated data annotation and quantitation software, MultiGlycan-ESI, was developed and utilized for glycan quantitation. Data generated by the software from LC/MS analysis of permethylated N-glycans derived from fetuin were initially validated by manual integration to assess the performance of the software. The performance of MultiGlycan-ESI was then assessed for the quantitation of permethylated fetuin N-glycans analyzed at different concentrations or spiked with permethylated N-glycans derived from human blood serum.The relative abundance differences between data generated by the software and those generated by manual integration were less than 5%, indicating the reliability of MultiGlycan-ESI in quantitation of permethylated glycans analyzed by LC/MS. Automated quantitation resulted in a linear relationship for all six N-glycans derived from 50 ng to 400 ng fetuin with correlation coefficients (R(2) ) greater than 0.93. Spiking of permethylated fetuin N-glycans at different concentrations in permethylated N-glycan samples derived from a 0.02 ?L of HBS also exhibited linear agreement with R(2) values greater than 0.9.With a variety of options, including mass accuracy, merged adducts, and filtering criteria, MultiGlycan-ESI allows automated annotation and quantitation of LC/ESI-MS N-glycan data. The software allows the reliable quantitation of glycan LC/MS data. The software is reliable for automated glycan quantitation, thus facilitating rapid and reliable high-throughput glycomics studies.
Project description:Permethylation is a common derivatization method for MS-based glycomic analyses. Permethylation enhances glycan ionization efficiency in positive MS analysis and improves glycan structural stability. Recent biological glycomic studies have added to the growing body of knowledge and suggest the need for complete structural analysis of glycans. However, reverse phase LC analysis of permethylated glycans usually results in poor isomeric separation. To achieve isomeric separation of permethylated glycans, a porous graphitic carbon (PGC) column was used. PGC columns are well-known for their isomeric separation capability for hydrophilic analyses. In this study, we have optimized temperature conditions to overcome the issues encountered while separating permethylated glycans on a PGC column and found that the highest temperature examined, 75 °C, was optimal. Additionally, we utilized tandem MS to elucidate detailed structural information for the isomers separated. Glycan standards were also utilized to facilitate structural identifications through MS/MS spectra and retention time comparison. The result is an efficient and sensitive method capable of the isomeric separation of permethylated glycans. This method was successfully applied for the isomeric characterization of N-glycans released from the breast cancer cell lines MDA-MB-231 and MDA-MB-231BR (brain seeking). A total of 127 unique glycan structures were identified with 39 isobaric structures, represented as 106 isomers, with 21 nonisomeric glycans. Thirty seven structures exhibited significant differences in isomeric distribution (P < 0.05). Additionally, alterations in the distribution of isomeric sialylated glycans, structures known to be involved in cell attachment to the blood-brain barrier during brain metastasis, were observed.
Project description:Various glycomic analysis methods have been developed due to the essential roles of glycans in biological processes as well as the potential application of glycomics in biomarker discovery in many diseases. Permethylation is currently considered to be one of the most common derivatization methods in MS-based glycomic analysis. Permethylation not only improves ionization efficiency and stability of sialylated glycans in positive mode but also allows for enhanced separation performance on reversed-phase liquid chromatography (RPLC). Recently, RPLC-MS analysis of permethylated glycans exhibited excellent performance in sensitivity and reproducibility and became a widely-applied comprehensive strategy in glycomics. However, separating permethylated glycans by RPLC always suffers from peak broadening for high-molecular-weight branched glycans, which probably due to the low exchange rate between the stationary phase and mobile phase limited by intermolecular interactions of the methyl groups associated with the branching of the glycan structures. In this study, we employed high separation temperature conditions for RPLC of permethylated glycans, thus achieving enhanced peak capacity, improving peak shape, and enhancing separation efficiency. Additionally, partial isomeric separation were observed in RPLC of permethylated glycans at high-temperature. Mathematical processing of the correlation between retention time and molecular weight also revealed the advantage of high-temperature LC method for both manual and automatic glycan identification.
Project description:Mass spectrometry based comparative glycomics is essential for disease biomarker discovery. However, developing a reliable quantification method is still a challenging task.We here report an isotopic labeling strategy employing stable isotopic iodomethane for comparative glycomic profiling by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). N-Glycans released from model glycoproteins and blood serum samples were permethylated with iodomethane ('light') and iodomethane-d1 or -d3 ('heavy') reagents. Permethylated samples were then mixed at equal volumes prior to LC/ESI-MS analysis.Peak intensity ratios of N-glycans isotopically permethylated (Heavy/Light, H/L) were almost equal to the theoretical values. Observed differences were mainly related to the purity of 'heavy' iodomethane reagents (iodomethane-d1 or -d3). The data suggested the efficacy of this strategy to simultaneously quantify N-glycans derived from biological samples representing different cohorts. Accordingly, this strategy is effective in comparing multiple samples in a single LC/ESI-MS analysis. The potential of this strategy for defining glycomic differences in blood serum samples representing different esophageal diseases was explored.LC/ESI-MS comparative glycomic profiling of isotopically permethylated N-glycans derived from biological samples and glycoproteins reliably defined glycan changes associated with biological conditions or glycoproteins expression. As a biological application, this strategy permitted the reliable quantification of glycomic changes associated with different esophageal diseases, including high grade dysplasia, Barrett's disease, and esophageal adenocarcinoma.
Project description:Oligosaccharides in milk not only provide nutrition to the infants but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat, and human milks using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat, and human milk samples (without isomeric consideration) were 11, 8, and 11, respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat, and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by porous graphitic carbon LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using porous graphitic carbon column. Permethylation of the glycan structures facilitated the interpretation of MS/MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers.
Project description:We have previously developed the enabling techniques for sulfoglycomics based on mass spectrometry (MS) analysis of permethylated glycans, which preserves the attractive features of more reliable MS/MS sequencing compared with that performed on native glycans, while providing an easy way to separate and hence enrich the sulfated glycans. Unlike LC-MS/MS analysis of native glycans in negative ion mode that has been more widely in use, the characteristics and potential benefits of similar applications based on permethylated sulfated glycans have not been fully investigated. We report here the important features of reverse phase-based nanoLC-MS/MS analysis of permethylated sulfated glycans in negative ion mode and demonstrate that complementary sets of diagnostic fragment ions afforded can allow rapid identification of various fucosylated, sialylated, sulfated glycotopes and definitive determination of the location of sulfate in a way difficult to achieve by other means. A parallel acquisition of both higher collision energy and trap-based MS(2) coupled with a product dependent MS(3) is conceivably the most productive sulfoglycomic workflow currently possible and the manually curated fragmentation characteristics presented here will allow future developments in automating data analysis.
Project description:The occurrence of numerous structural isomers in glycans from biological sources presents a severe challenge for structural glycomics. The subtle differences among isomeric structures demand analytical methods that can provide structural details while working efficiently with on-line glycan separation methods. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool for mixture analysis, the commonly utilized collision-induced dissociation (CID) method often does not generate a sufficient number of fragments at the MS2 level for comprehensive structural characterization. Here, we studied the electronic excitation dissociation (EED) behaviors of metal-adducted, permethylated glycans, and identified key spectral features that could facilitate both topology and linkage determinations. We developed an EED-based, nanoscale, reversed phase (RP)LC-MS/MS platform, and demonstrated its ability to achieve complete structural elucidation of up to five structural isomers in a single LC-MS/MS analysis. Graphical Abstract.
Project description:An ion mobility quadrupole time-of-flight mass spectrometer was used to examine the gas-phase structures of a set of glycopeptides resulting from proteolytic digestion of the well-characterized glycoproteins bovine ribonuclease B, human transferrin, bovine fetuin and human ?1-acid glycoprotein, the corresponding deglycosylated peptides, and the glycans released by the endoglycosidase PNGase F. When closely related glycoforms did not occur naturally, exoglycosidases were used to achieve stepwise removal of individual saccharide units from the nonreducing termini of the multiantennary structures. Collision cross sections (CCS) were calculated and plotted as a function of mass-to-charge ratio. Linear trendlines were observed for the glycoforms of individual N-linked glycopeptides, the deglycosylated peptides, and the released, deutero-reduced permethylated glycans. For the glycoforms of a given glycopeptide or set of derivatized glycans, the slope of the line connecting CCS values remained similar for the [M+3H]3+ ions observed as the glycan antennae were shortened by stepwise exoglycosidase treatments; this trend was consistent regardless of the peptide length or the saccharide removed. The results form the basis for a database of CCS values and the CCS increments that correspond to changes in glycoform compositions.
Project description:LC-MS/MS is one of the most powerful tools for N-glycan structure elucidation; however, it is still challenging to identify some glycan structures with low abundance. In this study, we investigated the chromatographic behavior of permethylated N-glycans. The relationship between retention times versus molecular weight of dextran, dextrin, and model glycans was investigated. Also, the nonpolar surface area of glycans was calculated and compared to their experimental retention times. Both retention time and nonpolar surface area trends are similar when the intermolecular interaction is included in the calculation. Moreover, retention time corresponds to glycan types and branch types. The N-glycans analysis model, which combines high mass accuracy and retention time, was applied to confirm serum N-glycans. In total, there were 78 N-glycan compositions identified. A linear relationship between retention times and molecular weights were observed for each subgroup of glycan structures, for example, R(2) value for complex N-glycans was determined to be > 0.98. Moreover, the retention time could be further applied to distinguish between structural isomers as well as linkage isomers. MS/MS data were used to confirm the structural isomers.
Project description:Because routine preparation of glycan samples involves multiple reaction and cleaning steps at which sample loss occurs, glycan analysis is typically performed using large tissue samples. This type of analysis yields no detailed molecular spatial information and requires special care to maintain proper storage and shipping conditions. We describe here a new glycan sample preparation protocol using minimized sample preparation steps and optimized procedures. Tissue sections and spotted samples first undergo on-surface enzymatic digestion to release N-glycans. The released glycans are then reduced and permethylated prior to online purification and LC-electrospray ionization (ESI)-MS analysis. The efficiency of this protocol was initially evaluated using model glycoproteins and human blood serum (HBS) spotted on glass or Teflon slides. The new protocol permitted the detection of permethylated N-glycans derived from 10 ng RNase B. On the other hand, 66 N-glycans were identified when injecting the equivalent of permethylated glycans derived from a 0.1-?L aliquot of HBS. On-tissue enzymatic digestion of nude mouse brain tissue permitted the detection of 43 N-glycans. The relative peak areas of these 43 glycans were comparable to those from a C57BL/6 mouse reported by the Consortium for Functional Glycomics (CFG). However, the sample size analyzed in the protocol described here was substantially smaller than for the routine method (submicrogram vs mg). The on-tissue N-glycan profiling method permits high sensitivity and reproducibility and can be widely applied to assess the spatial distribution of glycans associated with tissue sections, and may be correlated with immunoflourescence imaging when adjacent tissue sections are analyzed.