RESUMO
To date, few tools are available for the analysis of the glycome without derivatization, a process which is known to introduce issues such as differential loss of sialic acid and incomplete labeling. We have previously reported the use of ion chromatography-mass spectrometry (IC-MS) to analyze native sialylated and sulfated glycans. Here, we introduce improvements to IC column technology, enabling the separation of neutral glycans while maintaining charge separation capabilities. When implemented in an IC-MS workflow, this enables the structural characterization of a broad array of chemically distinct glycans. With the newly developed IC column and modified IC-MS instrumentation configuration, we qualitatively investigated O-glycome profiles in bovine fetuin and porcine gastric mucins. The improved chromatographic resolution in combination with high-resolution MS data present a powerful tool for glycan structural identification.
Assuntos
Glicômica/métodos , Leite/química , Polissacarídeos/análise , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Fetuínas/química , Mucinas/química , Ácido N-Acetilneuramínico/análise , Sulfatos/análise , Suínos , Espectrometria de Massas em TandemRESUMO
Glycans play an important physiological role and are drawing attention as biomarkers that capture pathophysiological changes. Glycans can be detected by mass spectrometry, but recently matrix-assisted laser desorption/ionization- mass spectrometry imaging (MALDI-MSI) has enabled the visualization of glycans distribution on tissues. In this study, focusing on sialylated glycan (sialoglycans), we investigated the amidation reaction used to visualize glycans distribution, and developed a method of sialic acid derivatization by benzylamidation which is more sensitive than conventional amidation. Furthermore, we adapted this method for visualizing glycans in formalin-fixed paraffin-embedded (FFPE) liver tissue from normal mice and non-alcoholic steatohepatitis (NASH) model mice using MALDI-MSI. As a result, an increase in the distribution of glycan N-Acetylneuraminic acid-(NeuAc) ions was observed in the NASH mouse liver, and the change in glycan structure in the NASH model was clarified.
Assuntos
Fígado/química , Ácido N-Acetilneuramínico/química , Hepatopatia Gordurosa não Alcoólica/patologia , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Fetuínas/química , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Inclusão em Parafina , Polissacarídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Fixação de Tecidos/métodosRESUMO
O-glycosylation is a highly diverse and complex form of protein post-translational modification. Mucin-type O-glycosylation is initiated by the transfer of N-acetyl-galactosamine (GalNAc) to the hydroxyl group of serine, threonine and tyrosine residues through catalysis by a family of glycosyltransferases, the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (E.C. 2.4.1.41) that are conserved across metazoans. In the last decade, structural characterization of glycosylation has substantially advanced due to the development of analytical methods and advances in mass spectrometry. However, O-glycosite mapping remains challenging since mucin-type O-glycans are densely packed, often protecting proteins from cleavage by proteases. Adding to the complexity is the fact that a given glycosite can be modified by different glycans, resulting in an array of glycoforms rising from one glycosite. In this study, we investigated conditions of solid phase extraction (SPE) enrichment, protease digestion, and Electron-transfer/Higher Energy Collision Dissociation (EThcD) fragmentation to optimize identification of O-glycosites in densely glycosylated proteins. Our results revealed that anion-exchange stationary phase is sufficient for glycopeptide enrichment; however, the use of a hydrophobic-containing sorbent was detrimental to the binding of polar-hydrophilic glycopeptides. Different proteases can be employed for enhancing coverage of O-glycosites, while derivatization of negatively charged amino acids or sialic acids would enhance the identification of a short O-glycopeptides. Using a longer than normal electron transfer dissociation (ETD) reaction time, we obtained enhanced coverage of peptide bonds that facilitated the localization of O-glycosites. O-glycosite mapping strategy via proteases, cut-off filtration and solid-phase chemoenzymatic processing. Glycopeptides are enriched by SPE column, followed by release of N-glycans, collection of higher MW O-glycopeptides via MW cut-off filter, O-glycopeptide release via O-protease, and finally detected by LC-MS/MS using EThcD.
Assuntos
Glicopeptídeos/análise , Glicopeptídeos/química , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Aminoácidos/química , Animais , Bovinos , Fracionamento Químico , Cromatografia Líquida , Fetuínas/análise , Fetuínas/química , Fetuínas/metabolismo , Glicopeptídeos/metabolismo , Glicosilação , Mucinas/análise , Mucinas/química , Mucinas/metabolismo , Ácido N-Acetilneuramínico/química , Peptídeo Hidrolases/química , Glândula Submandibular/químicaRESUMO
The analysis of N-linked glycans using liquid chromatography and mass spectrometry (LC-MS) presents significant challenges, particularly owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods have been developed to facilitate improved ionization and detection sensitivity. One such method, the Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ strategy for labeling glycans, has previously been utilized in the analysis of N- and O-linked glycans in biological samples. To assess the maximum sensitivity and separability of the INLIGHT™ preparation and analysis pipeline, several critical steps were investigated. First, recombinant and nonrecombinant sources of PNGase F were compared to assess variations in the released glycans. Second, modifications in the INLIGHT™ derivatization step were evaluated including temperature optimization, solvent composition changes, reaction condition length and tag concentration. Optimization of the modified method resulted in 20-100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase compared with the standard method. Furthermore, the identification of low-abundance glycans, such as (Fuc)1(Gal)2(GlcNAc)4(Man)3(NeuAc)1 and (Gal)3(GlcNAc)5(Man)3(NeuAc)3, was possible. Finally, the optimal LC setup for the INLIGHT™ derivatized N-linked glycan analyses was found to be a C18 reverse-phase (RP) column with mobile phases typical of RPLC.
Assuntos
Glicômica/métodos , Polissacarídeos/análise , Cromatografia de Fase Reversa/métodos , Feminino , Fetuínas/química , Glicosilação , Humanos , Masculino , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Espectrometria de Massas em Tandem/métodosRESUMO
Sialylation, an important form of glycosylation, is involved in many biological processes and plays an important role in the development of diseases. However, due to the low abundance among various glycosylation and lack of efficient enrichment method with high specificity, the study of sialylation remains a challenge. Herein, multi-histidine modified microspheres (MHM) were synthesized to enrich sialylated glycopeptides. It was found that MHM could selectively enrich sialylated glycopeptides from over 100 times of non-sialylated glycopeptides, which indicated MHM possessed good enrichment specificity towards sialylated glycopeptides. Furthermore, MHM were utilized to the large-scale analysis of protein sialylation, and 510 intact glycopeptides were identified with over 94.5% sialylated glycopeptide specificity from 4 µL human serum. The good specificity could be attributed to the synergistic effect by the electrostatic interaction and hydrophilic interaction. Hence, MHM could provide an alternative approach for the analysis of site-specific sialylation at proteome level from complex biological samples.
Assuntos
Glicopeptídeos/análise , Histidina/química , Ácido N-Acetilneuramínico/química , Sequência de Aminoácidos , Avidina/química , Fetuínas/química , Glicopeptídeos/sangue , Glicosilação , Humanos , Microesferas , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
O-Glycoprotein analysis has been historically challenging due, in part, to a dearth of available enzymes active in the release of O-glycans. Moreover, chemical releasing methods, such as ß-elimination/Michael addition, are not specific to O-glycan release and can also eliminate phosphoryl substitutions. Both of these events leave behind deaminated serine and threonine and thus can lead to ambiguous structural conclusions. Recently, the O-protease OpeRATOR, derived from intestinal bacteria and expressed in Escherichia coli, has become commercially available. The digestion of O-glycoprotein yields O-glycopeptides cleaved at the N-terminal end of serine and threonine, with O-glycan remaining intact. The enzyme has a broad substrate specificity and includes mammalian cores 1-8. However, OpeRATOR is not fully active toward sialylated glycoproteins, and it has been suggested that this acidic residue be removed prior to digestion, thus sacrificing structural information. In this study, we investigated the performance of OpeRATOR under a range of conditions, including buffer selection, varying pH, sialic acid modification, and digestion temperature, in order to optimize the enzymatic activity, with a special emphasis on sialylated glycosites. Conditions derived in this work facilitate the OpeRATOR digestion of fully sialylated O-glycopeptides that are mass tagged to identify the sialyl linkage, thus facilitating the analysis of these charged O-glycopeptides, which are often important in biological processes.
Assuntos
Endopeptidases/química , Glicopeptídeos/análise , Glicoproteínas/análise , Polissacarídeos/análise , Ácidos Siálicos/química , Animais , Sequência de Carboidratos , Bovinos , Escherichia coli/enzimologia , Etildimetilaminopropil Carbodi-Imida/química , Fetuínas/análise , Fetuínas/química , Glicoproteínas/química , Lactoferrina/análise , Lactoferrina/química , Mucinas/análise , Mucinas/química , Polissacarídeos/química , Triazóis/químicaRESUMO
Protein glycosylation is involved in many biological processes and physiological functions. Despite the recent advances in LC-MS/MS methodologies, the profiling of site-specific glycosylation is one of the major analytical challenges of glycoprotein analysis. Herein, we report that the separation of glycopeptide isomers on porous graphitic carbon (PGC)-LC was significantly improved by elevating the separation temperature under basic mobile phases. These findings permitted the isomeric separation of glycopeptides resulting from highly specific enzymatic digestions. The selectivity for different glycan types was studied using bovine fetuin, asialofetuin, IgG, ribonuclease B, and alpha-1 acid glycoprotein (AGP) by PGC-LC-MS. Comprehensive structural isomeric separation of glycopeptides was observed by high-resolution MS and confirmed by MS/MS. The specific structures of the glycopeptide isomers were identified and confirmed through exoglycosidase digestions. Glycosylation analysis of human AGP revealed the potential use of PGC-LC-MS for extensive glycoprotein analysis for biomarker discovery. This newly developed separation technique was shown as a reproducible and useful analytical method to study site-specific isomeric glycosylation.
Assuntos
Carbono/química , Cromatografia Líquida/métodos , Glicopeptídeos/química , Glicoproteínas/química , Espectrometria de Massas em Tandem/métodos , Fracionamento Químico , Fetuínas/química , Glicopeptídeos/metabolismo , Glicoproteínas/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
Precise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics and an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of α2,3/α2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, we developed an innovative glycosyltransferase labeling assisted mass spectrometry (GLAMS) strategy. After specific enzymatic labeling, oxonium ions from higher-energy C-trap dissociation (HCD) fragmentation of α2,3-sailoglycopeptides then generate unique reporters to distinctly differentiate those of α2,6-sailoglycopeptide isomers. With this strategy, a total of 1236 linkage-specific sialoglycopeptides were successfully identified from 161 glycoproteins in human serum.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Sialoglicoproteínas/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Azidas/química , Azidas/metabolismo , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Campylobacter jejuni/enzimologia , Sequência de Carboidratos , Bovinos , Cromatografia Líquida de Alta Pressão , Fetuínas/química , Fetuínas/metabolismo , Glicosilação , Hexosaminas/química , Hexosaminas/metabolismo , Humanos , Isomerismo , Sialoglicoproteínas/metabolismoRESUMO
Like sialylation, fucose usually locates at the nonreducing ends of various glycans on glycoproteins and constitutes important glycan epitopes. Detecting the substrate glycans of fucosyltransferases is important for understanding how these glycan epitopes are regulated in response to different growth conditions and external stimuli. Here we report the detection of these glycans on glycoproteins as well as in their free forms via enzymatic incorporation of fluorophore-conjugated fucose using FUT2, FUT6, FUT7, FUT8 and FUT9. Specifically, we describe the detection of the substrate glycans of these enzymes on fetal bovine fetuin, recombinant H1N1 viral neuraminidase and therapeutic antibodies. The detected glycans include complex and high-mannose N-glycans. By establishing a series of precursors for the synthesis of Lewis X and sialyl Lewis X structures, we not only provide convenient electrophoresis methods for studying glycosylation but also demonstrate the substrate specificities and some kinetic features of these enzymes. Our results support the notion that fucosyltransferases are key targets for regulating the synthesis of Lewis X and sialyl Lewis X structures.
Assuntos
Corantes Fluorescentes/química , Fucose/química , Fucosiltransferases/química , Polissacarídeos/análise , Animais , Bovinos , Eletroforese , Fetuínas/química , Fetuínas/metabolismo , Corantes Fluorescentes/metabolismo , Fucose/metabolismo , Fucosiltransferases/metabolismo , Polissacarídeos/metabolismo , Especificidade por SubstratoRESUMO
The study of protein glycosylation is important to deepening the current understanding of its biological functions as well as to elucidate novel biomarkers of glycosylation. However, glycopeptides must be enriched prior to analysis due to their naturally low abundance. In this study, tryptophan functionalized polymer materials (denoted as Poly-Trp) were synthesized to serve as a novel biomimetic polymers. The resulting materials were characterized by various methods including scanning electron microscopy, thermal analysis, and infrared spectrometry, validating the successful synthesis of Poly-Trp. The retention of glycopeptides on Poly-Trp was investigated revealing that the retention ability decreased as the acetonitrile content of the mobile phase decreased and its acidity increased. Poly-Trp exhibited higher enrichment selectivity for glycopeptides from bovine fetuin than the commercial material ZIC-HILIC, and aminated silica which could defect the interference of 100-fold amount of substance ratios of bovine serum albumin. Poly-Trp is expected to have further applications in the purification of biological samples for the study of glycosylation.
Assuntos
Biomimética , Glicopeptídeos/química , Polímeros/química , Animais , Bovinos , Fetuínas/química , Glicosilação , Dióxido de SilícioRESUMO
RATIONALE: Porous graphic carbon chromatography (PGC) has a different mechanism in the retention of tryptic peptides compared with reversed-phase chromatography and in this study we show that coupling PGC with tandem mass spectrometry offer advantages for the quantitation of phosphorylation stoichiometry and characterization of site-specific glycosylation. METHODS: Digests of protein standards (horse myoglobin, bovine fetuin and ß-casein) were analyzed with a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) system by coupling an Agilent 1100 HPLC system to a Synapt G2-Si HDMS (Waters). Peptides were separated using a HyperCarb PGC column (300 µm i.d. × 100 mm) packed with 3 µm particles. MS/MS data were collected in data-dependent mode and three MS/MS scans were acquired after the full MS scan. RAW data were transformed to .mgf by PLGS (Waters) and searched against the Swissprot database by Mascot. Chromatograms and MS/MS spectra of identified compounds were extracted with Masslynx (Waters) and imported to Origin for analysis. Glycan composition and peptide sequence were manually annotated. RESULTS: PGC/MS/MS enabled accurate quantitation of the stoichiometry of specific phosphorylation sites from ß-casein by efficient separation of the phosphopeptide and its non-phosphorylated counterpart, which cannot be achieved by reversed-phase chromatography. PGC/MS/MS also enabled comprehensive characterization of protein sialoglycosylation as isomeric glycopeptides with different combinations of α2-3- and α2-6-linked sialic acids can be separated and the ratios of each combination were verified by exoglycosidase digestion. CONCLUSIONS: PGC has demonstrated superior separation of peptides with phosphorylation and glycosylation and can be used as an alternative in the proteomic characterization of post-translational modifications (PTMs) by polar groups.
Assuntos
Caseínas/química , Cromatografia/métodos , Fetuínas/química , Mioglobina/química , Animais , Carbono/química , Bovinos , Cromatografia/instrumentação , Glicosilação , Cavalos , Peptídeos/química , Fosforilação , Polissacarídeos/química , Porosidade , Espectrometria de Massas em TandemRESUMO
A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry (named SiO2-DVS-GSH). The successful synthesis of the SiO2-DVS-GSH material was confirmed by FT-IR, elemental analysis, and zeta potential analysis. The effects of water content, pH value, and salt concentration in the mobile phase on the model compound (uracil, uridine, cytosine, cytidine, guanosine, xanthosine, orotic acid) retention was studied, and a hydrophilic interaction liquid chromatography (HILIC) retention feature together with electrostatic interaction of the SiO2-DVS-GSH material was observed. The prepared stationary phase was further applied for the separation of oligosaccharide. In addition, the SiO2-DVS-GSH material displayed remarkable selectivity and specificity for the sialylated N-glycopeptides' enrichment from bovine fetuin tryptic digests, even at a mass ratio of 1:1000 (w/w) to bovine serum albumin (BSA, non-glycosylated protein), showing superior performance compared to commercial ZIC-HILIC material. Moreover, the SiO2-DVS-GSH material behaved well in the N-glycopeptides' enrichment from human serum, demonstrating its promising potential for glycoproteomics of complex biological samples. Graphical Abstract A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry, and it shows remarkable selectivity and specificity for the sialylated N-glycopeptides' enrichment.
Assuntos
Glutationa/química , Glicopeptídeos/química , Ácido N-Acetilneuramínico/química , Dióxido de Silício/química , Cromatografia Líquida/métodos , Fetuínas/química , Concentração de Íons de Hidrogênio , Mapeamento de Peptídeos , Espectroscopia de Infravermelho com Transformada de Fourier , Tripsina/químicaRESUMO
The difficulty in uncovering detailed information about protein glycosylation stems from the complexity of glycans and the large amount of material needed for the experiments. Here we report a method that gives information on the isomeric variants of glycans in a format compatible with analyzing low-abundance proteins. On-chip glycan modification and probing (on-chip gmap) uses sequential and parallel rounds of exoglycosidase cleavage and lectin profiling of microspots of proteins, together with algorithms that incorporate glycan-array analyses and information from mass spectrometry, when available, to computationally interpret the data. In tests on control proteins with simple or complex glycosylation, on-chip gmap accurately characterized the relative proportions of core types and terminal features of glycans. Subterminal features (monosaccharides and linkages under a terminal monosaccharide) were accurately probed using a rationally designed sequence of lectin and exoglycosidase incubations. The integration of mass information further improved accuracy in each case. An alternative use of on-chip gmap was to complement the mass spectrometry analysis of detached glycans by specifying the isomers that comprise the glycans identified by mass spectrometry. On-chip gmap provides the potential for detailed studies of glycosylation in a format compatible with clinical specimens or other low-abundance sources.
Assuntos
Biologia Computacional/métodos , Fetuínas/química , Polissacarídeos/química , Transferrina/química , Algoritmos , Animais , Bovinos , Glicosilação , Humanos , Espectrometria de Massas , Análise Serial de ProteínasRESUMO
We used an electron-transfer carbohydrate-mimetic peptide (YYYYC) to construct an electrochemical cytosensing system. Magnetic beads were modified with either asialofetuin (ASF) or soybean agglutinin (SBA) to evaluate the effect on cell sensing. Because SBA binds to the galactose residue that exists at the terminals of the carbohydrate chains in ASF, the target protein was accumulated on the protein magnetic beads. SBA is an example of N-acetylgalactosamine- and galactose-binding proteins that readily combine with YYYYC. When the peptides and protein-immobilized beads competed for a target protein, the peak current of the peptides changed according to the concentration of the protein at the 10-12â¯M level. Next, human myeloid leukemia cells (K562â¯cell) were measured using the peptide and the carbohydrate chains on the cell surface that recognize SBA. The electrode response was linear to the number of K562â¯cells and ranged from 1.0â¯×â¯102 to 5.0â¯×â¯103â¯cells mL-1. In addition, detection of a human liver cancer cell (HepG2 cell) was carried out using interactions with the peptide, the ASF receptors in HepG2 cells, and the carbohydrate chains of ASF. The peak currents were proportional and ranged between 5.0â¯×â¯101 and 1.5â¯×â¯103â¯cells mL-1. When the values estimated from an electrochemical process were compared with those obtained by ELISA, the results were within the acceptable range of measurement error.
Assuntos
Aglutininas/química , Assialoglicoproteínas/química , Proteínas de Ligação ao Cálcio/química , Fetuínas/química , Proteínas de Transporte de Monossacarídeos/química , Proteínas Periplásmicas de Ligação/química , Lectinas de Plantas/química , Proteínas de Soja/química , Técnicas Biossensoriais , Técnicas Eletroquímicas , Transporte de Elétrons , Células Hep G2 , Humanos , Células K562 , Células Tumorais CultivadasRESUMO
Lectin inspired polymers were prepared through modification of silica microspheres with Ser-Asp (SD). This functional polymer showed distinct adsorption and retention towards different disaccharides and demonstrated high-efficiency enrichment of glycopeptides.
Assuntos
Materiais Biomiméticos/química , Glicopeptídeos/química , Microesferas , Peptídeos/química , Dióxido de Silício/química , Adsorção , Animais , Materiais Biomiméticos/síntese química , Bovinos , Dissacarídeos/química , Fetuínas/química , Lectinas/química , Fragmentos de Peptídeos/química , Peptídeos/síntese química , Soroalbumina Bovina/química , Dióxido de Silício/síntese químicaRESUMO
Glycosylation analysis of proteins from biological sources utilizing mass spectrometry based approaches is challenging due to the relatively low abundance of glycopeptides, the structural diversity of glycans, and the coexisting matrices. In this study, a customized dextran-bonded silica-based stationary phase was introduced for selective enrichment of glycopeptides and glycans from complex biological samples. This material has exhibited superior selectivity and broader glycosylation site coverage over commercial Sepharose in glycoproteomic evaluation. Additionally, the glycomic analysis of fetuin, α1 -acid glycoprotein, and human serum N-glycome also indicated the relatively higher sensitivity, selectivity, and glycoform coverage of dextran-bonded silica than that of Sepharose and porous graphitized carbon. Therefore, the dextran-bonded silica is expected to make contributions in the fields of glycoproteomics and glycomics.
Assuntos
Dextranos/química , Glicopeptídeos/química , Polissacarídeos/química , Adulto , Animais , Carbono/química , Bovinos , Fetuínas/química , Glicômica , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Espectrometria de Massas , Peptídeos/química , Proteômica , Sefarose/química , Soro/química , Dióxido de Silício/químicaRESUMO
The lack of a highly sensitive and simple method for the quantitative analysis of glycan has impeded the exploration of protein glycosylation patterns (glycomics), evaluation of antibody drug stability, and screening of disease glycan biomarkers. In this study, we describe a novel and simplified quantitative glycomics strategy. Quantitation by mutant enzyme reaction stable isotope labeling (QMERSIL) to label the N-glycans with either a nondeuterated (d0-) or deuterated (d8-) 4-(2,4-Dinitro-5-piperazin-1-yl-phenyl)-1,1-dimethyl-piperazin-1-ium (MPDPZ)-Boc-asparaginyl-N-acetyl-d-glucosamine (Boc-Asn-GlcNAc) acceptor of a positive charge structure through the glycosynthase (Endo-M-N175Q) transglycosylation reaction with mass spectrometry facilitates comparative glycomics. The sialylglycopeptide (SGP) of the complex type was used to demonstrate that QMERSIL facilitates the relative quantitation over a linear dynamic range (up to d0/d8=0.02:20) of 3 orders of magnitude. The area ratios of the N-glycan peaks from the QMERSIL method showed a good linearity (d0/d8, R2=0.9999; d8/d0, R2=0.9978). The reproducibility and accuracy assay precisions were all less than 6.12%, and the mean recoveries (%) of SGP spiked in the human plasma were 97.34%. Moreover, the QMERSIL using LC-MS/MS was evaluated with various molar ratios (1:1, 1:5, 5:1) of d0(d8)- MPDPZ-Boc-Asn-GlcNAc-labeled glycans from ribonuclease B, bovine fetuin, and ovalbumin. The ratios of the relative intensity between the isotopically MPDPZ-Boc-Asn-GlcNAc labeled N-glycans were almost equal a close to the theoretical values (1:1, 1:5, 5:1). Finally, this method was used for the relative quantitative comparison of the N-Linked oligosaccharides in human plasma.
Assuntos
Glicômica/métodos , Marcação por Isótopo/métodos , Polissacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto , Calibragem , Cromatografia Líquida de Alta Pressão/métodos , Deutério/química , Fetuínas/química , Fetuínas/metabolismo , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Voluntários Saudáveis , Humanos , Masculino , Ovalbumina/química , Ovalbumina/metabolismo , Polissacarídeos/química , Reprodutibilidade dos Testes , Ribonucleases/química , Ribonucleases/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas F-Box/química , Glicopeptídeos/química , Proteínas do Tecido Nervoso/química , Polissacarídeos/química , Eletroforese em Gel de Poliacrilamida , Fetuínas/química , Variação Genética , Glicoproteínas/química , Glicosilação , Humanos , Imunoglobulina G/química , Lectinas/química , Manose/química , Mutagênese , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Proteômica , Ribonucleases/química , Sais/química , Espectrometria de Massas em Tandem , Tripsina/químicaRESUMO
The ability to resolve glycans while attached to tryptic peptides would greatly facilitate glycoproteomics, as this would enable site-specific glycan characterization. Peptide/glycopeptide separations are typically performed using reversed-phase liquid chromatography (RPLC), where retention is driven by hydrophobic interaction. As the hydrophilic glycans do not interact significantly with the RPLC stationary phase, it is difficult to resolve glycopeptides that differ only in their glycan structure, even when these differences are large. Alternatively, glycans interact extensively with the stationary phases used in hydrophilic interaction chromatography (HILIC), and consequently, differences in glycan structure have profound chromatographic shifts in this chromatographic mode. Here, we evaluate HILIC for the separation of isomeric glycopeptide mixtures that have the same peptide backbone but isomeric glycans. Hydrophilic functional groups on both the peptide and the glycan interact with the HILIC stationary phase, and thus, changes to either of these moieties can alter the chromatographic behavior of a glycopeptide. The interactive processes permit glycopeptides to be resolved from each other based on differences in their amino acid sequences and/or their attached glycans. The separations of glycans in HILIC are sufficient to permit resolution of isomeric N-glycan structures, such as sialylated N-glycan isomers differing in α2-3 and α2-6 linkages, while these glycans remain attached to peptides.
Assuntos
Fetuínas/isolamento & purificação , Glicopeptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Bovinos , Cromatografia em Gel , Cromatografia de Fase Reversa , Fetuínas/química , Glicopeptídeos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Ligação Proteica , Sensibilidade e EspecificidadeRESUMO
Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS-based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (aminoxyTMT) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophageal diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.