RESUMEN
BACKGROUND: In sharp contrast with analysis of N-glycan that can be prepared by PNGase F, O-glycan analysis remains challenging due to a lack of versatile and simple procedures, especially those mediating cleavage of O-glycans from proteins. Most N-glycans and O-glycans are modified with sialic acids at the non-reducing end and their glycosidic linkages are labile, making it difficult to measure glycans by mass spectrometric analysis. In addition, sialic acid residues present on glycan chains via α2,3-, α2,6-, and α2,8-linkages as structural isomers. RESULTS: In this study, we firstly established a direct and linkage-specific derivatization method for sialylated O-glycans on proteins via linkage-specific lactone-opening aminolysis. In this procedure, labile sialylated glycans were not only stabilized, but also allowed distinguishing between sialyl linkages. Furthermore, we revealed that general reductive ß-elimination was not useful for O-glycan cleavages with undesirable degradations of resulting methyl amides. Using ß-elimination in the presence of pyrazolone (PMP), with low pH despite alkali base concentration, SALSA-derivatized O-glycans could be cleaved with minimal degradations. Cleaved and PMP-labeled O-glycans could be efficiently prepared in an open reaction system at high temperature (evaporative BEP reaction) and detected by simple liquid-phase extraction. Moreover, in the evaporative BEP reaction by changing the alkali solution with LiOH, the lithiated O-glycans could be observed and provided a lot of fragment information reflecting the complex structure of the O-glycans. SIGNIFICANCE: Direct sialic acid linkage-specific derivatization of O-glycans on glycoproteins is simple protocol containing in-solution aminolysis-SALSA and acetonitrile precipitation for removal of excess reagents. Evaporative ß-elimination with pyrazolone makes possible intact O-linked glycan analysis just by liquid-phase extraction. These analytical methods established by the appropriate combination of direct-SALSA and evaporative ß-elimination will facilitate O-glycomic studies in various biological samples.
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Polisacáridos , Ácidos Siálicos , Polisacáridos/química , Ácidos Siálicos/químicaRESUMEN
Chronic metabolic stress paradoxically elicits pro-tumorigenic signals that facilitate cancer stem cell (CSC) development. Therefore, elucidating the metabolic sensing and signaling mechanisms governing cancer cell stemness can provide insights into ameliorating cancer relapse and therapeutic resistance. Here, we provide convincing evidence that chronic metabolic stress triggered by hyaluronan production augments CSC-like traits and chemoresistance by partially impairing nucleotide sugar metabolism, dolichol lipid-linked oligosaccharide (LLO) biosynthesis and N-glycan assembly. Notably, preconditioning with either low-dose tunicamycin or 2-deoxy-D-glucose, which partially interferes with LLO biosynthesis, reproduced the promoting effects of hyaluronan production on CSCs. Multi-omics revealed characteristic changes in N-glycan profiles and Notch signaling activation in cancer cells exposed to mild glycometabolic stress. Restoration of N-glycan assembly with glucosamine and mannose supplementation and Notch signaling blockade attenuated CSC-like properties and further enhanced the therapeutic efficacy of cisplatin. Therefore, our findings uncover a novel mechanism by which tolerable glycometabolic stress boosts cancer cell resilience through altered N-glycosylation and Notch signaling activation.
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Ácido Hialurónico , Resiliencia Psicológica , Humanos , Glicosilación , Ácido Hialurónico/metabolismo , Recurrencia Local de Neoplasia/metabolismo , Polisacáridos/metabolismo , Suplementos Dietéticos , Células Madre Neoplásicas/metabolismoRESUMEN
Characterization of O-glycans linked to serine or threonine residues in glycoproteins has mostly been achieved using chemical reaction approaches because there are no known O-glycan-specific endoglycosidases. Most O-glycans are modified with sialic acid residues at the non-reducing termini through various linkages. In this study, we developed a novel approach for sialic acid linkage-specific O-linked glycan analysis through lactone-driven ester-to-amide derivatization combined with non-reductive ß-elimination in the presence of hydroxylamine. O-glycans released by non-reductive ß-elimination were efficiently purified using glycoblotting via chemoselective ligation between carbohydrates and a hydrazide-functionalized polymer, followed by modification of methyl or ethyl ester groups of sialic acid residues on solid-phase. In-solution lactone-driven ester-to-amide derivatization of ethyl-esterified O-glycans was performed, and the resulting sialylated glycan isomers were discriminated by mass spectrometry. In combination with PNGase F digestion, we carried out simultaneous, quantitative, and sialic acid linkage-specific N- and O-linked glycan analyses of a model glycoprotein and human cartilage tissue. This novel glycomic approach will facilitate detailed characterization of biologically relevant sialylated N- and O-glycans on glycoproteins.
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Ésteres , Ácido N-Acetilneuramínico , Humanos , Ácido N-Acetilneuramínico/química , Glicoproteínas/química , Polisacáridos/química , LactonasRESUMEN
Glycosphingolipids (GSLs) are glycolipids with ceramide and carbohydrate head groups that play an important role in numerous biological processes. Previously, we performed GSL-glycan analysis of various cell lines and virus-infected cells using a glycoblotting approach. Recently, we developed several methods for sialic acid linkage-specific chemical modification to distinguish sialylated glycan isomers by mass spectrometry. In this chapter, we describe a method for analyzing GSL-glycans in human serum/plasma using glycoblotting combined with aminolysis-SALSA (sialic acid linkage-specific alkylamidation) and lactone-driven ester-to-amide derivatization (LEAD)-SALSA for comprehensive and detailed structural glycomics.
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Ácido N-Acetilneuramínico , Esfingolípidos , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Glicoesfingolípidos/metabolismo , Polisacáridos/químicaRESUMEN
Avian or human influenza A viruses bind preferentially to avian- or human-type sialic acid receptors, respectively, indicating that receptor tropism is an important factor for determining the viral host range. However, there are currently no reliable methods for analyzing receptor tropism biologically under physiological conditions. In this study, we established a novel system using MDCK cells with avian- or human-type sialic acid receptors and with both sialic acid receptors knocked out (KO). When we examined the replication of human and avian influenza viruses in these KO cells, we observed unique viral receptor tropism that could not be detected using a conventional solid-phase sialylglycan binding assay, which directly assesses physical binding between the virus and sialic acids. Furthermore, we serially passaged an engineered avian-derived H4N5 influenza virus, whose PB2 gene was deleted, in avian-type receptor KO cells stably expressing PB2 to select a mutant with enhanced replication in KO cells; however, its binding to human-type sialylglycan was undetectable using the solid-phase binding assay. These data indicate that a panel of sialic acid receptor KO cells could be a useful tool for determining the biological receptor tropism of influenza A viruses. Moreover, the PB2KO virus experimental system could help to safely and efficiently identify the mutations required for avian influenza viruses to adapt to human cells that could trigger a new influenza pandemic. IMPORTANCE The acquisition of mutations that allow avian influenza A virus hemagglutinins to recognize human-type receptors is mandatory for the transmission of avian viruses to humans, which could lead to a pandemic. In this study, we established a novel system using a set of genetically engineered MDCK cells with knocked out sialic acid receptors to biologically evaluate the receptor tropism for influenza A viruses. Using this system, we observed unique receptor tropism in several virus strains that was undetectable using conventional solid-phase binding assays that measure physical binding between the virus and artificially synthesized sialylglycans. This study contributes to elucidation of the relationship between the physical binding of virus and receptor and viral infectivity. Furthermore, the system using sialic acid knockout cells could provide a useful tool to explore the sialic acid-independent entry mechanism. In addition, our system could be safely used to identify mutations that could acquire human-type receptor tropism.
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Virus de la Influenza A , Ácido N-Acetilneuramínico , Receptores de Superficie Celular , Receptores Virales , Tropismo Viral , Internalización del Virus , Animales , Aves/virología , Perros , Técnicas de Inactivación de Genes , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/metabolismo , Gripe Aviar/virología , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Ácido N-Acetilneuramínico/metabolismo , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Virales/química , Receptores Virales/genética , Receptores Virales/metabolismoRESUMEN
Sialic acids are unique sugars with negative charge and exert various biological functions such as regulation of immune systems, maintenance of nerve tissues and expression of malignant properties of cancers. Alpha 2,6 sialylated N-glycans, one of representative sialylation forms, are synthesized by St6gal1 or St6gal2 gene products in humans and mice. Previously, it has been reported that St6gal1 gene is ubiquitously expressed in almost all tissues. On the other hand, St6gal2 gene is expressed mainly in the embryonic and perinatal stages of brain tissues. However, roles of St6gal2 gene have not been clarified. Expression profiles of N-glycans with terminal α2,6 sialic acid generated by St6gal gene products in the brain have never been directly studied. Using conventional lectin blotting and novel sialic acid linkage-specific alkylamidationmass spectrometry method (SALSA-MS), we investigated the function and expression of St6gal genes and profiles of their products in the adult mouse brain by establishing KO mice lacking St6gal1 gene, St6gal2 gene, or both of them (double knockout). Consequently, α2,6-sialylated N-glycans were scarcely detected in adult mouse brain tissues, and a majority of α2,6-sialylated glycans found in the mouse brain were O-linked glycans. The majority of these α2,6-sialylated O-glycans were shown to be disialyl-T antigen and sialyl-(6)T antigen by mass spectrometry analysis. Moreover, it was revealed that a few α2,6-sialylated N-glycans were produced by the action of St6gal1 gene, despite both St6gal1 and St6gal2 genes being expressed in the adult mouse brain. In the future, where and how sialylated O-linked glycoproteins function in the brain tissue remains to be clarified.
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Encéfalo/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/metabolismo , Sialiltransferasas/genética , Animales , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sialiltransferasas/deficiencia , Sialiltransferasas/metabolismo , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
Sialic acid attached to nonreducing ends of glycan chains via different linkages is associated with specific interactions and physiological events. Linkage-specific derivatization of sialic acid is of great interest for distinguishing sialic acids by mass spectrometry, specifically for events governed by sialyl linkage types. In the present study, we demonstrate that α-2,3/8-sialyl linkage-specific amidation of esterified sialyloligosaccharides can be achieved via an intramolecular lactone. The method of lactone-driven ester-to-amide derivatization for sialic acid linkage-specific alkylamidation, termed LEAD-SALSA, employs in-solution ester-to-amide conversion to directly generate stable and sialyl linkage-specific glycan amides from their ester form by mixing with a preferred amine, resulting in the easy assignments of sialyl linkages by comparing the signals of esterified and amidated glycan. Using this approach, we demonstrate the accumulation of altered N-glycans in cardiac muscle tissue during mouse aging. Furthermore, we find that the stability of lactone is important for ester-to-amide conversion based on experiments and density functional theory calculations of reaction energies for lactone formation. By using energy differences of lactone formation, the LEAD-SALSA method can be used not only for the sialyl linkage-specific derivatization but also for distinguishing the branching structure of galactose linked to sialic acid. This simplified and direct sialylglycan discrimination will facilitate important studies on sialylated glycoconjugates.
RESUMEN
Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrPSc) after conversion of the cellular prion protein (PrPC). The glycosylphosphatidylinositol (GPI) anchor of PrPC is involved in prion disease pathogenesis, and especially sialic acid in a GPI side chain reportedly affects PrPC conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrPC Moreover, the sialic acid linkage type in the GPI side chain has not been determined for any GPI-anchored protein. Here we report GPI glycan structures of human PrPC isolated from human brains and from brains of a knock-in mouse model in which the mouse prion protein (Prnp) gene was replaced with the human PRNP gene. LC-electrospray ionization-MS analysis of human PrPC from both biological sources indicated that Gly229 is the ω site in PrPC to which GPI is attached. Gly229 in human PrPC does not correspond to Ser231, the previously reported ω site of Syrian hamster PrPC We found that â¼41% and 28% of GPI anchors in human PrPCs from human and knock-in mouse brains, respectively, have N-acetylneuraminic acid in the side chain. Using a sialic acid linkage-specific alkylamidation method to discriminate α2,3 linkage from α2,6 linkage, we found that N-acetylneuraminic acid in PrPC's GPI side chain is linked to galactose through an α2,3 linkage. In summary, we report the GPI glycan structure of human PrPC, including the ω-site amino acid for GPI attachment and the sialic acid linkage type.
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Glicosilfosfatidilinositoles , Ácido N-Acetilneuramínico , Proteínas PrPC , Proteínas Priónicas , Animales , Conformación de Carbohidratos , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/genética , Glicosilfosfatidilinositoles/metabolismo , Humanos , Masculino , Mesocricetus , Ratones , Ratones Noqueados , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/genética , Ácido N-Acetilneuramínico/metabolismo , Proteínas PrPC/química , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Proteínas Priónicas/química , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismoRESUMEN
Mass spectrometry (MS) is a well-accepted means for analyzing glycans. Before glycan analysis by MS, several chemical derivatizations are generally carried out. These are classified into three categories; (1) labeling of the reducing end of glycans, (2) permethylation, and (3) sialic acid derivatization. Because sialic acid residues are unstable, they are easily lost during pretreatment and during or after ionization in a mass spectrometer. Sialic acid derivatization can prevent the loss of this residue. Recently, new types of sialic acid derivatization techniques have been developed, which allow straight-forward sialic acid linkage analysis (α2,3-/α2,6-linkages) as well as residue stabilization. This review summarizes the developments in sialic acid derivatization techniques, especially the varied methods of sialic acid linkage-specific derivatization.
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Espectrometría de Masas , Polisacáridos/análisis , Ácidos Siálicos/química , Animales , Glicopéptidos/química , Humanos , Lactonas/química , Espectrometría de Masas/métodos , Estructura Molecular , Oxidación-ReducciónRESUMEN
Sialic acids form the terminal sugars in glycan chains on glycoproteins via α2,3, α2,6, or α2,8 linkages, and structural isomers of sialyl linkages play various functional roles in cell recognition and other physiological processes. We recently developed a novel procedure based on sialic acid linkage-specific alkylamidation via lactone ring opening (aminolysis-SALSA). Herein, we have investigated an isotope labeling of α2,3-linked sialic acid residues (iSALSA) using amine hydrochloride salts. One limitation of SALSA using amine hydrochloride salts may be solved by adding only tert-butylamine (t-BA) as an acid scavenger, and comparative and quantitative glycomic analyses can be performed using iSALSA. We also developed quantitative glycomic analysis using dual isotope-labeled glycans by derivatizing with aminooxy-functionalized tryptophanylarginine methyl ester (aoWR) and iSALSA at the reducing and nonreducing end, respectively. Furthermore, we demonstrate that the amount of α2,3-linked sialoglycans in serum are altered during liver fibrosis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography MS (LC/MS) analyses. We revealed that the ratio of A33,6,6 to A3F3,6,6 was gradually decreased along with liver fibrosis progression. Therefore, these glycan alterations are potential diagnostic markers of nonalcoholic steatohepatitis (NASH) fibrosis progression.
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Glicómica/métodos , Ácido N-Acetilneuramínico/química , Polisacáridos/química , Aminas/química , Biomarcadores , Glicoproteínas/química , Humanos , Marcaje Isotópico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Polisacáridos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Membrane proteins (such as ion channels, transporters, and receptors) and secreted proteins are essential for cellular activities. N-linked glycosylation is involved in stability and function of these proteins and occurs at Asn residues. In several organs, profiles of N-glycans have been determined by comprehensive analyses. Nevertheless, the cochlea of the mammalian inner ear, a tiny organ mediating hearing, has yet to be examined. Here, we focused on the stria vascularis, an epithelial-like tissue in the cochlea, and characterised N-glycans by liquid chromatography with mass spectrometry. This hypervascular tissue not only expresses several ion transporters and channels to control the electrochemical balance in the cochlea but also harbours different transporters and receptors that maintain structure and activity of the organ. Seventy-nine N-linked glycans were identified in the rat stria vascularis. Among these, in 55 glycans, the complete structures were determined; in the other 24 species, partial glycosidic linkage patterns and full profiles of the monosaccharide composition were identified. In the process of characterisation, several sialylated glycans were subjected sequentially to two different alkylamidation reactions; this derivatisation helped to distinguish α2,3-linkage and α2,6-linkage sialyl isomers with mass spectrometry. These data should accelerate elucidation of the molecular architecture of the cochlea.
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Cóclea/metabolismo , Polisacáridos/análisis , Estría Vascular/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Glicosilación , Polisacáridos/química , Ratas , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Sialic acids occur widely as glycoconjugates at the nonreducing ends of glycans. Glycosphingolipids (GSLs) include a large number of sialyl-linked glycan isomers with α2,3-, α2,6-, and α2,8-linked polysialic acids. Thus, it is difficult to distinguish structural isomers with the same mass by mass spectrometry. The sialic acid linkage specific alkylamidation (SALSA) method has been developed for discriminating between α2,3- and α2,6-linked isomers, but sequential amidation of linkage-specific sialic acids is generally complicated and time-consuming. Moreover, analysis of GSL-glycans containing α2,8-linked polysialic acids using solid-phase SALSA has not been reported. Herein, we report a novel SALSA method focused on ring-opening aminolysis (aminolysis-SALSA), which shortens the reaction time and simplifies the experimental procedures. We demonstrate that aminolysis-SALSA can successfully distinguish serum GSL-glycan isomers by mass spectrometry. In addition, ring-opening aminolysis can easily be applied to amine and hydrazine derivatives.
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Gangliósidos/sangre , Glicómica/métodos , Lactonas/química , Polisacáridos/sangre , Ácidos Siálicos/química , Animales , Bovinos , Fenómenos Químicos , Gangliósidos/química , Isomerismo , Polisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Mass spectrometry (MS) has become an indispensable tool for analyzing post translational modifications of proteins, including N-glycosylated molecules. Because most glycosylation sites carry a multitude of glycans, referred to as "glycoforms," the purpose of an N-glycosylation analysis is glycoform profiling and glycosylation site mapping. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has unique characteristics that are suited for the sensitive analysis of N-glycosylated products. However, the analysis is often hampered by the inherent physico-chemical properties of N-glycans. Glycans are highly hydrophilic in nature, and therefore tend to show low ion yields in both positive- and negative-ion modes. The labile nature and complicated branched structures involving various linkage isomers make structural characterization difficult. This review focuses on MALDI-MS-based approaches for enhancing analytical performance in N-glycosylation research. In particular, the following three topics are emphasized: (1) Labeling for enhancing the ion yields of glycans and glycopeptides, (2) Negative-ion fragmentation for less ambiguous elucidation of the branched structure of N-glycans, (3) Derivatization for the stabilization and linkage isomer discrimination of sialic acid residues.
RESUMEN
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for high-throughput glycan profiling analysis. In spite of the biological importance of sialic acids on nonreducing ends of glycans, it is still difficult to analyze glycans containing sialic acid residues due to their instability and the presence of linkage isomers. In this Article, we describe a one-pot glycan purification/derivatization method employing a newly developed linkage-specific sialic acid derivatization for MS-based glycan profiling with differentiation of sialyl linkage isomer. The derivatization, termed sialic acid linkage specific alkylamidation (SALSA), consists of sequential two-step alkylamidations. As a result of the reactions, α2,6- and α2,3-linked sialic acids are selectively amidated with different length of alkyl chains, allowing distinction of α2,3-/α2,6-linkage isomers from given mass spectra. Our studies using N-glycan standards with known sialyl linkages proved high suitability of SALSA for reliable relative quantification of α2,3-/α2,6-linked sialic acids compared with existing sialic acid derivatization approaches. SALSA fully stabilizes both α2,3- and α2,6-linked sialic acids by alkylamidation; thereby, it became possible to combine SALSA with existing glycan analysis/preparation methods as follows. The combination of SALSA and chemoselective glycan purification using hydrazide beads allows easy one-pot purification of glycans from complex biological samples, together with linkage-specific sialic acid stabilization. Moreover, SALSA-derivatized glycans can be labeled via reductive amination without causing byproducts such as amide decomposition. This solid-phase SALSA followed by glycan labeling has been successfully applied to human plasma N-glycome profiling.
RESUMEN
Dissociation of gas-phase peptide ions through interaction with low-energy hydrogen (H) radical (â¼0.15 eV) was observed with a quadrupole ion trap mass spectrometry. The H radical generated by thermal dissociation of H2 molecules passing through a heated tungsten capillary (â¼2000 °C) was injected into the ion trap containing target peptide ions. The fragmentation spectrum showed abundant c-/z- and a-/x-type ions, attributable to H attachment/abstraction to/from peptide ion. Because the low-energy neutral H radical initiated the fragmentation, the charge state of the precursor ion was maintained during the dissociation. As a result, precursor ions of any charge state, including singly charged positive and negative ions, could be analyzed for amino acid sequence. The sequence coverage exceeding 90% was obtained for both singly protonated and singly deprotonated substance P peptide. This mass spectrometry also preserved labile post-translational modification bonds. The modification sites of triply phosphorylated peptide (kinase domain of insulin receptor) were identified with the sequence coverage exceeding 80%.
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Hidrógeno/química , Péptidos/análisis , Espectrometría de Masas en Tándem/métodos , Secuencia de AminoácidosRESUMEN
UNLABELLED: Tandem mass spectrometry (MS/MS or MS(n)) is a potent technique for characterizing N-glycan structures. GlycanAnalysis searches a glycan database to support the identification of glycan structures from MS/MS spectra. It also calculates diagnostic ions of glycan structures registered in a glycan database (GlycomeDB or KEGG GLYCAN) and searches for MS/MS spectra of N-glycans that match diagnostic ions to determine the structures. This program functions as a plug-in for Mass++, a freeware mass spectrum visualization and analysis program. AVAILABILITY AND IMPLEMENTATION: The executable files of Mass++ are available for free at http://www.first-ms3d.jp/english/. The GlycanAnalysis plug-in is included in the standard package of Mass++ for Windows. CONTACT: k-morimt@shimadzu.co.jp or nishikaz@shimadzu.co.jp or acyshzw@shimadzu.co.jp SUPPLEMENTARY INFORMATION: Supplementary material are available at Bioinformatics online.
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Bases de Datos Factuales , Glicopéptidos/análisis , Polisacáridos/análisis , Motor de Búsqueda , Programas Informáticos , Espectrometría de Masas en Tándem/métodos , Glicopéptidos/química , Glicosilación , Humanos , Espectrometría de Masas , Polisacáridos/química , Proteómica/métodosRESUMEN
Tandem mass spectrometry (MS/MS or MS(n)) is a powerful tool for characterizing N-linked glycopeptide structures. However, it is still difficult to obtain detailed structural information on the glycan moiety directly from glycopeptide ions. Here, we propose a new method for in-depth analysis of the glycopeptide structure using MS/MS. This method involves complete derivatization of carboxyl groups in glycopeptides. Methylamidation using PyAOP as a condensing reagent has been optimized for derivatizing all carboxyl groups in glycopeptides. By derivatizing carboxyl groups on the peptide moiety (i.e., Asp, Glu, and C-terminus), the glycopeptides efficiently produce informative glycan fragment ions, including the nonreducing end of the glycan moiety under negative-ion collision-induced dissociation (CID) conditions. These glycan fragment ions can define detailed structural features on the glycan moiety (e.g., the specific composition of the two antennae, the location of fucose residues, and the presence/absence of bisecting GlcNAc residues). For sialylated glycopeptides, carboxyl groups on sialic acid residues are simultaneously derivatized using methylamidation, suppressing preferential loss of residues during MS analysis. As a result, both sialylated and nonsialylated glycopeptides can be analyzed in the same manner. Positive-ion CID of methylamine-derivatized glycopeptides mainly provides information on peptide sequence and glycan composition, whereas negative-ion CID provides in-depth structural information on the glycan moiety. The derivatization step can be readily incorporated into conventional pretreatment for glycopeptide MS analysis without loss of sensitivity, making derivatization suitable for practical use.
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Glicopéptidos/química , Amidas/química , Indicadores y Reactivos , Fragmentos de Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
Glycopeptide structural analysis using tandem mass spectrometry is becoming a common approach for elucidating site-specific N-glycosylation. The analysis is generally performed in positive-ion mode. Therefore, fragmentation of protonated glycopeptides has been extensively investigated; however, few studies are available on deprotonated glycopeptides, despite the usefulness of negative-ion mode analysis in detecting glycopeptide signals. Here, large sets of glycopeptides derived from well-characterized glycoproteins were investigated to understand the fragmentation behavior of deprotonated N-linked glycopeptides under low-energy collision-induced dissociation (CID) conditions. The fragment ion species were found to be significantly variable depending on their amino acid sequence and could be classified into three types: (i) glycan fragment ions, (ii) glycan-lost fragment ions and their secondary cleavage products, and (iii) fragment ions with intact glycan moiety. The CID spectra of glycopeptides having a short peptide sequence were dominated by type (i) glycan fragments (e.g., (2,4)AR, (2,4)AR-1, D, and E ions). These fragments define detailed structural features of the glycan moiety such as branching. For glycopeptides with medium or long peptide sequences, the major fragments were type (ii) ions (e.g., [peptide + (0,2)X0-H](-) and [peptide-NH3-H](-)). The appearance of type (iii) ions strongly depended on the peptide sequence, and especially on the presence of Asp, Asn, and Glu. When a glycosylated Asn is located on the C-terminus, an interesting fragment having an Asn residue with intact glycan moiety, [glycan + Asn-36](-), was abundantly formed. Observed fragments are reasonably explained by a combination of existing fragmentation rules suggested for N-glycans and peptides.
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Glicopéptidos/química , Fragmentos de Péptidos/química , Polisacáridos/química , Secuencia de Aminoácidos , Aminoácidos/química , Iones/química , Espectrometría de Masas , Peso MolecularRESUMEN
Glycosylation and phosphorylation are important post-translational modifications in biological processes and biomarker research. The difficulty in analyzing these modifications is mainly their low abundance and dissociation of labile regions such as sialic acids or phosphate groups. One solution in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is to improve matrices for glycopeptides, carbohydrates, and phosphopeptides by increasing the sensitivity and suppressing dissociation of the labile regions. Recently, a liquid matrix 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA) (3-AQ/CHCA), introduced by Kolli et al. in 1996, has been reported to increase sensitivity for carbohydrates or phosphopeptides, but it has not been systematically evaluated for glycopeptides. In addition, 3-AQ/CHCA enhances the dissociation of labile regions. In contrast, a liquid matrix 1,1,3,3-tetramethylguanidium (TMG, G) salt of p-coumaric acid (CA) (G3CA) was reported to suppress dissociation of sulfate groups or sialic acids of carbohydrates. Here we introduce a liquid matrix 3-AQ/CA for glycopeptides, carbohydrates, and phosphopeptides. All of the analytes were detected as [M + H](+) or [M - H](-) with higher or comparable sensitivity using 3-AQ/CA compared with 3-AQ/CHCA or 2,5-dihydroxybenzoic acid (2,5-DHB). The sensitivity was increased 1- to 1000-fold using 3-AQ/CA. The dissociation of labile regions such as sialic acids or phosphate groups and the fragmentation of neutral carbohydrates were suppressed more using 3-AQ/CA than using 3-AQ/CHCA or 2,5-DHB. 3-AQ/CA was thus determined to be an effective MALDI matrix for high sensitivity and the suppression of dissociation of labile regions in glycosylation and phosphorylation analyses.