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1.
Anal Chem ; 94(38): 12954-12959, 2022 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-36098998

RESUMEN

Isomeric N-glycans often vastly differ in their biological activities, hence the need for methods that allow resolving and structurally characterizing them in biological material. Here, we established a zero flow approach using capillary electrophoresis in combination with (tandem) mass spectrometry to allow structural characterization of isomeric N-glycans at high sensitivity. Additionally, diagnostic fragment ion ratios were identified, indicative for the antenna carrying specifically linked sialic acids. In total, 208 N-glycans were characterized in human plasma, with 57 compositions showing multiple isomers.


Asunto(s)
Electroforesis Capilar , Espectrometría de Masas en Tándem , Electroforesis Capilar/métodos , Humanos , Isomerismo , Polisacáridos/química , Ácidos Siálicos , Espectrometría de Masas en Tándem/métodos
2.
Anal Chem ; 93(18): 6919-6923, 2021 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-33914523

RESUMEN

The desolvation and ionization process of analytes can significantly be improved by enriching the nebulizing gas with a dopant (dopant enriched nitrogen (DEN) gas) in the electrospray source. However, for the analysis of released glycans in negative ion mode, the usage of DEN gas remains largely unexplored. For this purpose, we investigated the effect of different polar protic solvents (methanol, ethanol, and isopropanol) as well as using solely the nebulizing gas or ambient air on the ionization and charge state distribution of released N- and O-glycans. Compared to the standard acetonitrile enriched nitrogen gas, isopropanol showed the highest increase in regards to peak areas. Moreover, it showed large benefits for the identification of glycan structures at high sensitivity as the increased precursor intensities subsequently resulted in higher intensities in tandem MS mode. While similar effects are noted for both neutral and sialylated species, the most significant effect was observed for early eluting glycans where very low acetonitrile concentrations were present in the eluent. The best results in terms of S/N ratios were obtained with methanol, with less effect on the MS/MS signal enhancement. Therefore, the use of this dopant would be particularly beneficial for high sensitivity MS-mode applications. In conclusion, isopropanol enriched DEN gas greatly improves the detection of both N-and O-glycan species and their tandem mass spectra, particularly for the early eluting species whose ionization in the absence of DEN gas is hindered by low organic concentrations.


Asunto(s)
Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Nitrógeno , Polisacáridos
3.
J Allergy Clin Immunol ; 146(3): 652-666.e11, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32445838

RESUMEN

BACKGROUND: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of inflammatory (auto)immune disorders and have also been linked to protection against viral infection and discussed in the context of vaccine-induced protection. In contrast, sialylated IgG Abs have shown immunosuppressive effects. OBJECTIVE: We sought to investigate IgG glycosylation programming during the germinal center (GC) reaction following immunization of mice with a foreign protein antigen and different adjuvants. METHODS: Mice were analyzed for GC T-cell, B-cell, and plasma cell responses, as well as for antigen-specific serum IgG subclass titers and Fc glycosylation patterns. RESULTS: Different adjuvants induce distinct IgG+ GC B-cell responses with specific transcriptomes and expression levels of the α2,6-sialyltransferase responsible for IgG sialylation that correspond to distinct serum IgG Fc glycosylation patterns. Low IgG Fc sialylation programming in GC B cells was overall highly dependent on the Foxp3- follicular helper T (TFH) cell-inducing cytokine IL-6, here in particular induced by water-in-oil adjuvants and Mycobacterium tuberculosis. Furthermore, low IgG Fc sialylation programming was dependent on adjuvants that induced IL-27 receptor-dependent IFN-γ+ TFH1 cells, IL-6/IL-23-dependent IL-17A+ TFH17 cells, and high ratios of TFH cells to Foxp3+ follicular regulatory T cells. Here, the 2 latter were dependent on M tuberculosis and its cord factor. CONCLUSION: This study's findings regarding adjuvant-dependent GC responses and IgG glycosylation programming may aid in the development of novel vaccination strategies to induce IgG Abs with both high affinity and defined Fc glycosylation patterns in the GC.


Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígenos/administración & dosificación , Centro Germinal/inmunología , Inmunoglobulina G/inmunología , Compuestos de Alumbre/administración & dosificación , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Citocinas/inmunología , Femenino , Adyuvante de Freund/administración & dosificación , Glicosilación , Lipopolisacáridos/administración & dosificación , Ratones Endogámicos C57BL , Ratones Noqueados , Aceite Mineral/administración & dosificación , Mycobacterium tuberculosis/inmunología , Ovalbúmina/administración & dosificación , Polisorbatos/administración & dosificación , Escualeno/administración & dosificación , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Vacunación
4.
Nat Commun ; 10(1): 2137, 2019 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-31086181

RESUMEN

The in-depth, high-sensitivity characterization of the glycome from complex biological samples, such as biofluids and tissues, is of utmost importance in basic biological research and biomarker discovery. Major challenges often arise from the vast structural diversity of glycans in combination with limited sample amounts. Here, we present a method for the highly sensitive characterization of released N-glycans by combining a capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) approach with linkage-specific derivatization of sialic acids and uniform cationic reducing end labelling of all glycans. This method allows the analysis of glycans at the attomole level, provides information on sialic acid isomers and enables the in-depth characterization of complex samples, even when available in minute amounts.


Asunto(s)
Glicómica/métodos , Polisacáridos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Proteínas Sanguíneas/química , Electroforesis Capilar/métodos , Glicosilación , Humanos , Isomerismo , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Ácidos Siálicos/química
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