B Subunit Monomers of Cholera Toxin Bind G1 Ganglioside Class as Revealed by Chip-Nanoelectrospray Multistage Mass Spectrometry

Capitan, Florina; Robu, Adrian; Popescu, Laurentiu; Flangea, Corina; Vukelić, Željka; Zamfir, Alina D.

doi:10.1080/07328303.2015.1085061

Cited by 8 publications

(6 citation statements)

References 39 publications

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“…The CaR-ESI-MS assay has also been combined with picodiscs (PDs) [14], which are smaller lipid-transporting macromolecular complexes composed of the human sphingolipid activator protein, saposin A (SapA), and phospholipids, for the detection of protein-GL complexes [22,23]. More recently, Zamfir and coworkers reported on the detection of interactions between proteins and gangliosides (which are glycosphingolipids that contain sialic acid) in aqueous solution using direct ESI-MS analysis [24]. Using this approach, in which the gangliosides presumably form GL micelles (glycomicelles) in solution, the authors identified the interactions between B subunits of cholera toxin (CTB) and GM1, GD1, GT1, GQ1, GP1, as well as the fucosylated GD1, GT1, and GQ1 [24].…”

Section: Introductionmentioning

confidence: 99%

“…More recently, Zamfir and coworkers reported on the detection of interactions between proteins and gangliosides (which are glycosphingolipids that contain sialic acid) in aqueous solution using direct ESI-MS analysis [24]. Using this approach, in which the gangliosides presumably form GL micelles (glycomicelles) in solution, the authors identified the interactions between B subunits of cholera toxin (CTB) and GM1, GD1, GT1, GQ1, GP1, as well as the fucosylated GD1, GT1, and GQ1 [24]. However, it is notable that these measurements were carried out at acidic pH (5.8), conditions under which the native homopentameric structure of CTB (i.e., CTB 5 ) disassembles into individual subunits.…”

Section: Introductionmentioning

confidence: 99%

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Detecting Protein–Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS

Han

Kitova

Klassen

2016

J. Am. Soc. Mass Spectrom.

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Abstract. This study reports on the use of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay, combined with glycomicelles, as a method for detecting specific interactions between water-soluble proteins and glycolipids (GLs) in aqueous solution. The B subunit homopentamers of cholera toxin (CTB 5 ) and Shiga toxin type 1 B (Stx1B 5 ) and the gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2 served as model systems for this study. The CTB 5 exhibits broad specificity for gangliosides and binds to GM1, GM2, GM3, GD1a, GD1b, and GT1b; Stx1B 5 does not recognize gangliosides. The CaR-ESI-MS assay was used to analyze solutions of CTB 5 or Stx1B 5 and individual gangliosides (GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2) or mixtures thereof. The high affinity interaction of CTB 5 with GM1 was successfully detected. However, the apparent affinity, as determined from the mass spectra, is significantly lower than that of the corresponding pentasaccharide or when GM1 is presented in model membranes such as nanodiscs. Interactions between CTB 5 and the low affinity gangliosides GD1a, GD1b, and GT1b, as well as GD2, which served as a negative control, were detected; no binding of CTB 5 to GM2 or GM3 was observed. The CaR-ESI-MS results obtained for Stx1B 5 reveal that nonspecific protein-ganglioside binding can occur during the ESI process, although the extent of binding varies between gangliosides. Consequently, interactions detected for CTB 5 with GD1a, GD1b, and GT1b are likely nonspecific in origin. Taken together, these results reveal that the CaR-ESI-MS/glycomicelle approach for detecting protein-GL interactions is prone to false positives and false negatives and must be used with caution.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Detecting Protein–Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS

Han

Kitova

Klassen

2016

J. Am. Soc. Mass Spectrom.

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“…In ganglioside research, microfluidics‐MS in general and NanoMate‐MS in particular, have proven a remarkable ionization efficiency with the beneficial characteristic of multicharged ion formation, versatility in both negative and positive ion mode for a wide variety of solvents and mass analyzers and a reduced in‐source fragmentation of labile carbohydrate or non‐carbohydrate groups constituting the ganglioside molecule, such as Neu5Ac, Fuc, and O ‐Ac [40]. Over the years, all these features have led to de novo discovery and structural characterization in details of compounds associated to various brain regions [58,59] and biomarkers of neurodegenerative diseases [60], primary tumors of the central [61,62] and peripheral [63] NS, and brain metastases [10], or for the study of structural and functional interactions involving gangliosides [42,64].…”

Section: Microfluidics‐msmentioning

confidence: 99%

Developments and applications of separation and microfluidics methods coupled to electrospray mass spectrometry in glycomics of nervous system gangliosides

2021

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Gangliosides are particularly abundant in the nervous system (NS) where their pattern and structure in a certain milieu or a defined region exhibit a pronounced specificity. Since gangliosides are useful biomarkers for diagnosis of NS ailments, a clear‐cut mapping of individual components represents a prerequisite for designing ganglioside‐based diagnostic procedures, treatments, or vaccines. These bioclinical aspects and the high diversity of ganglioside species claim for development of specific analytical strategies. This review summarizes the state‐of‐the‐art in the implementation of separation techniques and microfluidics coupled to MS, which have contributed significantly to the advancement of the field. In the first part, the review discusses relevant approaches based on HPLC MS and CE coupled to ESI MS and their applications in the characterization of gangliosides expressed in healthy and diseased NS. A considerable section is dedicated to microfluidics MS and ion mobility separation MS, developed for the study of brain gangliosidome and its changes triggered by various factors, as well as for ganglioside biomarker discovery in neurodegenerative diseases and brain cancer. In the last part of the review, the benefits and perspectives in ganglioside research of these high‐performance techniques are presented.

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“…Researchers have been probing protein–carbohydrate interactions for biomarker discovery with novel MS methods. Complexes formed with gangliosides and the B subunit of cholera toxin (CTB) were studied with nano-ESI multistage MS by Capitan et al, and their specific noncovalent interactions were revealed by multistage CID and ETD fragmentation . An intriguing new technique for analyzing protein–GSL interactions is catch-and-release electrospray ionization-mass spectrometry (CaR-ESI-MS).…”

Section: Glycosphingolipids As Cancer Markersmentioning

confidence: 99%

“…Complexes formed with gangliosides and the B subunit of cholera toxin (CTB) were studied with nano-ESI multistage MS by Capitan et al, and their specific noncovalent interactions were revealed by multistage CID and ETD fragmentation. 137 An intriguing new technique for analyzing protein−GSL interactions is catch-and-release electrospray ionization-mass spectrometry (CaR-ESI-MS). The method was developed by Li et al and involves the assembly of picodiscs of proteins, lipids, and GSLs in vitro.…”

Section: ■ Glycosphingolipids As Cancer Markersmentioning

confidence: 99%