Picodiscs for Facile Protein-Glycolipid Interaction Analysis

Leney, Aneika C.; Darestani, Reza Rezaei; Jun, Li; Nikjah, Sanaz; Kitova, Elena N.; Zou, Chunxia; Cairo, Christopher W.; Xiong, Zi Jian; Privé, Gilbert G.; Klassen, John S.

doi:10.1021/acs.analchem.5b00170

Cited by 28 publications

(55 citation statements)

References 46 publications

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“…However, significantly higher GL concentrations are required in order to achieve the same extent of binding compared with the corresponding GL oligosaccharide or when using NDs (or PDs) to solubilize the GLs [20,22,37]. The next step was to establish whether low affinity protein-GL interactions could be detected.…”

Section: Resultsmentioning

confidence: 99%

“…The successful detection of both high and low affinity protein-GL interactions using this approach has been reported [21]. 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].…”

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: Resultsmentioning

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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“…Overall, although the debate whether native protein structure is maintained in the gas phase continues, the future is bright for structural MS analysis of proteins and their complexes, as illustrated by a growing body of work of, for instance, protein–lipid [26, 74, 75], protein–RNA [76], protein–DNA [77], protein–drug [78], and protein–protein complexes [79, 80]. …”

Section: Protein Structure In the Gas Phasementioning

confidence: 99%

Native Mass Spectrometry: What is in the Name?

Leney

Heck

2016

J. Am. Soc. Mass Spectrom.

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516

480

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Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein–protein and protein–ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as “native MS,” has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure–function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by “native MS,” when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions. Graphical Abstractᅟ

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“…Building from this initial method, nanodiscs were then made from lipid mixtures extracted from a human epithelial cell line known to contain GM1, which enabled investigation of the interactions of the protein-GSL complexes in an environment that more closely mimicked in vivo conditions [50]. Additional research from Klassen and co-workers has improved quantitation with the method [51], extended these approaches to Saposin A-based picodiscs [52], and employed catch-and-release assays for the investigation of various carbohydrate-binding proteins with a panel of glycolipids [53,54]. Similarly, the first applications of nMS to integral membrane proteins in nanodiscs by Hopper et al [34] used collisional activation to release membrane proteins from the nanodiscs.…”

Section: Use Of Nanodiscs For the Study Of Peripheral Membrane Proteimentioning

confidence: 99%

Scratching the surface: native mass spectrometry of peripheral membrane protein complexes

Sahin

Reid

Marty

et al. 2020

Biochemical Society Transactions

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A growing number of integral membrane proteins have been shown to tune their activity by selectively interacting with specific lipids. The ability to regulate biological functions via lipid interactions extends to the diverse group of proteins that associate only peripherally with the lipid bilayer. However, the structural basis of these interactions remains challenging to study due to their transient and promiscuous nature. Recently, native mass spectrometry has come into focus as a new tool to investigate lipid interactions in membrane proteins. Here, we outline how the native MS strategies developed for integral membrane proteins can be applied to generate insights into the structure and function of peripheral membrane proteins. Specifically, native MS studies of proteins in complex with detergent-solubilized lipids, bound to lipid nanodiscs, and released from native-like lipid vesicles all shed new light on the role of lipid interactions. The unique ability of native MS to capture and interrogate protein–protein, protein–ligand, and protein–lipid interactions opens exciting new avenues for the study of peripheral membrane protein biology.

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Picodiscs for Facile Protein-Glycolipid Interaction Analysis

Cited by 28 publications

References 46 publications

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

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

Native Mass Spectrometry: What is in the Name?

Scratching the surface: native mass spectrometry of peripheral membrane protein complexes

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