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

2020 ◽  
Vol 48 (2) ◽  
pp. 547-558 ◽  
Author(s):  
Cagla Sahin ◽  
Deseree J. Reid ◽  
Michael T. Marty ◽  
Michael Landreh
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Vesicles ◽  
Native Mass Spectrometry ◽  
Integral Membrane Proteins ◽  
Structural Basis ◽  
Lipid Interactions ◽  
Native Ms ◽  
Peripheral Membrane Protein

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.

Membrane Protein–Lipid Interactions Probed Using Mass Spectrometry

2019 ◽  
Vol 88 (1) ◽  
pp. 85-111 ◽  
Author(s):  
Jani Reddy Bolla ◽  
Mark T. Agasid ◽  
Shahid Mehmood ◽  
Carol V. Robinson
Keyword(s):  
Mass Spectrometry ◽  
Membrane Protein ◽  
Lipid Bilayers ◽  
Protein Interactions ◽  
Native Mass Spectrometry ◽  
Lipid Interactions ◽  
X Ray Crystallography ◽  
Native Ms ◽  
Molecular Aspects ◽  
Lipid Protein Interactions

Membrane proteins that exist in lipid bilayers are not isolated molecular entities. The lipid molecules that surround them play crucial roles in maintaining their full structural and functional integrity. Research directed at investigating these critical lipid–protein interactions is developing rapidly. Advancements in both instrumentation and software, as well as in key biophysical and biochemical techniques, are accelerating the field. In this review, we provide a brief outline of structural techniques used to probe protein–lipid interactions and focus on the molecular aspects of these interactions obtained from native mass spectrometry (native MS). We highlight examples in which lipids have been shown to modulate membrane protein structure and show how native MS has emerged as a complementary technique to X-ray crystallography and cryo–electron microscopy. We conclude with a short perspective on future developments that aim to better understand protein–lipid interactions in the native environment.

scholarly journals Extending native mass spectrometry approaches to integral membrane proteins

2015 ◽  
Vol 396 (9-10) ◽  
pp. 991-1002 ◽  
Author(s):  
Albert Konijnenberg ◽  
Jeroen F. van Dyck ◽  
Lyn L. Kailing ◽  
Frank Sobott
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Gas Phase ◽  
Conformational Changes ◽  
Drug Targets ◽  
Native Mass Spectrometry ◽  
Lipid Binding ◽  
Drug Binding ◽  
Integral Membrane Proteins ◽  
Oligomeric State

Abstract Recent developments in native mass spectrometry and ion mobility have made it possible to analyze the composition and structure of membrane protein complexes in the gas-phase. In this short review we discuss the experimental strategies that allow to elucidate aspects of the dynamic structure of these important drug targets, such as the structural effects of lipid binding or detection of co-populated conformational and assembly states during gating on an ion channel. As native mass spectrometry relies on nano-electrospray of natively reconstituted proteins, a number of commonly used lipid- and detergent-based reconstitution systems have been evaluated for their compatibility with this approach, and parameters for the release of intact, native-like folded membrane proteins studied in the gas-phase. The strategy thus developed can be employed for the investigation of the subunit composition and stoichiometry, oligomeric state, conformational changes, and lipid and drug binding of integral membrane proteins.

scholarly journals Native mass spectrometry goes more native: investigation of membrane protein complexes directly from SMALPs

2018 ◽  
Vol 54 (97) ◽  
pp. 13702-13705 ◽  
Author(s):  
Nils Hellwig ◽  
Oliver Peetz ◽  
Zainab Ahdash ◽  
Igor Tascón ◽  
Paula J. Booth ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Maleic Acid ◽  
Protein Complexes ◽  
Native Mass Spectrometry ◽  
Direct Extraction ◽  
Membrane Protein Complexes

Other than more widely used methods, the use of styrene maleic acid copolymers allows the direct extraction of membrane proteins from the lipid bilayer into SMALPs keeping it in its native lipid surrounding.

scholarly journals Measuring Membrane Protein-Lipid Interactions in Nanodiscs with Native Mass Spectrometry

2020 ◽  
Vol 118 (3) ◽  
pp. 240a
Author(s):  
James E. Keener ◽  
Julia Townsend ◽  
Megan Mowad ◽  
Michael T. Marty
Keyword(s):  
Mass Spectrometry ◽  
Membrane Protein ◽  
Native Mass Spectrometry ◽  
Lipid Interactions

scholarly journals Development of a target identification approach using native mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miaomiao Liu ◽  
Wesley C. Van Voorhis ◽  
Ronald J. Quinn
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Target Identification ◽  
Native Mass Spectrometry ◽  
Pharmaceutical Drugs ◽  
Ligand Complex ◽  
Experimental Conditions ◽  
Protein Mixture ◽  
Native Ms ◽  
Drug Target Identification

AbstractA key step in the development of new pharmaceutical drugs is the identification of the molecular target and distinguishing this from all other gene products that respond indirectly to the drug. Target identification remains a crucial process and a current bottleneck for advancing hits through the discovery pipeline. Here we report a method, that takes advantage of the specific detection of protein–ligand complexes by native mass spectrometry (MS) to probe the protein partner of a ligand in an untargeted method. The key advantage is that it uses unmodified small molecules for binding and, thereby, it does not require labelled ligands and is not limited by the chemistry required to tag the molecule. We demonstrate the use of native MS to identify known ligand–protein interactions in a protein mixture under various experimental conditions. A protein–ligand complex was successfully detected between parthenolide and thioredoxin (PfTrx) in a five-protein mixture, as well as when parthenolide was mixed in a bacterial cell lysate spiked with PfTrx. We provide preliminary data that native MS could be used to identify binding targets for any small molecule.

scholarly journals Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP) and Stoichiometry of GroEL/GroES Complexes

2021 ◽  
Author(s):  
Thomas E Walker ◽  
Mehdi Shirzadeh ◽  
He Mirabel Sun ◽  
Jacob W McCabe ◽  
Andrew Roth ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Folding ◽  
Cell Function ◽  
Atp Hydrolysis ◽  
Variable Temperature ◽  
Native Mass Spectrometry ◽  
Solution Temperature ◽  
Temperature Dependent ◽  
Chemical Action ◽  
Native Ms

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature-electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL/GroES complexes. The results show clear evidences for de-stabilization of both GroEL14 and GroES7 at temperatures of 50 oC and 45 oC, respectively, substantially below the pre-viously reported melting temperature (Tm ~ 70 oC). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESx-ATPy, where x = 1, 2, 8 and y = 0, 1, 2, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry re-veals new insights about the stability of GroEL in response to several environmental effects: (i) temperature-dependent ATP binding to GroEL (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiome-try of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and, (iii) a change in the temper-ature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 to 56 oC. The similarities between results obtained using native MS and cryo-EM (Clare et al., An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-11; Ranson et al., Allosteric signaling of ATP hydrolysis in GroEL–GroES complexes. Nat. Struct. Mol. Biol. 2006, 13, 147-152.) underscores the utility of native MS for investiga-tions of molecular machines as well as identification of key intermediates involved in the chaperone-assisted protein folding cycle.

Top-down mass spectrometry of integral membrane proteins

2006 ◽  
Vol 3 (6) ◽  
pp. 585-596 ◽  
Author(s):  
Julian Whitelegge ◽  
Frederic Halgand ◽  
Puneet Souda ◽  
Vlad Zabrouskov
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Integral Membrane Proteins ◽  
Top Down ◽  
Top Down Mass Spectrometry

Enhancing the mass spectrometric identification of membrane proteins by combining chemical and enzymatic digestion methods

2015 ◽  
Vol 7 (17) ◽  
pp. 7220-7227 ◽  
Author(s):  
Johanna M. Smeekens ◽  
Weixuan Chen ◽  
Ronghu Wu
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Analysis ◽  
Enzymatic Digestion ◽  
Mass Spectrometric ◽  
Spectrometric Identification ◽  
Mass Spectrometric Identification ◽  
Digestion Methods

Combining chemical and enzymatic digestion methods proved to be extremely effective for mass-spectrometry (MS)-based membrane protein analysis.

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