Variable-Temperature Native Mass Spectrometry for Studies of Protein Folding, Stabilities, Assembly, and Molecular Interactions

2021 ◽  
Vol 51 (1) ◽  
Author(s):  
Arthur Laganowsky ◽  
David E. Clemmer ◽  
David H. Russell
Keyword(s):  
Mass Spectrometry ◽  
Dynamic Range ◽  
Protein Complexes ◽  
Noncovalent Interactions ◽  
Variable Temperature ◽  
Regulatory Protein ◽  
Conformational Dynamics ◽  
Native Mass Spectrometry ◽  
Annual Review ◽  
Publication Date

The structures and conformational dynamics of proteins, protein complexes, and their noncovalent interactions with other molecules are controlled specifically by the Gibbs free energy (entropy and enthalpy) of the system. For some organisms, temperature is highly regulated, but the majority of biophysical studies are carried out at room, nonphysiological temperature. In this review, we describe variable-temperature electrospray ionization (vT-ESI) mass spectrometry (MS)-based studies with unparalleled sensitivity, dynamic range, and selectivity for studies of both cold- and heat-induced chemical processes. Such studies provide direct determinations of stabilities, reactivities, and thermodynamic measurements for native and non-native structures of proteins and protein complexes and for protein–ligand interactions. Highlighted in this review are vT-ESI-MS studies that reveal 40 different conformers of chymotrypsin inhibitor 2, a classic two-state (native → unfolded) unfolder, and thermochemistry for a model membrane protein system binding lipid and its regulatory protein. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Native Mass Spectrometry: Recent Progress and Remaining Challenges

2022 ◽  
Vol 51 (1) ◽  
Author(s):  
Kelly R. Karch ◽  
Dalton T. Snyder ◽  
Sophie R. Harvey ◽  
Vicki H. Wysocki
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Structural Biology ◽  
Recent Progress ◽  
Native Mass Spectrometry ◽  
Structure And Function ◽  
Annual Review ◽  
Publication Date ◽  
Analysis Software ◽  
And Function

Native mass spectrometry (nMS) has emerged as an important tool in studying the structure and function of macromolecules and their complexes in the gas phase. In this review, we cover recent advances in nMS and related techniques including sample preparation, instrumentation, activation methods, and data analysis software. These advances have enabled nMS-based techniques to address a variety of challenging questions in structural biology. The second half of this review highlights recent applications of these technologies and surveys the classes of complexes that can be studied with nMS. Complementarity of nMS to existing structural biology techniques and current challenges in nMS are also addressed. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

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.

scholarly journals Insight to Functional Conformation and Noncovalent Interactions of Protein-Protein Assembly Using MALDI Mass Spectrometry

Molecules ◽  
2020 ◽  
Vol 25 (21) ◽  
pp. 4979
Author(s):  
Marco Giampà ◽  
Elvira Sgobba
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Noncovalent Interactions ◽  
Conformational Dynamics ◽  
High Sensitivity ◽  
Maldi Mass Spectrometry ◽  
Noncovalent Interaction ◽  
Ionization Mass ◽  
Label Free

Noncovalent interactions are the keys to the structural organization of biomolecule e.g., proteins, glycans, lipids in the process of molecular recognition processes e.g., enzyme-substrate, antigen-antibody. Protein interactions lead to conformational changes, which dictate the functionality of that protein-protein complex. Besides biophysics techniques, noncovalent interaction and conformational dynamics, can be studied via mass spectrometry (MS), which represents a powerful tool, due to its low sample consumption, high sensitivity, and label-free sample. In this review, the focus will be placed on Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) and its role in the analysis of protein-protein noncovalent assemblies exploring the relationship within noncovalent interaction, conformation, and biological function.

scholarly journals Advantages of Molecular Weight Identification during Native MS Screening

Planta Medica ◽  
2018 ◽  
Vol 84 (16) ◽  
pp. 1201-1212
Author(s):  
Ahad Khan ◽  
Anne Bresnick ◽  
Sean Cahill ◽  
Mark Girvin ◽  
Steve Almo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Fourier Transform ◽  
T Cell ◽  
Electrospray Ionization ◽  
Fourier Transform Mass Spectrometry ◽  
Protein Complexes ◽  
Native Mass Spectrometry ◽  
Biochanin A ◽  
Electrospray Ionization Fourier Transform

AbstractNative mass spectrometry detection of ligand-protein complexes allowed rapid detection of natural product binders of apo and calcium-bound S100A4 (a member of the metal binding protein S100 family), T cell/transmembrane, immunoglobulin (Ig), and mucin protein 3, and T cell immunoreceptor with Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains precursor protein from extracts and fractions. Based on molecular weight common hits were detected binding to all four proteins. Seven common hits were identified as apigenin 6-C-β-D-glucoside 8-C-α-L-arabinoside, sweroside, 4′,5-dihydroxy-7-methoxyflavanone-6-C-rutinoside, loganin acid, 6-C-glucosylnaringenin, biochanin A 7-O-rutinoside and quercetin 3-O-rutinoside. Mass guided isolation and NMR identification of hits confirmed the mass accuracy of the ligand in the ligand-protein MS complexes. Thus, molecular weight ID from ligand-protein complexes by electrospray ionization Fourier transform mass spectrometry allowed rapid dereplication. Native mass spectrometry using electrospray ionization Fourier transform mass spectrometry is a tool for dereplication and metabolomics analysis.

scholarly journals Mechanical Frequency Tuning by Sensory Hair Cells, the Receptors and Amplifiers of the Inner Ear

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Pascal Martin ◽  
A.J. Hudspeth
Keyword(s):  
Hopf Bifurcation ◽  
Hair Cells ◽  
Force Feedback ◽  
Dynamic Range ◽  
Frequency Tuning ◽  
Hair Bundle ◽  
Annual Review ◽  
Publication Date ◽  
Mechanical Waves ◽  
Myosin Motors

We recognize sounds by analyzing their frequency content. Different frequency components evoke distinct mechanical waves that each travel within the hearing organ, or cochlea, to a frequency-specific place. These signals are detected by hair cells, the ear's sensory receptors, in response to vibrations of mechanically sensitive antennas termed hair bundles. An active process enhances the sensitivity, sharpens the frequency tuning, and broadens the dynamic range of hair cells through several mechanisms, including active hair-bundle motility. A dynamic interplay between negative stiffness mediated by ion channels’ gating forces and delayed force feedback owing to myosin motors and channel reclosure by calcium ions brings the hair bundle to the vicinity of an oscillatory instability—a Hopf bifurcation. Operation near a Hopf bifurcation provides nonlinear generic features that are characteristic of hearing. Multiple gradients at molecular, cellular, and supercellular scales tune hair cells to characteristic frequencies that cover our auditory range. Expected final online publication date for the Annual Review of Condensed Matter Physics, Volume 12 is March 10, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Insights Into the Polerovirus–Plant Interactome Revealed by Coimmunoprecipitation and Mass Spectrometry

2015 ◽  
Vol 28 (4) ◽  
pp. 467-481 ◽  
Author(s):  
Stacy L. DeBlasio ◽  
Richard Johnson ◽  
Jaclyn Mahoney ◽  
Alexander Karasev ◽  
Stewart M. Gray ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Carbon Fixation ◽  
Dynamic Range ◽  
High Resolution Mass Spectrometry ◽  
Protein Complexes ◽  
Interaction Network ◽  
Host Protein ◽  
Host Proteins ◽  
Leafroll Virus ◽  
Readthrough Protein

Identification of host proteins interacting with the aphidborne Potato leafroll virus (PLRV) from the genus Polerovirus, family Luteoviridae, is a critical step toward understanding how PLRV and related viruses infect plants. However, the tight spatial distribution of PLRV to phloem tissues poses challenges. A polyclonal antibody raised against purified PLRV virions was used to coimmunoprecipitate virus-host protein complexes from Nicotiana benthamiana tissue inoculated with an infectious PLRV cDNA clone using Agrobacterium tumefaciens. A. tumefaciens-mediated delivery of PLRV enabled infection and production of assembled, insect-transmissible virus in most leaf cells, overcoming the dynamic range constraint posed by a systemically infected host. Isolated protein complexes were characterized using high-resolution mass spectrometry and consisted of host proteins interacting directly or indirectly with virions, as well as the nonincorporated readthrough protein (RTP) and three phosphorylated positional isomers of the RTP. A bioinformatics analysis using ClueGO and STRING showed that plant proteins in the PLRV protein interaction network regulate key biochemical processes, including carbon fixation, amino acid biosynthesis, ion transport, protein folding, and trafficking.

scholarly journals Native Tandem and Ion Mobility Mass Spectrometry Highlight Structural and Modular Similarities in Clustered-Regularly-Interspaced Shot-Palindromic-Repeats (CRISPR)-associated Protein Complexes From Escherichia coli and Pseudomonas aeruginosa

2012 ◽  
Vol 11 (11) ◽  
pp. 1430-1441 ◽  
Author(s):  
Esther van Duijn ◽  
Ioana M. Barbu ◽  
Arjan Barendregt ◽  
Matthijs M. Jore ◽  
Blake Wiedenheft ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Ion Mobility ◽  
Protein Complexes ◽  
Acquired Resistance ◽  
Native Mass Spectrometry ◽  
Ion Mobility Mass Spectrometry ◽  
E Coli ◽  
Ribonucleoprotein Complexes ◽  
Specific Association

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.

scholarly journals Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

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