Direct Detection of Iron Clusters in L Ferritins through ESI MS Experiments

2021 ◽  
Author(s):  
Lara Massai ◽  
Silvia Ciambellotti ◽  
Lucrezia Cosottini ◽  
Luigi Messori ◽  
Paola Turano ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Direct Detection ◽  
Nucleation Site ◽  
Iron Clusters ◽  
Esi Ms ◽  
Ferroxidase Center

Human cytoplasmic ferritins are heteropolymers of H and L subunits containing a catalytic ferroxidase center and a nucleation site for iron biomineralization, respectively. Here, ESI-MS successfully detected labile metal-protein interactions...

scholarly journals Universal Screening Methods and Applications of ThermoFluor®

2006 ◽  
Vol 11 (7) ◽  
pp. 854-863 ◽  
Author(s):  
Maxwell D. Cummings ◽  
Michael A. Farnum ◽  
Marina I. Nelen
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Protein Unfolding ◽  
Direct Detection ◽  
Functional Characterization ◽  
Screening Methods ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Bacterial Enzyme ◽  
Research Problems

The genomics revolution has unveiled a wealth of poorly characterized proteins. Scientists are often able to produce milligram quantities of proteins for which function is unknown or hypothetical, based only on very distant sequence homology. Broadly applicable tools for functional characterization are essential to the illumination of these orphan proteins. An additional challenge is the direct detection of inhibitors of protein-protein interactions (and allosteric effectors). Both of these research problems are relevant to, among other things, the challenge of finding and validating new protein targets for drug action. Screening collections of small molecules has long been used in the pharmaceutical industry as 1 method of discovering drug leads. Screening in this context typically involves a function-based assay. Given a sufficient quantity of a protein of interest, significant effort may still be required for functional characterization, assay development, and assay configuration for screening. Increasingly, techniques are being reported that facilitate screening for specific ligands for a protein of unknown function. Such techniques also allow for function-independent screening with better characterized proteins. ThermoFluor®, a screening instrument based on monitoring ligand effects on temperature-dependent protein unfolding, can be applied when protein function is unknown. This technology has proven useful in the decryption of an essential bacterial enzyme and in the discovery of a series of inhibitors of a cancer-related, protein-protein interaction. The authors review some of the tools relevant to these research problems in drug discovery, and describe our experiences with 2 different proteins.

Do electrospray mass spectra reflect the ligand binding state of proteins in solution?

2005 ◽  
Vol 83 (11) ◽  
pp. 1953-1960 ◽  
Author(s):  
Belal M Hossain ◽  
Douglas A Simmons ◽  
Lars Konermann
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Protein Interactions ◽  
Noncovalent Complex ◽  
Deuterium Exchange ◽  
Solution Phase ◽  
Hydrogen Deuterium Exchange ◽  
Esi Ms ◽  
Protein Ions ◽  
Hydrogen Deuterium

Electrospray ionization (ESI) mass spectrometry (MS) has become a popular tool for monitoring ligand–protein and protein–protein interactions. Due to the "gentle" nature of the ionization process, it is often possible to transfer weakly bound complexes into the gas phase, thus making them amenable to MS detection. One problem with this technique is the potential occurrence of fragmentation events during ESI. Also, some analytes tend to cluster together during ionization, thus forming nonspecific gas-phase assemblies that do not represent solution-phase complexes. In this work, we implemented a hydrogen–deuterium exchange (HDX) approach that can reveal whether or not the free and (or) bound constituents of a complex observed in ESI-MS reflect the binding situation in solution. Proteins are subjected to ESI immediately following an isotopic labeling pulse; only ligand-free and ligand-bound protein ions that were formed directly from the corresponding solution-phase species showed different HDX levels. Using myoglobin as a model system, it is demonstrated that this approach can readily distinguish scenarios where the heme–protein interactions were disrupted in solution from those where dissociation of the complex occurred in the gas phase. Experiments on cytochrome c strongly suggest that dimeric protein ions observed in ESI-MS reflect aggregates that were formed in solution.Key words: electrospray mass spectrometry, ligand–protein interaction, noncovalent complex, hydrogen–deuterium exchange, protein folding.

2003 Fred Beamish Award Lecture — Exploring the dynamics of biological systems by mass spectrometry

2004 ◽  
Vol 82 (11) ◽  
pp. 1565-1580 ◽  
Author(s):  
Lars Konermann
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Structural Changes ◽  
Structural Information ◽  
Ionization Mass ◽  
Time Resolved ◽  
Rapid Mixing ◽  
Esi Ms ◽  
Compound Libraries

This review describes the use of electrospray ionization mass spectrometry (ESI-MS) in conjunction with on-line rapid mixing techniques. This combination, termed "time-resolved" ESI-MS, provides a powerful approach for studying solution-phase reactions on timescales as short as a few milliseconds. Of particular interest is the application of this technique for monitoring protein folding reactions. Time-resolved ESI-MS can provide detailed information on structural changes of the polypeptide chain, while at the same time probing the occurrence of noncovalent ligand–protein interactions. Especially when used in combination with hydrogen–deuterium pulse labeling, these measurements yield valuable structural information on short-lived folding intermediates. Similar approaches can be used to monitor the dynamics of proteins under equilibrium conditions. Another important application of time-resolved ESI-MS are mechanistic studies on enzyme-catalyzed processes. These reactions can be monitored under presteady-state conditions, without requiring artificial chromophoric substrates or radioactive labeling. We also discuss the use of ESI-MS for monitoring noncovalent ligand–protein interactions by diffusion measurements. In contrast to conventional MS-based techniques, this approach does not rely on the preservation of noncovalent interactions in the gas phase. It appears that diffusion measurements by ESI-MS could become an interesting alternative to existing methods for the high throughput screening of compound libraries in the context of drug discovery.Key words: reaction intermediate, rapid mixing, kinetics, protein conformation, protein function.

Development of a direct ESI-MS method for measuring the tannin precipitation effect of proline-rich peptides and in silico studies on the proline role in tannin-protein interactions

Fitoterapia ◽  
2019 ◽  
Vol 136 ◽  
pp. 104163
Author(s):  
Giovanna Baron ◽  
Alessandra Altomare ◽  
Laura Fumagalli ◽  
Cristiano Rumio ◽  
Marina Carini ◽  
...  
Keyword(s):  
Protein Interactions ◽  
In Silico ◽  
Esi Ms ◽  
Precipitation Effect ◽  
In Silico Studies

Multipronged ESI–MS Approach for Studying Glycan-Binding Protein Interactions with Glycoproteins

2019 ◽  
Vol 91 (3) ◽  
pp. 2140-2147 ◽  
Author(s):  
Yilin Wang ◽  
Heajin Park ◽  
Hong Lin ◽  
Elena N. Kitova ◽  
John S. Klassen
Keyword(s):  
Protein Interactions ◽  
Binding Protein ◽  
Esi Ms

Direct Detection of Key Intermediates in Rhodium(I)-Catalyzed [2+2+2] Cycloadditions of Alkynes by ESI-MS

2012 ◽  
Vol 18 (41) ◽  
pp. 13097-13107 ◽  
Author(s):  
Magda Parera ◽  
Anna Dachs ◽  
Miquel Solà ◽  
Anna Pla-Quintana ◽  
Anna Roglans
Keyword(s):  
Direct Detection ◽  
Esi Ms

Metallothionein Cd4S11 cluster formation dominates in the protection of carbonic anhydrase

Metallomics ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 767-783 ◽  
Author(s):  
Amelia T. Yuan ◽  
Natalie C. Korkola ◽  
Daisy L. Wong ◽  
Martin J. Stillman
Keyword(s):  
Carbonic Anhydrase ◽  
Protein Interactions ◽  
Cluster Formation ◽  
Stopped Flow ◽  
Protein Protein Interactions ◽  
Esi Ms ◽  
Stopped Flow Kinetics

Results from ESI-MS and stopped flow kinetics show that apo-MT protects from toxic metalation of apo-CA with Cd2+ due to the protein–protein interactions in solution.

scholarly journals Thermodynamic Evaluation of the Interactions between Anticancer Pt(II) Complexes and Model Proteins

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2376
Author(s):  
Chiara Pelosi ◽  
Francesca Saitta ◽  
Caterina Zerino ◽  
Giovanni Canil ◽  
Tarita Biver ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Anticancer Agents ◽  
Covalent Bond ◽  
Binding Mode ◽  
Critical Issue ◽  
Rnase A ◽  
Ionization Mass ◽  
Thermodynamic Evaluation ◽  
Scanning Calorimetry ◽  
Esi Ms

In this work, we have analysed the binding of the Pt(II) complexes ([PtCl(4′-phenyl-2,2′:6′,2″-terpyridine)](CF3SO3) (1), [PtI(4′-phenyl-2,2′:6′,2″-terpyridine)](CF3SO3) (2) and [PtCl(1,3-di(2-pyridyl)benzene) (3)] with selected model proteins (hen egg-white lysozyme, HEWL, and ribonuclease A, RNase A). Platinum coordination compounds are intensively studied to develop improved anticancer agents. In this regard, a critical issue is the possible role of Pt-protein interactions in their mechanisms of action. Multiple techniques such as differential scanning calorimetry (DSC), electrospray ionization mass spectrometry (ESI-MS) and UV-Vis absorbance titrations were used to enlighten the details of the binding to the different biosubstrates. On the one hand, it may be concluded that the affinity of 3 for the proteins is low. On the other hand, 1 and 2 strongly bind them, but with major binding mode differences when switching from HEWL to RNase A. Both 1 and 2 bind to HEWL with a non-specific (DSC) and non-covalent (ESI-MS) binding mode, dominated by a 1:1 binding stoichiometry (UV-Vis). ESI-MS data indicate a protein-driven chloride loss that does not convert into a covalent bond, likely due to the unfavourable complexes’ geometries and steric hindrance. This result, together with the significant changes of the absorbance profiles of the complex upon interaction, suggest an electrostatic binding mode supported by some stacking interaction of the aromatic ligand. Very differently, in the case of RNase A, slow formation of covalent adducts occurs (DSC, ESI-MS). The reactivity is higher for the iodo-compound 2, in agreement with iodine lability higher than chlorine.

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