Establish an automated flow injection ESI-MS method for the screening of fragment based libraries: Application to Hsp90

Sirtori, Federico Riccardi; Caronni, Dannica; Colombo, Maristella; Dalvit, Claudio; Paolucci, Mauro; Regazzoni, Luca; Visco, Carlo; Fogliatto, Gianpaolo

doi:10.1016/j.ejps.2015.05.001

Cited by 13 publications

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References 65 publications

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“…A fairly good agreement has been demonstrated between the K a values derived by native ESI-MS and by earlier established analytical methods for a multitude of protein complexes. − Direct native ESI-MS titration data allow for adequate characterization of complex protein–ligand binding equilibria involving multiple binding steps and cooperative effects, − which is often helpful in order to validate or refine the binding model as well as to increase the accuracy of K a value determination . Native ESI-MS is also an attractive technique for drug discovery − via screening of compound or fragment libraries. − However, significant deviations in the results are also rather common, both between MS and other methods − as well as between different research laboratories . Clearly, closer attention is needed toward the controversial phenomena encountered in native MS in order to better understand the capabilities of this approach for the correct spectral interpretation of noncovalent protein complexes. , …”

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confidence: 78%

Charge-State-Dependent Variation of Signal Intensity Ratio between Unbound Protein and Protein–Ligand Complex in Electrospray Ionization Mass Spectrometry: The Role of Solvent-Accessible Surface Area

Chingin

Barylyuk

2018

Anal. Chem.

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Native electrospray ionization mass spectrometry (ESI-MS) is nowadays widely used for the direct and sensitive determination of protein complex stoichiometry and binding affinity constants ( K). A common yet poorly understood phenomenon in native ESI-MS is the difference between the charge-state distributions (CSDs) of the bound protein-ligand complex (PL) and unbound protein (P) signals. This phenomenon is typically attributed to experimental artifacts such as nonspecific binding or in-source dissociation and is considered highly undesirable, because the determined K values display strong variation with charge state. This situation raises serious concerns regarding the reliability of ESI-MS for the analysis of protein complexes. Here we demonstrate that, contrary to the common belief, the CSD difference between P and PL ions can occur without any loss of complex integrity, simply due to a change in the solvent-accessible surface area (ΔSASA) of the protein upon ligand binding in solution. The experimental CSD shifts for PL and P ions in ESI-MS are explained in relation to the magnitude of ΔSASA for diverse protein-ligand systems using a simple model based on the charged residue mechanism. Our analysis shows that the revealed ΔSASA factor should be considered rather general and be given attention for the correct spectral interpretation of protein complexes.

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confidence: 78%

Charge-State-Dependent Variation of Signal Intensity Ratio between Unbound Protein and Protein–Ligand Complex in Electrospray Ionization Mass Spectrometry: The Role of Solvent-Accessible Surface Area

Chingin

Barylyuk

2018

Anal. Chem.

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“…If the known ligand is not binding in the same site as the fragment, as the known ligand concentration increases and the protein becomes saturated, the decrease in the fraction of the complex with the fragment is accompanied by a proportional increase in the fraction of the ternary complex between the protein, the fragment and the known ligand (Figure 4b). A recent example on the application of these kinds of experiments can be found on the work published by Sirtori et al, who used a reference inhibitor known to bind at the ATP binding site of heat shock protein 90 (Hsp90) to narrow down their fragment hit list to ATP-competitive fragments [85].…”

Section: Characterizing Noncovalent Protein-fragment Interactionsmentioning

confidence: 99%

Native Mass Spectrometry in Fragment-Based Drug Discovery

Pedro

Quinn

2016

Molecules

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Abstract:The advent of native mass spectrometry (MS) in 1990 led to the development of new mass spectrometry instrumentation and methodologies for the analysis of noncovalent protein-ligand complexes. Native MS has matured to become a fast, simple, highly sensitive and automatable technique with well-established utility for fragment-based drug discovery (FBDD). Native MS has the capability to directly detect weak ligand binding to proteins, to determine stoichiometry, relative or absolute binding affinities and specificities. Native MS can be used to delineate ligand-binding sites, to elucidate mechanisms of cooperativity and to study the thermodynamics of binding. This review highlights key attributes of native MS for FBDD campaigns.

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Critical Evaluation of Native Electrospray Ionization Mass Spectrometry for Fragment‐Based Screening

et al. 2017

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Fragment-based screening presents a promising alternative to high-throughput screening and has gained great attention in recent years. So far, only a few studies have discussed mass spectrometry as a screening technology for fragments. Herein, we report the application of native electrospray ionization mass spectrometry (MS) for screening defined sets of fragments against four different target proteins. Fragments were selected from a primary screening conducted with a thermal shift assay (TSA) and represented different binding categories. Our data indicated that, beside specific complex formation, many fragments show extensive multiple binding and also charge-state shifts. Both of these factors complicate automated data analysis and decrease the attractiveness of native MS as a primary screening tool for fragments. A comparison of the hits identified by native MS and TSA showed good agreement for two of the proteins. Furthermore, we discuss general challenges, including the determination of an optimal fragment concentration and the question of how to rank fragment hits according to their affinity. In conclusion, we consider native MS to be a highly valuable tool for the validation and deeper investigation of promising fragment hits rather than a method for primary screening.

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Establish an automated flow injection ESI-MS method for the screening of fragment based libraries: Application to Hsp90

Cited by 13 publications

References 65 publications

Charge-State-Dependent Variation of Signal Intensity Ratio between Unbound Protein and Protein–Ligand Complex in Electrospray Ionization Mass Spectrometry: The Role of Solvent-Accessible Surface Area

Charge-State-Dependent Variation of Signal Intensity Ratio between Unbound Protein and Protein–Ligand Complex in Electrospray Ionization Mass Spectrometry: The Role of Solvent-Accessible Surface Area

Native Mass Spectrometry in Fragment-Based Drug Discovery

Critical Evaluation of Native Electrospray Ionization Mass Spectrometry for Fragment‐Based Screening

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