Reliable Determinations of Protein–Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?

Kitova, Elena N.; El-Hawiet, Amr; Schnier, Paul D.; Klassen, John S.

doi:10.1007/s13361-011-0311-9

Cited by 218 publications

(338 citation statements)

References 93 publications

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“…Abundant ion signal corresponding to tubulin dimer (d n+ ), at charge states +18 to +22, is evident; low abundance signal corresponding to tubulin tetramer ions, at charge states +32 to +29, was also [48,49] are in agreement with theoretical values (UniProt, P02550 for αI and P02554 for βI) of 101,677 Da and 203,354 Da, respectively. The presence of tetramer could be due to selfassociation of the tubulin dimer; nonspecific binding during the ESI process might also play a role in its formation [38,39]. Because the stock solutions of L1-L8 contained DMSO, ESI mass spectra were also acquired for aqueous solutions of tubulin in the presence of low concentrations of DMSO.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, the direct electrospray ionization mass spectrometry (ESI-MS) assay has emerged as a powerful tool for measuring the affinities of protein-ligand interactions in vitro [34][35][36][37][38][39]. The assay is based on direct measurement of the relative abundances of the ligand-bound and free protein ions measured by ESI-MS.…”

Section: Introductionmentioning

confidence: 99%

“…This assay has a number of strengths that make it a valuable technique for binding measurements, including simplicity (no labeling or immobilization is required), speed (measurements can be completed within a few minutes), and low sample consumption (typically <pmol per analysis). The ability to measure multiple binding equilibria simultaneously makes the ESI-MS assay well suited for screening libraries of compounds against target proteins [38,39]. Where direct detection of the protein-ligand complexes by ESI-MS is not possible due to factors such as high molecular weight (MW) or protein heterogeneity, the catch-and-release (CaR) ESI-MS assay can be employed [40][41][42][43][44][45][46][47].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

Darestani

Winter

Kitova

et al. 2016

J. Am. Soc. Mass Spectrom.

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Abstract. Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

Darestani

Winter

Kitova

et al. 2016

J. Am. Soc. Mass Spectrom.

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“…Shiga toxin type 1 B subunit homopentamer (Stx1B 5 , MW 38, 455 Da) was a gift from Professor G. Armstrong (University of Calgary). A single chain variable fragment (scFv, MW 26,539 Da) of the monoclonal antibody Se155-4, which served as a reference protein (P ref ) [31,32] for direct ESI-MS binding measurements, was produced using recombinant technology as described elsewhere [33]. To prepare stock solutions of CTB 5 and Stx1B 5 , each protein was dialyzed against 200 mM ammonium acetate (pH 6.8) using 0.5 mL Amicon microconcentrators (EMD Millipore, Billerica, MA, USA) with a 30 kDa MW cutoff.…”

Section: Methodsmentioning

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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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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“…3 Electrospray ionization mass spectrometry (ESI-MS) in native conditions helps deciphering the complexation equilibria. [4][5][6][7] Coupled to ion mobility spectrometry (IMS), native mass spectrometry makes it possible to reveal the ensuing conformational responses. [8][9][10] But before native ESI-IMS-MS can be applied to study nucleic acids conformations from solution, it is essential to understand to what extent the different types of DNA/RNA secondary structures are preserved, or affected, by the transition from the solution to the gas phase.…”

Section: Introductionmentioning

confidence: 99%

Compaction of Duplex Nucleic Acids upon Native Electrospray Mass Spectrometry

Porrini

Rosu

Rabin

et al. 2017

Preprint

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ABSTRACT:Native mass spectrometry coupled to ion mobility spectrometry is a promising tool for structural biology. Intact complexes can be transferred to the mass spectrometer and, if native conformations survive, collision cross sections give precious information on the structure of each species in solution. Based on several successful reports for proteins and their complexes, the conformation survival becomes more and more taken for granted. Here we report on the fate of nucleic acids conformation in the gas phase. Disturbingly, we found that DNA and RNA duplexes, at the electrospray charge states naturally obtained from native solution conditions (≥ 100 mM aqueous NH 4 OAc), are significantly more compact in the gas phase compared to the canonical solution structures. The compaction is observed for short (12-bp) and long (36-bp) duplexes, and for DNA and RNA alike. Molecular modeling (density functional calculations on small helices, semi-empirical calculations on up to 12-bp, and molecular dynamics on up to 36-bp duplexes) demonstrates that the compaction is due to phosphate group self-solvation prevailing over Coulomb-driven expansion. Molecular dynamics simulations starting from solution structures do not reproduce the experimental compaction. To be experimentally relevant, molecular dynamics sampling should reflect the progressive structural rearrangements occurring during desolvation. For nucleic acid duplexes, the compaction observed for low charge states results from novel phosphate-phosphate hydrogen bonds formed across both grooves at the very late stages of electrospray.

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Reliable Determinations of Protein–Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?

Cited by 218 publications

References 93 publications

Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

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

Compaction of Duplex Nucleic Acids upon Native Electrospray Mass Spectrometry

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