Automated Nano-Electrospray Mass Spectrometry for Protein-Ligand Screening by Noncovalent Interaction Applied to Human H-FABP and A-FABP

Benkestock, Kurt; Pelt, Colleen K. Van; Åkerud, Tomas; Sterling, Alistair; Edlund, Per-Olof; Roeraade, Johan

doi:10.1177/1087057103008003002

Cited by 41 publications

(51 citation statements)

References 31 publications

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“…Recently, we published the first report on the use of automated nano-ESI microchip technology for protein-ligand screening. 19 It has been stated in the literature that the equilibrium between molecules present in solution is better reflected by nano-ESI than ESI during noncovalent binding studies (a higher complex ratio is obtained by nano-ESI). 20 No systematic studies of the conditions for noncovalent interactions have been performed to support this statement.…”

Section: Introductionmentioning

confidence: 99%

Influence of droplet size, capillary–cone distance and selected instrumental parameters for the analysis of noncovalent protein–ligand complexes by nano‐electrospray ionization mass spectrometry

et al. 2004

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It has been suggested in the literature that nano-electrospray ionization (nano-ESI) mass spectrometry better reflects the equilibrium between complex and free protein in solution than pneumatically assisted electrospray ionization (ESI) in noncovalent interaction studies. However, no systematic studies of the effects of ionization conditions have been performed to support this statement. In the present work, different instrumental and sample-derived parameters affecting the stability of noncovalent complexes during analysis by nano-ESI were investigated. In general, increased values of parameters such as drying gas flow-rate, ion-source temperature, capillary tip voltage and buffer concentration lead to a dissociation of ribonuclease A (RNAse)-cytidine 2'-monophosphate (CMP) and cytidine 5'-triphosphate (CTP) complexes. The size of the electrosprayed droplets was shown to be an important issue. Increasing the capillary to cone distance yielded an increased complex to free protein ratio when a hydrophilic ligand was present and the reverse effect was obtained with a hydrophobic ligand. Important in this regard is the degree of sampling of ions originating from late-generation residue droplets, that is, ions present in the droplet bulk. Sampling of these ions increases with longer capillary-cone distance (flight time). Furthermore, when the sample flow-rate was increased by increasing the capillary internal tip i.d. from 4 to 30 microm, a decreased complex to free protein ratio for the RNAse-CTP system was observed. This behavior was consistent with the change in surface to volume ratio for flow-rates between 2 and 100 nl min(-1). Finally, polarity switching between positive and negative ion modes gave a higher complex to free protein ratio when the ligand and the protein had the same polarity.

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Section: Introductionmentioning

confidence: 99%

Influence of droplet size, capillary–cone distance and selected instrumental parameters for the analysis of noncovalent protein–ligand complexes by nano‐electrospray ionization mass spectrometry

et al. 2004

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“…The instrument is well described elsewhere [12] and has been successfully used for the analysis of noncovalent protein-protein and protein-nonmetal ligand interactions [13][14][15][16]. In the present work, we use it to analyze the zinc affinity of the wild-type CphA enzyme and of several mutated enzymes in a semi-high throughput fashion.…”

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confidence: 99%

Monitoring the zinc affinity of the metallo-β-lactamase CphA by automated nanoESI-MS

Vriendt

Driessche

Devreese

et al. 2006

J. Am. Soc. Mass Spectrom.

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Metallo-␤-lactamases are zinc containing enzymes that are able to hydrolyze and inactivate ␤-lactam antibiotics. The subclass B2 enzyme CphA of Aeromonas hydrophila is a unique metallo-␤-lactamase because it degrades only carbapenems efficiently and is only active when it has one zinc ion bound. A zinc titration experiment was used to study the zinc affinity of the wild-type and of several mutant CphA enzymes. It shows that a second Zn 2ϩ is also bound at high ion concentrations. All samples were analyzed using mass spectrometry in combination with an automated nanoESI source. The metal-free enzyme has a bimodal charge distribution indicative of two conformational states. A completely folded enzyme is detected when the apo-enzyme has bound the first zinc. Intensity ratios of the different enzyme forms were used to deduce the zinc affinities. CphA enzymes mutated in metal ligands show decreased zinc affinity compared to wild-type, especially D120 mutants. (J Am Soc Mass Spectrom 2006, 17, 180 -188)

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“…These ion sources have also enabled automated sample delivery into MS instruments, without risk of cross-contamination, in a high throughput fashion. The advantages of an automated chip-based ESI infusion have already been widely proven in proteomics, direct screening of drugs and drug discovery [1][2][3][4][5][6][7].…”

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Fully-automated chip-based nanoelectrospray tandem mass spectrometry of gangliosides from human cerebellum

Zamfir

Vukelić

Bîndilă

et al. 2004

J. Am. Soc. Mass Spectrom.

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The introduction of chip-based electrospray (ESI) ion sources into biological mass spectrometry (MS) addressed the fundamental issue of how to analyze minute amounts of complex biological systems. The automation of sample delivery into the MS combined with the chip-based ESI allows for high quality bioanalysis in a high-throughput fashion. These advantages have already been demonstrated in proteomics, direct screening of drugs and drug discovery. As part of our continuing effort to implement automated chip-based mass spectrometry into the field of complex carbohydrate analysis, we hereby report the development of a chipESI MS and MS/MS methodology for the screening of gangliosides. A strategy to characterize a complex ganglioside mixture from human cerebellar tissue, by automated ESIchip-quadrupole time-of-flight (QTOF) MS and MS/MS is presented here. The feasibility of this method, and the general experimental requirements for automated chipESI MS analysis of these carbohydrate species is

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Automated Nano-Electrospray Mass Spectrometry for Protein-Ligand Screening by Noncovalent Interaction Applied to Human H-FABP and A-FABP

Cited by 41 publications

References 31 publications

Influence of droplet size, capillary–cone distance and selected instrumental parameters for the analysis of noncovalent protein–ligand complexes by nano‐electrospray ionization mass spectrometry

Influence of droplet size, capillary–cone distance and selected instrumental parameters for the analysis of noncovalent protein–ligand complexes by nano‐electrospray ionization mass spectrometry

Monitoring the zinc affinity of the metallo-β-lactamase CphA by automated nanoESI-MS

Fully-automated chip-based nanoelectrospray tandem mass spectrometry of gangliosides from human cerebellum

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