On‐line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non‐covalent complexes and competitive binding studies

Benkestock, Kurt; Edlund, Per Olof; Roeraade, Johan

doi:10.1002/rcm.832

Cited by 27 publications

(30 citation statements)

References 38 publications

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“…20,30 Other larger molecules may also bind to a target molecule in a nonspecific manner. Jorgensen et al 31 recently showed nonspecific interactions between peptides and vancomycin.…”

Section: Nonspecific Versus Specific Protein-ligand Interactionmentioning

confidence: 99%

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

Benkestock¹,

Pelt²,

Åkerud³

et al. 2003

SLAS Discovery

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A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Furthermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by

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“…20,30 Other larger molecules may also bind to a target molecule in a nonspecific manner. Jorgensen et al 31 recently showed nonspecific interactions between peptides and vancomycin.…”

Section: Nonspecific Versus Specific Protein-ligand Interactionmentioning

confidence: 99%

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

Benkestock¹,

Pelt²,

Åkerud³

et al. 2003

SLAS Discovery

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“…Many proteins and macromolecules rapidly form metal adduct ions when analyzed in pure buffer solutions, which impairs their analysis by mass spectrometry. Unwanted adduct formation can be reduced by using microdialysis 15 or an in-line size-exclusion chromatography gel filtration cartridge. 16 Second, hydrophobic interactions are weaker in the gas phase than the liquid phase.…”

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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“…A few existing designs employ such injectors, with the smallest sample volumes of 5 to 6 µl reported. 7,16,27 Lutz et al used a similar microinjector, although their method included a sheath flow rate of 5 µl/min with a standard ESI source. 7 In comparison, we utilize an ESI-friendly solvent to carry the plug of injected sample and an NSI source, as discussed in the following section.…”

Section: Microdialysis Unit Designmentioning

confidence: 98%

Online microdialysis‐dynamic nanoelectrospray ionization‐mass spectrometry for monitoring neuropeptide secretion

2005

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Although mass spectrometric approaches offer a sensitive method for identifying cell-cell signaling peptides, the high salt-containing environment of extracellular solutions often complicates characterization of these microscale samples. Accordingly, we have developed a miniature hollow-fiber microdialysis device optimized for desalting small-volume neuronal samples online, with the device directly connected to a modified dynamic nanoelectrospray ionization assembly interfaced with an ion trap mass spectrometer. Improvements over existing designs include placement of a capillary insert within the microdialysis fiber to minimize volume, as well as the use of a microinjector that enables 1 microl sample injections. We present detailed evaluation of peptide recoveries within the microdialysis fiber by liquid chromatography-electrospray ionization-ion trap-mass spectrometry analysis of tissue homogenate in artificial seawater with and without microdialysis. Analyte recoveries after microdialysis ranged from 6 to 78% with higher recoveries of more hydrophilic peptides, while little correlation between mass and percentage recovery was observed in the range studied (2000 to 6000 Da). Recoveries of peptides were the lowest for the analytes with the highest initial mass spectrometry signal intensity. Finally, we illustrate the utility of this microdialysis device for desalting neuropeptides secreted from preparations of the peptidergic bag cell neurons of the marine mollusk, Aplysia californica. Without microdialysis, the high concentration of salts ( approximately 0.5 M) prevented detection of peptides, whereas following online microdialysis-dynamic nanoelectrospray mass spectrometry of stimulated releasate, three peptides (acidic peptide, acidic peptide 1-24 and delta-bag cell peptide) were detected.

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On‐line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non‐covalent complexes and competitive binding studies

Cited by 27 publications

References 38 publications

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

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

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

Online microdialysis‐dynamic nanoelectrospray ionization‐mass spectrometry for monitoring neuropeptide secretion

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