• Application Note

Quantitative Determination of Noncovalent Protein-Ligand Interactions Automatic Chip-Based Nanoelectrospray

Quantitative Determination of Noncovalent Protein-Ligand Interactions Automatic Chip-Based Nanoelectrospray
  • S. Zhang
  • C.K. Van Pelt
  • D. B. Wilson
  • Anal Chem
  • Waters Corporation
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Abstract

The purpose of this experiment is to demonstrate automated nanoESI/MS analysis to determine micromolar and submicromolar dissociation constants as well as to measure the solution binding constants for the Ribonuclease A (RNase) complexes with cytidilic acid ligands.

Benefits

  • Allows extended acquisition time for better data quality 
  • Increases sample throughput 
  • Improves spray stability and reproducibility 
  • Automates nanoelectrospray with one time spray optimization 
  • No carryover

Introduction

Automated Chip-Based Nanoelectrospray

Advion BioSciences, Inc. (Ithaca, NY) has developed a method and demonstrated the capabilities of the NanoMate System for quantitative determination of noncovalent interactions between proteins and ligands. The NanoMate 100 is a chip-based automated nanoelectrospray ionization system for mass spectrometry, and is readily integrated with the Waters Micromass Q-Tof micro.

Investigations of noncovalent protein-ligand interactions by nanoelectrospray ionization mass spectrometry (nanoESI/MS) are of great interest because of their relevance to molecular recognition and to combinatory ligand library searching. This application note from Advion Biosciences introduces an experiment where automated nano ESI/MS analysis has been used to determine micromolar and submicromolar dissociation constants as well as to measure the solution biding constants for the Ribonuclease A (RNase) complexes with cytidilic acid ligands.

Waters Micromass Q-Tof micro Mass Spectrometer and the Advion NanoMate 100.

Experimental

Determination of Noncovalent Protein-Ligand Interactions

RNase complexed with cytidine 2’-monophosphate and cytidine triphosphate (see Figure 1), a well characterized model system, was used to demonstrate the method.

Titration Experiments

RNase protein was maintained at 10 μM and 4 μM, respectively, in 10 mM ammonium acetate pH 6.8 for titration of 2’-CMP (1 to 20 μM) and CTP (1 to 8 μM), respectively. The solutions were then incubated at room temperature for 15 minutes prior to MS analysis.

Competitive Binding Experiments

Equimolar solutions of 2’-CMP and CTP (4 μM) were mixed with 4 μM of RNase in 10 mM ammonium acetate pH 6.8. The solutions were then incubated at room temperature for 15 minutes prior to nanoelectrospray MS analysis.

NanoESI/MS Analytical Conditions

Sample size:

3 μL

Flow rate:

100 nL/minute

Spray voltage:

1.5 kV

Pressure:

0.3 psi

Acquisition time:

2 minutes

Instrumentation:

NanoMate100 with ESI Chip Micromass Q-Tof micro

Sample cone voltage:

30 V

Source temp.:

45 °C

Results and Discussion

Each ligand was detected using the NanoMate100 System (Figures 2 and 3) and as a result titration and competitive binding experiments were performed (Figure 4). The results presented are in agreement with previously published results of circular dichroism (CD).1

Figure 1. Protein and ligand structures.
Figure 2. NanoESI mass spectra.
Figure 3. Deconvoluted mass spectra of the RNase-Ligand complexes.
Figure 4. Titration assay for RNase (10mM) with 2’-CMP.
Table 1. Summary of binding assay for RNase and cytidine nucleotide ligands using automated NanoESI/MS.

Conclusion

The NanoMate100 System can be used to determine micromolar and submicromolar dissociation constants. In addition, an automated nanoESI/MS method can be used to measure solution binding constants for the RNase complexes with cytidilic acid ligands.

References

  1. Jones, C.L.; Fish, F.; Muccio, D.D. Anal BioChem 2002, 302, 184–190.
  2. Application note based on Zhang, S.; Van Pelt, C.K.; Wilson, D.B. Anal Chem 2003, 75, 3010–3018.

720000871, May 2004

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