Examination of Various Metal Ion Sources for Reducing Nonspecific Zinc finger-Zn2+Complex Formation in ESI Mass Spectrometry

Soo-Jin Park,Sun-Hee Park,Sang-Yun Han,Kyu-Bong Jo,Joo-Yeon Oh,Han-Bin Oh

doi:10.5478/msl.2012.3.3.82

Abstract

The formation of zinc finger peptide−Zn2+ complexes in electrospray ionization mass spectrometry (ESI-MS) wasexamined using three different metal ion sources: ZnCl2, Zn(CH3COO)2, and Zn(OOC(CHOH)2COO). For the four zinc fingerpeptides (Sp1-1, Sp1-3, CF2II-4, and CF2II-6) that bind only a single Zn2+ in the native condition, electrospray of apo-zinc fingerin solution containing ZnCl2 or Zn(CH3COO)2 resulted in the formation of zinc finger−Zn2+ complexes with multiple zincions. This result suggests the formation of nonspecific zinc finger−Zn2+ complexes. Zn(tartrate), Zn(OOC(CHOH)2COO),mainly produced specific zinc finger−Zn2+ complexes with a single zinc ion. This study clearly indicates that tartrate is an excellentcounter ion in ESI-MS studies of zinc finger−Zn2+ complexes, which prevents the formation of nonspecific zinc finger−Zn2+complexes.

Highlights

Electrospray ionization mass spectrometry (ESI-MS) has been widely used to characterize specific noncovalent biological interactions in solution, including multi-protein assemblies, protein-/peptide-ligand, and DNA-/RNA-drug complexes.[1−9] In practice, ESI-MS has been used to determine binding stoichiometry,[5,10−12] and to measure relative[13,14] and absolute[15−17] association constants.While ESI-MS is clearly a powerful bioanalytical tool, this technique does have certain limitations
The formation of nonspecific noncovalent complexes may lead to misinterpretation of ESI-MS results and obscure the binding stoichiometry of specific complexes
We demonstrate that tartrate is most effective in reducing nonspecific Zn2+ adduction for zinc finger peptides, i.e., minimizing the formation of zinc finger−Zn2+ complexes with multiple zinc ions

Summary

Introduction

Electrospray ionization mass spectrometry (ESI-MS) has been widely used to characterize specific noncovalent biological interactions in solution, including multi-protein assemblies, protein-/peptide-ligand, and DNA-/RNA-drug complexes.[1−9] In practice, ESI-MS has been used to determine binding stoichiometry,[5,10−12] and to measure relative[13,14] and absolute[15−17] association constants (binding affinities).While ESI-MS is clearly a powerful bioanalytical tool, this technique does have certain limitations. A few different metal sources that contain different counter ions have been evaluated for use in minimizing nonspecific protein-metal ion adduct formation in ESI-MS experiments. ESI-MS can be successfully used to examine the binding specificity of zinc finger peptides to DNA with a cognate sequence.[26] In this study, we demonstrate that tartrate is most effective in reducing nonspecific Zn2+ adduction for zinc finger peptides, i.e., minimizing the formation of zinc finger−Zn2+ complexes with multiple zinc ions.

Results

Conclusion