Liquid extraction surface analysis for native mass spectrometry: Protein complexes and ligand binding

Victor A Mikhailov,Rian L Griffiths,Helen J Cooper

doi:10.1016/j.ijms.2016.09.011

Abstract

Native liquid extraction surface analysis (LESA) mass spectrometry enables the direct sampling of protein complexes from a solid surface. We have previously demonstrated native LESA mass spectrometry of holomyoglobin (∼17kDa) from glass slides and tetrameric haemoglobin (∼64kDa) from dried blood spots and thin tissue sections. Here, we further explore the capabilities of this emerging technique by investigating a range of proteins which exist in various oligomeric states in vivo. Tetrameric avidin (∼64kDa), octameric (∼190kDa) and hexadecameric (∼380kDa) CS2 hydrolase, and tetradecameric GroEL (∼800kDa) were all detected by native LESA mass spectrometry. Moreover, trimeric AmtB, a membrane protein, could also be observed by native LESA mass spectrometry. The suitability of LESA mass spectrometry for probing protein-ligand binding was also investigated. Non-covalent complexes of the ligand biotin with the proteins avidin, haemoglobin and bovine serum albumin were detected. The results indicate that non-specific binding is minimal and that native LESA mass spectrometry is a promising tool for the investigation of biologically significant ligand binding.

Highlights

Knowledge of protein tertiary and quaternary structure is generally required for understanding protein function or malfunction
Mass spectra of the charge states (14+ to 18+) of the avidin tetramer obtained on the Nanomate/Synapt G2S apparatus by direct infusion demonstrate peak broadening similar to liquid extraction surface analysis (LESA) mass spectrometry (MS) (Fig. S1B versus Fig. 1A)
Collision induced dissociation (CID) of the avidin tetramer reveals multiple peaks for each charge state of the fragment monomer (Fig. S1B), indicating the presence of multiple modifications on the protein backbone. These results suggest the avidin tetramer ions observed in LESA MS have the same structure as those observed in ‘standard’ native MS

Summary

Introduction

Knowledge of protein tertiary and quaternary structure is generally required for understanding protein function or malfunction. Experimental methods that provide this knowledge are capable of maintaining noncovalent bonds, such as hydrogen bonds and ␲-␲ interactions, preserving folded proteins in their native structure and the noncovalent bonding with those species with which they interact. Native electrospray mass spectrometry (MS) enables analysis of folded proteins and non-covalent protein complexes in the gas phase by choosing a non-denaturing electrospray buffer and careful optimisation of electric fields and pressures in the mass spectrometer [1,2,3,4]. A native MS approach has been developed for membrane protein complexes [15,16]. Stabilisation of these complexes in solution and the gas-phase requires that they are purified and electrosprayed in detergent micelles. The native MS approach allows investigation of interactions of membrane protein complexes with lipids and drugs [17,18]

Methods

Results

Conclusion