Gas-Phase Collisions with Trimethylamine-N-Oxide Enable Activation-Controlled Protein Ion Charge Reduction

Margit Kaldmäe,Michael Landreh,Nicklas Österlund,Nina Kronqvist,Cagla Sahin,Erik G Marklund,Danai Lianoudaki,Peter Bergman,Leopold L Ilag,Tomas Nyman,Timothy M Allison

doi:10.1007/s13361-019-02177-8

Abstract

Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine-N-oxide (TMAO). Based on experiments carried out on proteins ranging from 4.5 to 35 kDa, we find that when combined with collisional activation, TMAO removes approximately 60% of the charges acquired under native conditions. Ion mobility measurements furthermore show that TMAO-mediated charge reduction produces the same end charge state and arrival time distributions for native-like and denatured protein ions. Our results suggest that gas-phase collisions between the protein ions and TMAO result in proton transfer, in line with previous findings for dimethyl- and trimethylamine. By adjusting the energy of the collisions experienced by the ions, it is possible to control the degree of charge reduction, making TMAO a highly dynamic charge reducer that opens new avenues for manipulating protein charge states in ESI-MS and for investigating the relationship between protein charge and conformation.ᅟ

Highlights

The analysis of intact proteins by electrospray ionization (ESI)-mass spectrometry (MS) relies on the generation of multiply charged ions from solution, where protein solution structure and ion charge are intricately related
To test whether charge reduction by TMAO is dependent on the solution pH, we repeated the experiment in the presence of 10% formic acid, which results in a solution pH of about 1.5
We show that TMAO in combination with collisional activation produces the same end charge state for folded and denatured proteins independent of solution pH

Summary

Introduction

The analysis of intact proteins by electrospray ionization (ESI)-mass spectrometry (MS) relies on the generation of multiply charged ions from solution, where protein solution structure and ion charge are intricately related. We find that activation-dependent charge reduction by TMAO produces the same end charge states and arrival time distributions for denatured and native-like protein ions. The average charge again dropped to 2.2+ (Fig. 1a), suggesting that the charge-reducing effect is independent of the protonation state of TMAO in solution.

Results

Conclusion