Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry.

Mia Abramsson ,Cagla Sahin,Arthur Laganowsky,Jakob R Winther,Lisa Lang,Axel Leppert,Kaare Teilum,Jonathan T S Hopper,Michael Landreh,Carol V Robinson,Joana Costeira-Paulo,Timothy M Allison,Jens Danielsson,Mikael Oliveberg,Leopold L Ilag,Shane A Chandler,Justin L P Benesch,Nicklas Österlund,Mangmang Xu ,Erik Marklund ,Rui M Branca

doi:10.1021/jacsau.1c00458

Abstract

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

Highlights

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics
Protein charging in positive electrospray ionization (ESI) can be dependent or independent of basic residues: native-like proteins charge according to their solvent-accessible surface area (SASA) regardless of surface charge, whereas the maximum charge for unfolded proteins depends on the number of basic residues.[38,39]
Our results show that droplet charge at the final step of desolvation, which is governed by the Rayleigh limit of droplet stability,[50] determines the final charge of compact protein ions, not their proton affinity

Summary

Introduction

The charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. Informs about the molecular weight of protein complexes to reveal oligomeric states and ligand binding.[1−3] Combined with ion mobility (IM), it can be used to determine collision cross sections (CCS) and identify conformational changes.[4−6] If protein complexes are subjected to collisional activation inside the mass spectrometer, the resulting collisioninduced unfolding (CIU) can be followed by IM-MS, informing about their gas-phase stabilities.[7−10]. We clarify the role of ionizable residues in native MS using engineered proteins where the number of ionizable side chains can be altered without affecting their native structures

Methods

Results

Conclusion