Desalting large protein complexes during native electrospray mass spectrometry by addition of amino acids to the working solution

Abstract

Native mass spectrometry is a rapidly emerging field for characterising the structure of proteins and protein assemblies. The technique relies on electrospray ionisation (ESI) to efficiently ionise the protein analyte and transmit it into the gas phase with retention of protein structure, non-covalent protein-ligand and protein–protein interactions. In native ESI, both the ionisation efficiency and the resulting mass spectral signal is adversely effected by the presence of non-volatile inorganic salts, such as sodium chloride, which form extensive adducts with the protein ions. Consequently, there is great interest in finding experimental strategies that mitigate these phenomena. Here we report our findings that the addition of 10 mM L-serine to the ESI spray solution reduces the adverse effects of sodium adduction to proteins. In the analysis of bovine serum albumin (BSA; 66 kDa), 10 mM serine increased signal to noise ratio (S/N) ∼4 fold. This increase in sensitivity was accompanied by peak narrowing (∼10 fold), which allowed more precise assignment of molecular mass. Similar effects were observed when analysing protein complexes – serine palmitoyl transferase (SPT, a 92 kDa homodimer), enolase (a 93 kDa homodimer); and alcohol dehydrogenase (ADH, a 148 kDa tetramer). Reduction in sodium ion adduction occurs with no loss of the non-covalent protein–protein interactions, and with little effect on the overall observed charge state-distribution. As a consequence of increasing signal intensity, the addition of serine to the ESI spray solution greatly improved the quality of the data obtained from native top-down electron-capture dissociation (ECD) experiments. In ECD analysis of native BSA, we observed an increasing in the S/N of all ECD fragments upon addition of 10 mM L-serine. The number of ECD fragments we observed with S/N > 1.5 increased from 15 to 44 and the number of assigned c and z ions increased from 5 to 16. Finally we show that this phenomenon is not specific to L-serine, and occurs with several amino acids such as L-alanine. Our findings suggest that desalting may occur via binding of sodium ion to the amino acid in solution. This simple and inexpensive strategy has broad utility for improving the mass spectra obtained in a range of MS-based structural proteomic studies.