Sodiation of Enhanced Green Fluorescent Protein (EGFP) in Basic Solution Studied by Electrospray Mass Spectrometry

In our previous work, the sodiation of melittin, cytochrome c, and ubiquitin in a 1 mM NaOH water/methanol solution was studied by electrospray mass spectrometry. It was suggested that the α-helix is more resistant to sodiation than the β-sheet. In this study, sodiation of enhanced green fluorescent protein (EGFP) composed of a β-barrel was studied in 1% CH₃COOH (AcOH) or 1 mM NaOH water/methanol solution by electrospray mass spectrometry. Although EGFP was denatured in an acidic solution, it maintains a near-native structure in a basic solution. For the 1% AcOH solution, the protonated EGFP, [EGFP + nH - mH + mNa]ⁿ⁺, with n = 14 - 36 and m = 0 was detected. For 1 mM NaOH, the number n for [EGFP + nH - mH + mNa]ⁿ⁺ was found to increase with the sodiation number m and vice versa for [EGFP + nH - mH + mNa]^n-. Namely, Na⁺ adducts counteract the negative charges of deprotonated acidic residues. The protonated EGFP detected as major ions for basic 1 mM NaOH was ascribed to the more surface-active H₃O⁺(aq) than OH^-(aq).