Protein structure information from mass spectrometry? Selective titration of arginine residues by sulfonates

Abstract

Noncovalently bound complexes between basic sites of peptides/proteins and sulfonates are studied using Matrix Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry. Reactive sulfonate dyes such as Cibacron Blue F3G-A are known to bind to protonated amino groups on the exterior of a protein. In this work, we examine a wide range of other sulfonates with distinctly simpler structure and more predictable reactivity. Naphthalene-sulfonic acid derivatives were found to bind to arginine only, as opposed to expected binding to all basic sites (Arg, Lys and His). Detailed control experiments were designed to unambigously confirm this selectivity and to rule out nonspecific adduct formation in the gas phase. The data show that the number of complex adducts found equals the number of accessible arginine sites on the surface of folded peptides and proteins, plus the N-terminus. Lys and His are not complexed nor are buried residues with hindered access. MALDI-MS can therefore provide fast information related to the exposed surface of these biomolecules. Additional titration experiments with 1-anilino-naphthalene-8-sulfonic acid (ANS) revealed that this fluorescent dye, which was often hypothesized to bind to so-called molten globule states of proteins, behaved exactly like all other naphthalene-sulfonic acids. ANS binding thus occurs largely through the sulfonate group.