Conformational stability of adsorbed insulin studied with mass spectrometry and hydrogen exchange

A new method is described for direct monitoring of the conformational stabi lity of proteins that are physically adsorbed from solution onto a solid su bstrate. The adsorption-induced conformational changes of insulin are studi ed using a combination of hydrogen/deuterium (H/D) exchange and matrix-assi sted laser desorption/ionization time-of flight (MALDI-TOF) mass spectromet ry. The effect of the surface hydrophobicity on the adsorption-induced conf ormational changes in the insulin structure is probed by adsorbing insulin on a hydrophilic silica and a hydrophobic methylated silica surface before subjecting the insulin molecules to the isotopic exchange process. The pres ent study describes the experimental procedure of this new application of M ALDI. Results show that insulin is more highly and more irreversibly adsorb ed to a hydrophobic methylated silica surface than to a hydrophilic silica surface. Hydrogen-exchange experiments clearly demonstrate that the strong interaction of insulin with the hydrophobic surface is accompanied by a str ong increase in the H/D-exchange rates and thus in a reduction in the insul in native structural stability. In contrast, H/D-exchange rates of insulin are somewhat reduced upon adsorption on silica from solution.