Electrochemically induced pH changes resulting in protein unfolding in theion source of an electrospray mass spectrometer

The operation of an electrospray ion source in the positive ion mode involv es charge-balancing oxidation reactions at the liquid/metal interface of th e sprayer capillary. One of these reactions is the electrolytic oxidation o f water. The protons generated in this process acidify the analyte solution within the electrospray capillary. This work explores the effects of this acidification on the electrospray ionization (ESI) mass spectrum of the pro tein cytochrome c (cyt c). En aqueous solution containing 40% propanol, cyt c unfolds around pH 5.6. Mass spectra recorded under these conditions, usi ng a simple ESI series circuit, display a bimodal charge-state distribution that reflects an equilibrium mixture of folded and unfolded protein in sol ution. These spectra are not strongly affected by electrochemical acidifica tion. An "external loop" is added to the ESI circuit when the metal needle of the sample injection syringe is connected to ground. The resulting circu it represents two coupled electrolytic cells that share the ESI capillary a s a common anode. Under these conditions, the rate of charge-balancing oxid ation reactions is dramatically increased because the ion source has to sup ply electrons for both, the external circuit and the ESI circuit. The analy tical implications of this effect are briefly discussed. Mass spectra of cy t c recorded with the syringe needle grounded are shifted to higher charge states, indicating that electrochemical acidification has caused the protei n to unfold in the ion source. The acidification can be suppressed by incre asing the flow rate and lowering the electrolyte concentration of the solut ion and by using an electrolyte that acts as redox buffer. The observed aci dification is similar for sprayer capillaries made of platinum and stainles s steel. Removal of the protective oxide layer on the stainless steel surfa ce results in effective redox buffering for a few minutes.