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The exposure of solution-phase proteins to reactive oxygen species (ROS) causes oxidative modifications, giving rise to the formation of covalent +16 Da adducts. Electrospray ionization (ESI) mass spectrometry (MS) is the most widely used method for monitoring the extent of these modifications. Unfortunately, protein oxidation can also take place as an experimental artifact during ESI, such that it may be difficult to assess the actual level of oxidation in bulk solution. Previous work has demonstrated that ESI-induced oxidation is highly prevalent when operating at strongly elevated capillary voltage V(0) (e.g., +8 kV) and with oxygen nebulizer gas in the presence of a clearly visible corona discharge. Protein oxidation under these conditions is commonly attributed to OH radicals generated in the plasma of the discharge. On the other hand, charge balancing oxidation reactions are known to take place at the metal/liquid interface of the emitter. Previous studies have not systematically explored whether such electrochemical processes could be responsible for the formation of oxidative +16 Da adducts instead of (or in combination with) plasma-generated ROS. Using hemoglobin as a model system, this work illustrates the occurrence of extensive protein oxidation even under typical operating conditions (e.g., V(0) = 3.5 kV, N(2) nebulizer gas). Surprisingly, measurements of the current flowing in the ESI circuit demonstrate that a weak corona discharge persists for these relatively gentle settings. On the basis of comparative experiments with nebulizer gases of different dielectric strength, it is concluded that ROS generated under discharge conditions are solely responsible for ESI-induced protein oxidation. This result is corroborated through off-line electrolysis experiments designed to mimic the electrochemical processes taking place during ESI. Our findings highlight the necessity of using easily oxidizable internal standards in biophysical or biomedical ESI-MS studies where knowledge of protein oxidation in bulk solution is desired. Strategies for eliminating ESI-induced oxidation artifacts are discussed.

Potentiostatic polarization experiments were performed as a function of potential ͑200 to 700 mV Ag/AgCl ͒ and temperature ͑25-85°C͒ on the Ni-Cr-Mo alloys C22 and C276. X-ray photoelectron spectroscopy ͑XPS͒ and time-of-flight secondary ion mass spectrometry ͑TOF SIMS͒ were used to determine the chemical composition and thickness of the films formed. The currents recorded as a function of applied potential were due to dissolution, with only minor increases in film thicknesses observed. Measured currents for C22 were lower than for C276 and decayed over the entire period of measurement at each temperature. Those on C276 more closely approached steady state. The temperature dependence of the currents on C22 was significantly lower than that on C276. Surface analyses, performed on specimens anodically treated at one potential but after a sequence of temperatures up to 85°C, confirmed that the passive films on both alloys consisted of a Mo, Cr, and Ni oxide, with Cr present as Cr 3 ϩ and Mo present in several oxidation states. The passive films on C22 showed a distinct layered structure, consisting of an inner layer rich in Cr and Ni, and an outer layer enhanced in Mo. By contrast, the oxide films on C276 did not show such a clear separation into layers, and the relative Cr content was much lower. The increase in oxide thickness with increasing anodic potential and the low temperature dependence of the passive current observed for C22 are consistent with an oxide dissolution rate which is low compared to the rate of creation of oxygen vacancies leading to film growth. The absence of a dependence of film thickness on potential and the higher temperature dependence of the passive currents on C276 are consistent with control of the overall anodic process by ion-transfer at the oxide/solution interface.