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Analysis of the effects of oxidants such as hypochlorite and peroxynitrite on yeast alcohol dehydrogenase suggests that the oxidants primarily target the zinc-thiolate moiety of the dehydrogenase. Enzyme inactivation by peroxynitrite or hypochlorite is associated with zinc release and thiol/thiolate oxidation. The zinc-thiolate moiety of dehydrogenase shows structural similarity with that in zinc finger proteins, confirming that oxidants target the zinc finger moiety for attack.

Demonstrates the sensitivity of the essential zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite. Peroxynitrite synthesis; Determination of zinc content of yeast alcohol dehydrogenase; Treatment of yeast alcohol dehydrogenase with peroxynitrite.

Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) requires high-capacity separation and extensive gas-phase fragmentation of proteoforms. Herein, we coupled capillary zone electrophoresis (CZE) to electron-capture collision-induced dissociation (ECciD) on an Agilent 6545 XT quadrupole time-of-flight (Q-TOF) mass spectrometer for dTDP for the first time. During ECciD, the protein ions were first fragmented using ECD, followed by further activation and fragmentation by applying a CID potential. In this pilot study, we optimized the CZE-ECciD method for small proteins (lower than 20 kDa) regarding the charge state of protein parent ions for fragmentation and the CID potential applied to maximize the protein backbone cleavage coverage and the number of sequence-informative fragment ions. The CZE-ECciD Q-TOF platform provided extensive backbone cleavage coverage for three standard proteins lower than 20 kDa from only single charge states in a single CZE-MS/MS run in the targeted MS/MS mode, including ubiquitin (97%, +7, 8.6 kDa), superoxide dismutase (SOD, 87%, +17, 16 kDa), and myoglobin (90%, +16, 17 kDa). The CZE-ECciD method produced comparable cleavage coverage of small proteins (i.e. myoglobin) with direct-infusion MS studies using electron transfer dissociation (ETD), activated ion-ETD, and combinations of ETD and collision-based fragmentation on high-end orbitrap mass spectrometers. The results render CZE-ECciD a new tool for dTDP to enhance both separation and gas-phase fragmentation of proteoforms. [ABSTRACT FROM AUTHOR]

Electron-based fragmentation methods have revolutionized biomolecular mass spectrometry, in particular native and top-down protein analysis. Here, we report the use of a new electromagnetostatic cell to perform electron capture dissociation (ECD) within a quadrupole/ion mobility/time-of-flight mass spectrometer. This cell was installed between the ion mobility and time-of-flight regions of the instrument, and fragmentation was fast enough to be compatible with mobility separation. The instrument was already fitted with electron transfer dissociation (ETD) between the quadrupole and mobility regions prior to modification. We show excellent fragmentation efficiency for denatured peptides and proteins without the need to trap ions in the gas phase. Additionally, we demonstrate native top-down backbone fragmentation of noncovalent protein complexes, leading to comparable sequence coverage to what was achieved using the instrument's existing ETD capabilities. Limited collisional ion activation of the hemoglobin tetramer before ECD was reflected in the observed fragmentation pattern, and complementary ion mobility measurements prior to ECD provided orthogonal evidence of monomer unfolding within this complex. The approach demonstrated here provides a powerful platform for both top-down proteomics and mass spectrometry-based structural biology studies.

One challenge associated with the discovery and development of monoclonal antibody (mAb) therapeutics is the determination of heavy chain and light chain pairing. Advances in MS instrumentation and MS/MS methods have greatly enhanced capabilities for the analysis of large intact proteins yielding much more detailed and accurate proteoform characterization. Consequently, direct interrogation of intact antibodies or F(ab')2 and Fab fragments has the potential to significantly streamline therapeutic mAb discovery processes. Here, we demonstrate for the first time the ability to efficiently cleave disulfide bonds linking heavy and light chains of mAbs using electron capture dissociation (ECD) and 157 nm ultraviolet photodissociation (UVPD). The combination of intact mAb, Fab, or F(ab')2 mass, intact LC and Fd masses, and CDR3 sequence coverage enabled determination of heavy chain and light chain pairing from a single experiment and experimental condition. These results demonstrate the potential of top-down and middle-down proteomics to significantly streamline therapeutic antibody discovery.