Laminar Flow Effects During Laser-Induced Oxidative Labeling for Protein Structural Studies by Mass Spectrometry

Laser-induced oxidative labeling of proteins provides insights into biomolecular structures and interactions. In these experiments, the hydroxyl radical ((*)OH) formed by photolysis of H(2)O(2) generates covalent modifications that are detectable by mass spectrometry. Under conditions where individual protein molecules are irradiated only once, the short (*)OH lifetime ( approximately 1 mus) ensures that covalent modifications are formed before any oxidation-induced conformational changes take place. This feature implies that the method should be free of structural artifacts. It has been proposed that single-exposure conditions can be achieved by passing the solution through a capillary where successive laser pulses generate a string of irradiated flow segments that are well separated from one another. The current work explores the convection phenomena within the labeling capillary in more detail. The experiments are conducted at Reynolds numbers \u3c\u3c2000, resulting in laminar flow. The associated parabolic velocity profile causes a portion of each irradiated segment to remain in the labeling window during the subsequent laser pulse. Achieving a genuine single-exposure regime is, therefore, not possible. We estimate the fraction of labeled protein formed under laminar flow conditions, as well as the occurrence of multiple exposure events for any combination of experimental parameters (laser spot width, pulse frequency, and solution flow rate). A proper choice of these parameters provides extensive labeling, while keeping multiple exposure events at an acceptably low level. The theoretical framework developed here is supported by experimental data. Overall, this study reaffirms the feasibility of the use of flow devices for meaningful laser-induced oxidative labeling studies. At the same time, we provide a theoretical underpinning of this technique that goes beyond previously suggested plug flow models

Structural Characterization of an Integral Membrane Protein in Its Natural Lipid Environment by Oxidative Methionine Labeling and Mass Spectrometry

Membrane proteins represent formidable challenges for many analytical techniques. Studies on these systems are often carried out after surfactant solubilization. Unfortunately, such a non-natural protein environment can affect conformation and stability, and it offers only partial protection against aggregation. This work employs bacteriorhodopsin (BR) as a model system for in situ structural studies on a membrane protein in its natural lipid bilayer. BR-containing purple membrane suspensions were exposed to hydroxyl radicals, generated by nanosecond laser photolysis of dilute aqueous H(2)O(2). The experiments rely on the premise that oxidative labeling occurs mainly at solvent-exposed side chains, whereas sites that are sterically protected will react to a much lesser extent. Following .OH exposure, the protein was analyzed by tryptic peptide mapping and electrospray tandem mass spectrometry. Oxidative labeling of BR was found to occur only at its nine Met residues. This is in contrast to the behavior of previously studied water-soluble proteins, which generally undergo modifications at many different types of residues. In those earlier experiments the high reactivity of Met has hampered its use as a structural probe. In contrast, the Met oxidation pattern observed here is in excellent agreement with the native BR structure. Extensive labeling is seen for Met32, 68, and 163, all of which are located in solvent-exposed loops. The remaining six Met residues are deeply buried and show severalfold less oxidation. Our results demonstrate the usefulness of Met oxidative labeling for structural studies on membrane proteins, especially when considering that many of these species are methionine-rich. The introduction of additional Met residues as conformational probes, as well as in vivo structural investigations, represents exciting future extensions of the methodology described here