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 micros) 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 <<2000, 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.
Konermann
Lars
L
Department of Chemistry, The University of Western Ontario, London, ON, N6A 5B7, Canada. konerman@uwo.ca
Stocks
Bradley B
BB
Czarny
Tomasz
T
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D017665:Hydroxyl Radical / Q000737:chemistry; D007834:Lasers; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*
D011506:Proteins; D017665:Hydroxyl Radical