Footprinting of proteins by hydroxyl radicals generated on the millisecond to minute timescales to probe protein surfaces suffers from the uncertainty that radical reactions cause the protein to unfold, exposing residues that are protected in the native protein. To circumvent this possibility, we developed a method using a 248 nm KrF excimer laser to cleave hydrogen peroxide at low concentrations (15 mM, 0.04%), affording hydroxyl radicals that modify the protein in less than a microsecond. In the presence of a scavenger (20 mM glutamine), the radical lifetimes decrease to ϳ1 microsecond, yet the reaction timescales are sufficient to provide significant oxidation of the protein. These times are arguably faster than supersecondary protein structure can unfold as a result of the modification. The radical formation step takes place in a nanoliter flow cell so that only one laser pulse irradiates each bolus of sample. The oxidation sites are located using standard analytical proteomics, requiring less than a nanomole of protein. We tested the method with apomyoglobin and observed modifications in accord with solvent accessibility data obtained from the crystal structure of holomyoglobin. Additionally, the results indicate that the F-helix is conformationally flexible in apomyoglobin, in accord with NMR results. We also find that the binding pocket is resistant to modifications, indicating that the protein pocket closes in the absence of the heme group-conclusions that cannot be drawn from current structural methods. When developed further, this method may enable the determination of protein-ligand interfaces, affinity constants, folding pathways, and regions of conformational flexibility. , following the introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), has become an important means for the identification of proteins and the analytical tool of choice for proteomics. Given its sensitivity and speed, MS should also allow a more general characterization of activity, conformation, and interactions of proteins. Here we describe the development of an approach using pulsed-laser, hydroxyl-radical footprinting with product analysis by MS for determining solvent accessibility of a protein, and show early results indicating its potential utility for probing protein-ligand interfaces.Although a protein's chemical nature is not significantly altered when it binds with a ligand, one can determine the effects of binding by evaluating changes in solvent accessibility with and without the bound ligand [1]. The use of hydroxyl radicals for this purpose is attractive because they are sufficiently small to probe solvent accessibility and are highly reactive, as has been convincingly demonstrated by successful footprinting of DNA/protein interactions and RNA folding [2]. Others have advanced this idea for proteins, using both continuous and pulsed sources for the radicals and MS as the analytical tool. Chance and coworkers [3] have demonstrated in an extensive set of articles a more ...

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In biopharmaceutical process development, it is desirable to identify sites of covalent degradations to ensure product consistency. One characterization method used for therapeutic immunoglobulin gamma (IgG) 1 antibodies is limited LysC proteolysis followed by reversed-phase LC/MS. Limited LysC proteolysis leads to high efficiency cleavage at the C-terminal side of the hinge lysine 222 residue, generating Fab and Fc fragments. In this report, we show that IgG 1 samples incubated under mildly acidic conditions at elevated temperatures were partially resistant to LysC cleavage at the hinge and resulted in a species where one of the Fab arms remained connected to the Fc region (Fab-Fc). The growth of the Fab-Fc species was proportional to the duration and storage temperature of the incubation period and correlated with the amount of isomerization of the aspartic acid residue preceding lysine 222, determined by peptide mapping. The isomerization rates of samples stored for up to one year at 4 degrees C, 6 months at 29 or 37 degrees C, or 3 months at 45 degrees C were determined, and the activation energy for this conversion was calculated to be approximately 33 kJ mol(-1). The apparent isomerization rate constant was only 0.02 week(-1) for samples stored at 4 degrees C, which resulted in a modest increase from 5.1 to 6.0% isoD after twenty four weeks of storage and, hence, is not a significant concern under normal storage conditions typically used for monoclonal antibodies. However, when stored at 29 degrees C, the apparent rate constant of this reaction was found to be 0.06 week(-1) and resulted in an increase from 5.1 to 21.1% isoD after twenty four weeks of storage and is a major degradant in stressed IgG 1 antibodies.