A Fast Photochemical Oxidation of Proteins (FPOP) platform for free-radical reactions: the carbonate radical anion with peptides and proteins

Abstract

Fast Photochemical Oxidation of Protein (FPOP), based on a pulsed KrF laser (248 nm) for freeradical generation, is a biophysical method that utilizes hydroxyl radicals to footprint proteins in solution. FPOP has been recognized for structural proteomics investigations, including epitope mapping, protein-aggregation characterization, protein-folding monitoring, and binding-affinity determination. The distinct merits of the platform are: i) the use of a scavenger to control radical lifetime and allow fast ("snapshot") footprinting of solvent-accessible residues in a protein; ii) the employment of a flow system to enable single-shot irradiation of small plugs of the targeted sample; iii) the use of methionine and catalase after radical oxidation chemistry to prevent postoxidation with residual oxidizing species; and iv) the utilization of mature mass spectrometrybased proteomic methods to afford detailed analysis. In addition to • OH, other reactive reagents (e.g., carbenes, iodide, sulfate radical anion, and trifluoromethyl radical) can be implemented on this platform to increase the versatility and scope. In this study, we further elaborate the use of FPOP platform to generate secondary radicals and establish a workflow to answer fundamental questions regarding the intrinsic selectivity and reactivity of radicals that are important in biology. Carbonate radical anion is the example we chose owing to its oxidative character and important putative pathogenic roles in inflammation. This systematic study with model proteins/peptides gives consistent results with a previous study that evaluated reactivity with free amino acids and shows that methionine and tryptophan are the most reactive residues with CO 3 −•. Other aromatic amino acids (i.e., tyrosine, histidine and phenylalanine) exhibit moderate reactivity, whereas, aliphatic amino acids are inert, unlike with • OH. The outcome demonstrates this approach to be appropriate for studying the fast reactions of radicals with proteins.

Composite Conformational Changes of Signaling Proteins upon Ligand Binding Revealed by a Single Approach: Calcium-Calmodulin Study

“…Residue M51, however, is either involved with or near an allosteric site, as seen from its “up and down” modification behavior. The promise of increased spatial resolution motivates the development of new FPOP reagents that provide complementary labeling to OH radicals, − even of residues directly involved in binding.…”

Benefits of Ion Mobility Separation and Parallel Accumulation–Serial Fragmentation Technology on timsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis

“…The high dynamic range of analysis allowed single residue resolution of the site of modification for peptides spanning an intensity range of 3 orders of magnitude and is comparable to the previous reports. 17 , 18 Also, the introduction of the TIMS technology allows an additional parameter, namely, CCS. Although this parameter has not been widely implemented for peptide identifications, there is an avenue for the simplification/automation of data processing.…”

The Application of Fluorine‐Containing Reagents in Structural Proteomics

“…Instead, the latter are generated slowly by chemical reactions in solution. [44] Interest in the development of new reagents is growing as investigators search for broad coverage of the proteome.N o single reagent will be equally reactive with all the amino acid residues and, as ar esult, modifications concentrate on reactive residues.R eagents with complementary reactivity are needed for high coverage.…”