Fast photochemical oxidation of proteins for comparing solvent-accessibility changes accompanying protein folding: data processing and application to barstar.

Mass spectrometry-based protein footprinting reveals regional and even amino-acid structural changes and fills the gap for many proteins and protein interactions that cannot be studied by X-ray crystallography or NMR spectroscopy. Hydroxyl radical-mediated labeling has proven to be particularly informative in this pursuit because many solvent-accessible residues can be labeled by OH in a protein or protein complex, thus providing more coverage than does specific amino-acid modifications. Finding all the OH-labeling sites requires LC/MS/MS analysis of a proteolyzed sample, but data processing is daunting without the help of automated software. We describe here a systematic means for achieving a comprehensive residue-resolved analysis of footprinting data in an efficient manner, utilizing software common to proteomics core laboratories. To demonstrate the method and the utility of OH-mediated labeling, we show that FPOP easily distinguishes the buried and exposed residues of barstar in its folded and unfolded states. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

Oligonucleotide mapping via mass spectrometry to enable comprehensive primary structure characterization of an mRNA vaccine against SARS-CoV-2.

Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world's first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5' terminus capping and 3' terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general.

Fast photochemical oxidation of proteins and mass spectrometry follow submillisecond protein folding at the amino-acid level.

We report a study of submillisecond protein folding with amino-acid residue resolution achieved with a two-laser pump/probe experiment with analysis by mass spectrometry. The folding of a test protein, barstar, can be triggered by a laser-induced temperature jump (T jump) from ∼0 °C to ∼room temperature. Subsequent reactions via fast photochemical oxidation of proteins (FPOP) at various fractional millisecond points after the T jump lead to oxidative modification of solvent-accessible side chains whose "protection" changes with time and extent of folding. The modifications are identified and quantified by LC-MS/MS following proteolysis. Among all the segments that form secondary structure in the native state, helix(1) shows a decreasing trend of oxidative modification during the first 0.1-1 ms of folding while others do not change in this time range. Residues I5, H17, L20, L24 and F74 are modified less in the intermediate state than the denatured state, likely due to full or partial protection of these residues as folding occurs. We propose that in the early folding stage, barstar forms a partially solvent-accessible hydrophobic core consisting of several residues that have long-range interaction with other, more remote residues in the protein sequence. Our data not only are consistent with the previous conclusion that barstar fast folding follows the nucleation-condensation mechanism with the nucleus centered on helix(1) formed in a folding intermediate but also show the efficacy of this new approach to following protein folding on the submillisecond time range.

Fast photochemical oxidation of proteins for comparing structures of protein-ligand complexes: the calmodulin-peptide model system.

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based protein footprinting method that modifies proteins on the microsecond time scale. Highly reactive (•)OH, produced by laser photolysis of hydrogen peroxide, oxidatively modifies the side chains of approximately one-half the common amino acids on this time scale. Because of the short labeling exposure, only solvent-accessible residues are sampled. Quantification of the modification extent for the apo and holo states of a protein-ligand complex provides structurally sensitive information at the amino-acid level to compare the structures of unknown protein complexes with known ones. We report here the use of FPOP to monitor the structural changes of calmodulin in its established binding to M13 of the skeletal muscle myosin light chain kinase. We use the outcome to establish the unknown structures resulting from binding with melittin and mastoparan. The structural comparison follows a comprehensive examination of the extent of FPOP modifications as measured by proteolysis and LC-MS/MS for each protein-ligand equilibrium. The results not only show that the three calmodulin-peptide complexes have similar structures but also reveal those regions of the protein that became more or less solvent-accessible upon binding. This approach has the potential for relatively high throughput, information-dense characterization of a series of protein-ligand complexes in biochemistry and drug discovery when the structure of one reference complex is known, as is the case for calmodulin and M13 of the skeletal muscle myosin light chain kinase, and the structures of related complexes are not.

Sulfate radical anion as a new reagent for fast photochemical oxidation of proteins.

The focus is to expand the original design of fast photochemical oxidation of proteins (FPOP) and introduce SO(4)(-•), generated by 248 nm homolysis of low millimolar levels of persulfate, as a radical reactant in protein footprinting. FPOP is a chemical approach to footprinting proteins and protein complexes by "snapshot" reaction with free radicals. The radical used until now is the OH radical, and it provides a measure of residue-resolved solvent accessibility of the native protein. We show that FPOP can accommodate other reagents, increasing its versatility. The new persulfate FPOP system is a potent, nonspecific, and tunable footprinting method; 3-5 times less persulfate is needed to give the same global levels of modification as seen with OH radicals. Although solvent-exposed His and Tyr residues are more reactive with SO(4)(-•) than with (•)OH, oxidation of apomyoglobin and calmodulin shows that (•)OH probes smaller accessible areas than SO(4)(-•), with the possible exception of histidine. His64, an axial ligand in the heme-binding pocket of apomyoglobin, is substantially up-labeled by SO(4)(-•) relative to (•)OH. Nevertheless, the kinds of modification and residue selectivity for both reagent radicals are strikingly similar. Thus, the choice of these reagents relies on the physical properties, particularly the membrane permeability, of the radical precursors.

Fast photochemical oxidation of protein footprints faster than protein unfolding.

Fast photochemical oxidation of proteins (FPOP) is a chemical footprinting method whereby exposed amino-acid residues are covalently labeled by oxidation with hydroxyl radicals produced by the photolysis of hydrogen peroxide. Modified residues can be detected by standard trypsin proteolysis followed by LC/MS/MS, providing information about solvent accessibility at the peptide and even the amino-acid level. Like other chemical footprinting techniques, FPOP must ensure only the native conformation is labeled. Although oxidation via hydroxyl radical induces unfolding in proteins on a time scale of milliseconds or longer, FPOP is designed to limit (*)OH exposure to 1 micros or less by employing a pulsed laser for initiation to produce the radicals and a radical-scavenger to limit their lifetimes. We applied FPOP to three oxidation-sensitive proteins and found that the distribution of modification (oxidation) states is Poisson when a scavenger is present, consistent with a single conformation protein modification model. This model breaks down when a scavenger is not used and/or hydrogen peroxide is not removed following photolysis. The outcome verifies that FPOP occurs on a time scale faster than conformational changes in these proteins.