The utilization of the search engine, Bolt, to decrease search time and increase peptide identifications in hydroxyl radical protein footprinting-based workflows.

Hydroxyl radical protein footprinting (HRPF) utilizes hydroxyl radicals to covalently modify solvent exposed regions of proteins. When coupled with mass spectrometry, HRPF can provide insightful information on protein structural changes including inside cells. However, the complex mixture of proteins and modifications makes identification a complicated task. To search all of the HRPF-induced modification combinations across the full proteome, requires substantial computational power and still can take days to search. To drastically decrease processing time and improve identifications, a novel cloud-based search engine, Bolt, was used to search for HRPF modifications in comparison to a commonly used search engine, Sequest. A 35% increase in the identification of modified peptides was observed in Bolt compared to Sequest with a decrease in computation time.

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins.

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein structure, protein-protein interactions, or protein-ligand interactions. HRPF utilizes hydroxyl radicals (▪OH) to irreversibly label a protein's solvent accessible surface. The inherent complexity, cost, and hazardous nature of performing HRPF have substantially limited broad-based adoption in biopharma. These factors include: 1) the use of complicated, dangerous, and expensive lasers that demand substantial safety precautions; and 2) the irreproducibility of HRPF caused by background scavenging of ▪OH that limit comparative studies. This publication provides a protocol for operation of a laser-free HRPF system. This laser-free HRPF system utilizes a high energy, high-pressure plasma light source flash oxidation technology with in-line radical dosimetry. The plasma light source is safer, easier to use, and more efficient in generating hydroxyl radicals than laser-based HRPF systems, and the in-line radical dosimeter increases the reproducibility of studies. Combined, the laser-free HRPF system addresses and surmounts the mentioned shortcomings and limitations of laser-based techniques.

Flash Oxidation (FOX) System: A Novel Laser-Free Fast Photochemical Oxidation Protein Footprinting Platform.

Hydroxyl radical protein footprinting (HRPF) is a powerful and flexible technique for probing changes in protein topography. With the development of the fast photochemical oxidation of proteins (FPOP), it became possible for researchers to perform HRPF in their laboratory on a very short time scale. While FPOP has grown significantly in popularity since its inception, adoption remains limited due to technical and safety issues involved in the operation of a hazardous Class IV UV laser and irreproducibility often caused by improper laser operation and/or differential radical scavenging by various sample components. Here, we present a new integrated FOX (Flash OXidation) Protein Footprinting System. This platform delivers sample via flow injection to a facile and safe-to-use high-pressure flash lamp with a flash duration of 10 µs fwhm. Integrated optics collect the radiant light and focus it into the lumen of a capillary flow cell. An inline radical dosimeter measures the hydroxyl radical dose delivered and allows for real-time compensation for differential radical scavenging. A programmable fraction collector collects and quenches only the sample that received the desired effective hydroxyl radical dose, diverting the carrier liquid and improperly oxidized sample to waste. We demonstrate the utility of the FOX Protein Footprinting System by determining the epitope of TNFα recognized by adalimumab. We successfully identify the surface of the protein that serves as the epitope for adalimumab, identifying four of the five regions previously noted by X-ray crystallography while seeing no changes in peptides not involved in the epitope interface. The FOX Protein Footprinting System allows for FPOP-like experiments with real-time dosimetry in a safe, compact, and integrated benchtop platform.

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins.

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein's solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Insights on the Conformational Ensemble of Cyt C Reveal a Compact State during Peroxidase Activity.

Cytochrome c (cyt c) is known for its role in the electron transport chain but transitions to a peroxidase-active state upon exposure to oxidative species. The peroxidase activity ultimately results in the release of cyt c into the cytosol for the engagement of apoptosis. The accumulation of oxidative modifications that accompany the onset of the peroxidase function are well-characterized. However, the concurrent structural and conformational transitions of cyt c remain undercharacterized. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry is a protein footprinting technique used to structurally characterize proteins. FPOP coupled with native ion mobility separation shows that exposure to H2O2 results in the accumulation of a compact state of cyt c. Subsequent top-down fragmentation to localize FPOP modifications reveals changes in heme coordination between conformers. A time-resolved functional assay suggests that this compact conformer is peroxidase active. Altogether, combining FPOP, ion mobility separation, and top-down and bottom-up mass spectrometry allows us to discern individual conformations in solution and obtain a better understanding of the conformational ensemble and structural transitions of cyt c as it transitions from a respiratory role to a proapoptotic role.

Rosetta Protein Structure Prediction from Hydroxyl Radical Protein Footprinting Mass Spectrometry Data.

In recent years mass spectrometry-based covalent labeling techniques such as hydroxyl radical footprinting (HRF) have emerged as valuable structural biology techniques, yielding information on protein tertiary structure. These data, however, are not sufficient to predict protein structure unambiguously, as they provide information only on the relative solvent exposure of certain residues. Despite some recent advances, no software currently exists that can utilize covalent labeling mass spectrometry data to predict protein tertiary structure. We have developed the first such tool, which incorporates mass spectrometry derived protection factors from HRF labeling as a new centroid score term for the Rosetta scoring function to improve the prediction of protein tertiary structures. We tested our method on a set of four soluble benchmark proteins with known crystal structures and either published HRF experimental results or internally acquired data. Using the HRF labeling data, we rescored large decoy sets of structures predicted with Rosetta for each of the four benchmark proteins. As a result, the model quality improved for all benchmark proteins as compared to when scored with Rosetta alone. For two of the four proteins we were even able to identify atomic resolution models with the addition of HRF data.

Modifications generated by fast photochemical oxidation of proteins reflect the native conformations of proteins.

Hydroxyl radical footprinting (HRF) is a nonspecific protein footprinting method that has been increasingly used in recent years to analyze protein structure. The method oxidatively modifies solvent accessible sites in proteins, which changes upon alterations in the protein, such as ligand binding or a change in conformation. For HRF to provide accurate structural information, the method must probe the native structure of proteins. This requires careful experimental controls since an abundance of oxidative modifications can induce protein unfolding. Fast photochemical oxidation of proteins (FPOP) is a HRF method that generates hydroxyl radicals via photo-dissociation of hydrogen peroxide using an excimer laser. The addition of a radical scavenger to the FPOP reaction reduces the lifetime of the radical, limiting the levels of protein oxidation. A direct assay is needed to ensure FPOP is probing the native conformation of the protein. Here, we report using enzymatic activity as a direct assay to validate that FPOP is probing the native structure of proteins. By measuring the catalytic activity of lysozyme and invertase after FPOP modification, we demonstrate that FPOP does not induce protein unfolding.

Analyzing the structure of macromolecules in their native cellular environment using hydroxyl radical footprinting.

Hydroxyl radical footprinting (HRF) has been successfully used to study the structure of both nucleic acids and proteins. The method utilizes hydroxyl radicals to oxidize solvent accessible sites in macromolecules. In recent years, the method has shown some utility for live cell analysis. In this review, we will survey the current state of the field for footprinting macromolecules in living cells. The field is relatively new, particularly for protein studies, with only a few publications on the development and application of HRF on live cells. DNA-protein interaction sites and information on the secondary and tertiary structure of RNA has been characterized. In addition, the conformational changes of membrane-spanning channels upon opening and activation have also been studied by in-cell HRF. In this review, we highlight examples of these applications.