Effects of photochemical aging on the molecular composition of organic aerosols derived from agricultural biomass burning in whole combustion process.

Biomass burning organic aerosols (BBOA) are key components of atmospheric particulate matter, yet the effects of aging process on their chemical composition and related properties remain poorly understood. In this study, fresh smoke emissions from the combustion of three types of agricultural biomass residues (rice, maize, and wheat straws) were photochemically aged in an oxidation flow reactor. The changes in BBOA composition were characterized by offline analysis using ultrahigh performance liquid chromatography coupled with Orbitrap mass spectrometry. The BBOA molecular composition varied dramatically with biomass type and aging process. Fresh and aged BBOA were predominated by CHO and nitrogen-containing CHON, CHN, and CHONS species, while with very few CHOS and other non­oxygen species. The signal peak area variations revealed that individual molecular species underwent dynamic changes, with 77-81 % of fresh species decreased or even disappeared and 33-46 % of aged species being newly formed. A notable increase was observed in the number and peak area of CxHyO≥6 compounds in aged BBOA, suggesting that photochemical process served as an important source of highly oxygenated species. Heterocyclic CxHyN2 compounds mostly dominated in fresh CHN species, whereas CxHyN1 were more abundant in aged ones. Fragmentation and homologs oxidation by addition of oxygen-containing functional groups were important pathways for the BBOA aging. The changes in BBOA composition with aging would have large impacts on particle optical properties and toxicity. This study highlights the significance of photochemical aging process in altering chemical composition and related properties of BBOA.

Photochemical oxidation of VOCs and their source impact assessment on ozone under de-weather conditions in Western Taiwan.

The application of statistical models has excellent potential to provide crucial information for mitigating the challenging issue of ozone (O3) pollution by capturing its associations with explanatory variables, including reactive precursors (VOCs and NOX) and meteorology. Considering the large contribution of O3 in degrading the air quality of western Taiwan, three-year (2019-2021) hourly concentration data of VOC, NOX and O3 from 4 monitoring stations of western Taiwan: Tucheng (TC), Zhongming (ZM), Taixi (TX) and Xiaogang (XG), was evaluated to identify the effect of anthropogenic emissions on O3 formation. Owing to the high-ambient reactivity of VOCs on the underestimation of sources, photochemical oxidation was assessed to calculate the consumed VOC (VOCcons) which was followed by the source identification of their initial concentrations. VOCcons was observed to be highest in the summer season (16.7 and 22.7 ppbC) at north (TC and ZM) and in the autumn season (17.8 and 11.4 ppbC) in southward-located stations (TX and XG, respectively). Results showed that VOCs from solvents (25-27%) were the major source at northward stations whereas VOCs-industrial emissions (30%) dominated in south. Furthermore, machine learning (ML): eXtreme Gradient Boost (XGBoost) model based de-weather analysis identified that meteorological factors favor to reduce ambient O3 levels at TC, ZM and XG stations (-67%, -47% and -21%, respectively) but they have a major role in accumulating the O3 (+38%) at the TX station which is primarily transported from the upwind region of south-central Taiwan. Crucial insights using ML outputs showed that the finding of the study can be utilized for region-specific data-driven control of emission from VOCs-sources and prioritized to limit the O3-pollution at the study location-ns as well as their accumulation in distant regions.

Hydrogen Deuterium Exchange and other Mass Spectrometry-based Approaches for Epitope Mapping.

Antigen-antibody interactions are a fundamental subset of protein-protein interactions responsible for the "survival of the fittest". Determining the interacting interface of the antigen, called an epitope, and that on the antibody, called a paratope, is crucial to antibody development. Because each antigen presents multiple epitopes (unique footprints), sophisticated approaches are required to determine the target region for a given antibody. Although X-ray crystallography, Cryo-EM, and nuclear magnetic resonance can provide atomic details of an epitope, they are often laborious, poor in throughput, and insensitive. Mass spectrometry-based approaches offer rapid turnaround, intermediate structural resolution, and virtually no size limit for the antigen, making them a vital approach for epitope mapping. In this review, we describe in detail the principles of hydrogen deuterium exchange mass spectrometry in application to epitope mapping. We also show that a combination of MS-based approaches can assist or complement epitope mapping and push the limit of structural resolution to the residue level. We describe in detail the MS methods used in epitope mapping, provide our perspective about the approaches, and focus on elucidating the role that HDX-MS is playing now and in the future by organizing a discussion centered around several improvements in prototype instrument/applications used for epitope mapping. At the end, we provide a tabular summary of the current literature on HDX-MS-based epitope mapping.

Toward a MALDI in-source decay (ISD) method for top-down analysis of protein footprinting.

Irreversible protein footprinting is a mass spectrometry-based approach in which solvent-accessible sites of a protein are modified to assess high-order protein structure. Structural insights can be gained by determining the position and extents of modification. The usual approach to obtain the "footprint" is to analyze the protein through bottom-up LC-MS/MS. In this approach, the proteins are digested to yield a mixture of peptides that are then separated by LC before locating the modification sites by MS/MS. This process consumes substantial amounts of time and is difficult to accelerate for applications that require quick and high-throughput analysis. Here, we describe employing matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) to analyze a footprinted small test protein (ubiquitin) via a top-down approach. Matrix-assisted laser desorption/ionization is easily adapted for high-throughput analysis, and top-down strategies can avoid lengthy proteolysis and LC separation. We optimized the method with model peptides and then demonstrated its feasibility on ubiquitin submitted to two types of footprinting. We found that MALDI ISD can produce a comprehensive set of fragment ions for small proteins, affording footprinting information in a fast manner and giving results that agree with the established methods, and serve as a rough measure of protein solvent accessibility. To assist in the implementation of the MALDI approach, we developed a method of processing top-down ISD data.

Secondary aerosol formation in incense burning particles by O3 and OH oxidation via single particle mixing state analysis.

Incense burning is a common religious activity that emits abundant gaseous and particulate pollutants into the atmosphere. During their atmospheric lifetime, these gases and particles are subjected to oxidation, leading to the formation of secondary pollutants. We examined the oxidation of incense burning plumes under O3 exposure and dark condition using an oxidation flow reactor connected to a single particle aerosol mass spectrometer (SPAMS). Nitrate formation was observed in incense burning particles, mainly attributable to the ozonolysis of nitrogen-containing organic compounds. With UV on, nitrate formation was significantly enhanced, likely due to HNO3/HNO2/NOx uptake triggered by OH chemistry, which is more effective than ozone oxidation. The extent of nitrate formation is insensitive to O3 and OH exposure, possibly due to the diffusion limitation on interfacial uptake. The O3-UV-aged particles are more oxygenated and functionalized than O3-Dark-aged particles. Oxalate and malonate, two typical secondary organic aerosol (SOA) components, were found in O3-UV-aged particles. Our work reveals that nitrate, accompanied by SOA, can rapidly form in incense-burning particles upon photochemical oxidation in the atmosphere, which could deepen our understanding of air pollution caused by religious activities.

Advances in Mass Spectrometry on Membrane Proteins.

Understanding the higher-order structure of membrane proteins (MPs), which are vital for numerous biological processes, is crucial for comprehending their function. Although several biophysical approaches have been used to study the structure of MPs, limitations exist owing to the proteins' dynamic nature and heterogeneity. Mass spectrometry (MS) is emerging as a powerful tool for investigating membrane protein structure and dynamics. Studying MPs using MS, however, must meet several challenges including the lack of stability and solubility of MPs, the complexity of the protein-membrane system, and the difficulty of digestion and detection. To meet these challenges, recent advances in MS have engendered opportunities in resolving the dynamics and structures of MP. This article reviews achievements over the past few years that enable the study of MPs by MS. We first introduce recent advances in hydrogen deuterium exchange and native mass spectrometry for MPs and then focus on those footprinting methods that report on protein structure.

Evolution characteristic of atmospheric black carbon particles at a coastal site in the Pearl River Delta, China.

The mixing of black carbon (BC) with secondary materials is a major uncertainty source in assessing its radiative forcing. However, current understanding of the formation and evolution of various BC components is limited, particularly in the Pearl River Delta, China. This study measured submicron BC-associated nonrefractory materials and the total submicron nonrefractory materials using a soot particle aerosol mass spectrometer and a high-resolution time-of-flight aerosol mass spectrometer, respectively, at a coastal site in Shenzhen, China. Two distinct atmospheric conditions were also identified to further explore the distinctive evolution of BC-associated components: polluted period (PP) and clean period (CP). Comparing the components of two particles, we found that more-oxidized organic factor (MO-OOA) prefers to form on BC during PP rather CP. The formation of MO-OOA on BC (MO-OOABC) was affected by both enhanced photochemical processes and nocturnal heterogeneous processes. Enhanced photo-reactivity of BC, photochemistry during the daytime, and heterogeneous reaction at nighttime were potential pathways for MO-OOABC formation during PP. The fresh BC surface was favorable for the formation of MO-OOABC. Our study shows the evolution of BC-associated components under different atmospheric conditions, which should be considered in regional climate models to improve the assessment of the climate effects of BC.

MALDI Peptide Mapping for Fast Analysis in Protein Footprinting.

Although protein footprinting results are commonly obtained by ESI-based LC-MS/MS, a more rapid-turnaround alternative approach is desirable to expand the scope of protein footprinting and facilitate routine analysis such as monitoring protein high order structure in quality control or checking epitope maps. Considering that MALDI is a faster procedure that can be easily adapted for high-throughput analysis, we explore here the feasibility of developing a MALDI-based analysis "portfolio" of bottom-up peptide mass mapping for footprinting. The approach was applied to several model proteins that were submitted to two footprinting strategies, FPOP and GEE labeling, and their performance was evaluated. We found adequate coverage that can be improved with automatic off-line separation and spotting, demonstrating the capability to footprint accurately protein conformational change, showing that MALDI may be useful for selected applications in protein footprinting.

Structural Investigation of Therapeutic Antibodies Using Hydroxyl Radical Protein Footprinting Methods.

Commercial monoclonal antibodies are growing and important components of modern therapies against a multitude of human diseases. Well-known high-resolution structural methods such as protein crystallography are often used to characterize antibody structures and to determine paratope and/or epitope binding regions in order to refine antibody design. However, many standard structural techniques require specialized sample preparation that may perturb antibody structure or require high concentrations or other conditions that are far from the conditions conducive to the accurate determination of antigen binding or kinetics. We describe here in this minireview the relatively new method of hydroxyl radical protein footprinting, a solution-state method that can provide structural and kinetic information on antibodies or antibody-antigen interactions useful for therapeutic antibody design. We provide a brief history of hydroxyl radical footprinting, examples of current implementations, and recent advances in throughput and accessibility.

Electrochemical and Spectroscopic Characterization of Oxidized Intermediate Forms of Vitamin E.

Vitamin E, a collection of lipophilic phenolic compounds based on chroman-6-ol, has a rich and fascinating oxidative chemistry involving a range of intermediate forms, some of which are proposed to be important in its biological functions. In this review, the available electrochemical and spectroscopic data on these oxidized intermediates are summarized, along with a discussion on how their lifetimes and chemical stability are either typical of similar phenolic and chroman-6-ol derived compounds, or atypical and unique to the specific oxidized isomeric form of vitamin E. The overall electrochemical oxidation mechanism for vitamin E can be summarized as involving the loss of two-electrons and one-proton, although the electron transfer and chemical steps can be controlled to progress along different pathways to prolong the lifetimes of discreet intermediates by modifying the experimental conditions (applied electrochemical potential, aqueous or non-aqueous solvent, and pH). Depending on the environment, the electrochemical reactions can involve single electron transfer (SET), proton-coupled electron transfer (PCET), as well as homogeneous disproportionation and comproportionation steps. The intermediate species produced via chemical or electrochemical oxidation include phenolates, phenol cation radicals, phenoxyl neutral radicals, dications, diamagnetic cations (phenoxeniums) and para-quinone methides. The cation radicals of all the tocopherols are atypically long-lived compared to the cation radicals of other phenols, due to their relatively weak acidity. The diamagnetic cation derived from α-tocopherol is exceptionally long-lived compared to the diamagnetic cations from the other ß-, γ- and δ-isomers of vitamin E and compared with other phenoxenium cations derived from phenolic compounds. In contrast, the lifetime of the phenoxyl radical derived from α-tocopherol, which is considered to be critical in biological reactions, is typical for what is expected for a compound with its structural features. Over longer times via hydrolysis reactions, hydroxy para-quinone hemiketals and quinones can be formed from the oxidized intermediates, which can themselves undergo reduction processes to form intermediate anion radicals and dianions. Methods for generating the oxidized intermediates by chemical, photochemical and electrochemical methods are discussed, along with a summary of how the final products vary depending on the method used for oxidation. Since the intermediates mainly only survive in solution, they are most often monitored using UV-vis spectroscopy, FTIR or Raman spectroscopies, and EPR spectroscopy, with the spectroscopic techniques sometimes combined with fast photoinitiated excitation and time-resolved spectroscopy for detection of short-lived species.

Secondary organic aerosol formation from atmospheric reactions of anisole and associated health effects.

Anisole (methoxybenzene) represents an important marker compound of lignin pyrolysis and a starting material for many chemical products. In this study, secondary organic aerosols (SOA) formed by anisole via various atmospheric processes, including homogeneous photooxidation with varying levels of OH• and NOx and subsequent heterogeneous NO3• dark reactions, were investigated. The yields of anisole SOA, particle-bound organoperoxides, particle-induced oxidative potential (OP), and cytotoxicity were characterized in view of the atmospheric fate of the anisole precursor. Anisole SOA yields ranged between 0.12 and 0.35, depending on the reaction pathways and aging degrees. Chemical analysis of the SOA suggests that cleavage of the benzene ring is the main reaction channel in the photooxidation of anisole to produce low-volatility, highly oxygenated small molecules. Fresh anisole SOA from OH• photooxidation are more light-absorbing and have higher OP and organoperoxide content. The high correlation between SOA OP and organoperoxide content decreases exponentially with the degree of OH• aging. However, the contribution of organoperoxides to OP is minor (<4%), suggesting that other, non-peroxide oxidizers play a central role in anisole SOA OP. The particle-induced OP and particulate organoperoxides yield both reach a maximum value after ∼2 days' of photooxidation, implicating the potential long impact of anisole during atmospheric transport. NOx-involved photooxidation and nighttime NO3• reactions facilitate organic nitrate formation and enhance particle light absorption. High NOx levels suppress anisole SOA formation and organoperoxides yield in photooxidation, with decreased aerosol OP and cellular oxidative stress. In contrast, nighttime aging significantly increases the SOA toxicity and reactive oxygen species (ROS) generation in lung cells. These dynamic properties and the toxicity of anisole SOA advocate consideration of the complicated and consecutive aging processes in depicting the fate of VOCs and assessing the related effects in the atmosphere.

Synergy between Sunlight, Titanium Dioxide, and Microbes Enhances Cellulose Diacetate Degradation in the Ocean.

Sunlight chemically transforms marine plastics into a suite of products, with formulation─the specific mixture of polymers and additives─driving rates and products. However, the effect of light-driven transformations on subsequent microbial lability is poorly understood. Here, we examined the interplay between photochemical and biological degradation of fabrics made from cellulose diacetate (CDA), a biobased polymer used commonly in consumer products. We also examined the influence of ∼1% titanium dioxide (TiO2), a common pigment and photocatalyst. We sequentially exposed CDA to simulated sunlight and native marine microbes to understand how photodegradation influences metabolic rates and pathways. Nuclear magnetic resonance spectroscopy revealed that sunlight initiated chain scission reactions, reducing CDA's average molecular weight. Natural abundance carbon isotope measurements demonstrated that chain scission ultimately yields CO2, a newly identified abiotic loss term of CDA in the environment. Measurements of fabric mass loss and enzymatic activities in seawater implied that photodegradation enhanced biodegradation by performing steps typically facilitated by cellulase. TiO2 accelerated CDA photodegradation, expediting biodegradation. Collectively, these findings (i) underline the importance of formulation in plastic's environmental fate and (ii) suggest that overlooking synergy between photochemical and biological degradation may lead to overestimates of marine plastic persistence.

Fast photochemical oxidation of proteins coupled with mass spectrometry.

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical footprinting approach whereby radicals, produced by UV laser photolysis of hydrogen peroxide, induce oxidation of amino acid side-chains. Mass Spectrometry (MS) is employed to locate and quantify the resulting irreversible, covalent oxidations to use as a surrogate for side-chain solvent accessibility. Modulation of oxidation levels under different conditions allows for the characterisation of protein conformation, dynamics and binding epitopes. FPOP has been applied to structurally diverse and biopharmaceutically relevant systems from small, monomeric aggregation-prone proteins to proteome-wide analysis of whole organisms. This review evaluates the current state of FPOP, the progress needed to address data analysis bottlenecks, particularly for residue-level analysis, and highlights significant developments of the FPOP platform that have enabled its versatility and complementarity to other structural biology techniques.

Secondary organic aerosol formation at an urban background site on the coastline of South China: Precursors and aging processes.

Understanding the formation mechanisms of secondary organic aerosols (SOA) is an arduous task in atmospheric chemistry. In November 2018, a sampling campaign was conducted at an urban background site in Hong Kong for characterization of secondary air pollution. A high-resolution time-of-flight aerosol mass spectrometer was used to monitor the compositions of non-refractory submicron particulate matters (NR-PM1), and multiple online instruments provided us with comprehensive auxiliary data. Organic aerosol (OA) constituted the largest fraction (43.8%) of NR-PM1, and 86.5% of the organics was contributed by the oxygenated OA (OOA, secondary components). Formation mechanisms of a dominant and more variable component of the less-oxidized OOA (labelled as LO-OOA1 in this study) and the more-oxidized OOA (MO-OOA) were explored. Based on the multilinear regression with molecular markers of OA (e.g., hydroxybenzonic acids and 2,3-dihydroxy-4-oxopentanoic acid), we presumed that anthropogenic organic compounds, especially aromatics, were the most likely precursors of LO-OOA1. MO-OOA correlated well with odd oxygen (Ox), and its concentration responded positively to the increase of liquid water content (LWC) in NR-PM1, indicating that the formation of MO-OOA involved photochemical oxidation and aqueous processes. It exhibited the best correlation with malic acid which can be formed through the oxidation of various precursors. Moreover, it was plausible that LO-OOA1 was further oxidized to MO-OOA through aqueous processes, as indicated by the consistent diurnal variations of MO-OOA to LO-OOA1 ratio and LWC. This study highlights the important roles of anthropogenic emissions and aqueous processes in SOA formation in coastal areas downwind of cities.

Elevated Gaseous Oxidized Mercury Revealed by a Newly Developed Speciated Atmospheric Mercury Monitoring System.

Gaseous oxidized mercury (Hg2+) monitoring is one of the largest challenges in the mercury research field, where existing methods cannot simultaneously satisfy the measurement requirements of both accuracy and time precision, especially in high-particulate environments. Here, we verified that dual-stage cation exchange membrane (CEM) sampler is incapable of gaseous elemental mercury (Hg0) uptake even if particulate matter is trapped on CEM, whereas the Hg2+ capture efficiency of the sampler is more than 90%. We then developed a Cation Exchange Membrane-Coupled Speciated Atmospheric Mercury Monitoring System (CSAMS) by coupling the dual-stage CEM sampler with the commercial Tekran 2537/1130/1135 system and configuring a new sampling and analysis procedure, so as to improve the monitoring accuracy of Hg2+ and ensure the simultaneous measurement of Hg0, Hg2+, and Hgp in 2 h time resolution. We deployed the CSAMS in urban Beijing in September 2021 and observed an unprecedented elevated Hg2+ during the daytime with an average amplitude of 510 pg m-3. Using a zero-dimensional box model, the elevated Hg2+ production rate was attributed to high atmospheric oxidant concentrations, Hg0 heterogeneous and interfacial oxidation processes on the surface of atmospheric particles, or potential unknown oxidants.

Atmospheric pollutants response to the emission reduction and meteorology during the COVID-19 lockdown in the north of Africa (Morocco).

Climate and air quality change due to COVID-19 lockdown (LCD) are extremely concerned subjects of several research recently. The contribution of meteorological factors and emission reduction to air pollution change over the north of Morocco has been investigated in this study using the framework generalized additive models, that have been proved to be a robust technique for the environmental data sets, focusing on main atmospheric pollutants in the region including ozone (O3), nitrogen dioxide (NO2), sulfur dioxide (SO2), particulate matter (PM2.5 and PM10), secondary inorganic aerosols (SIA), nom-methane volatile organic compounds and carbon monoxide (CO) from the regional air pollution dataset of the Copernicus Atmosphere Monitoring Service. Our results, indicate that secondary air pollutants (PM2.5, PM10 and O3) are more influenced by metrological factors and the other air pollutants reported by this study (NO2 and SO2). We show a negative effect for PBHL, total precipitation and NW10M on PM (PM2.5 and PM10 ), this meteorological parameters contribute to decrease in PM2.5 by 9, 2 and 9% respectively, before LCD and 8, 1 and 5% respectively during LCD. However, a positive marginal effect was found for SAT, Irradiance and RH that contribute to increase PM2.5 by 9, 12 and 18% respectively, before LCD and 17, 54 and 34% respectively during LCD. We found also that meteorological factors contribute to O3, PM2.5, PM10 and SIA average mass concentration by 22, 5, 3 and 34% before LCD and by 28, 19, 5 and 42% during LCD respectively. The increase in meteorological factors marginal effect during LCD shows the contribution of photochemical oxidation to air pollution due to increase in atmospheric oxidant (O3 and OH radical) during LCD, which can explain the response of PM to emission reduction. This study indicates that PM (PM2.5, PM10) has more controlled by SO2 due to the formation of sulfate particles especially under high oxidants level. The positive correlation between westward wind at 10 m (WW10M), Northward Wind at 10 m (NW10M) and PM indicates the implication of sea salt particles transported from Mediterranean Sea and Atlantic Ocean. The Ozone mass concentration shows a positive trend with Irradiance, Total and SAT during LCD; because temperature and irradiance enhance tropospheric ozone formation via photochemical reaction.This study shows the contribution of atmospheric oxidation capacity to air pollution change. Supplementary Information: The online version contains supplementary material available at 10.1007/s00477-022-02224-z.

Advances in mass spectrometry-based epitope mapping of protein therapeutics.

Monoclonal antibody (mAb) has grown to be the major asset in protein therapeutics market since its initial introduction in 1980s. To identify a suitable mAb as a drug candidate for development requires deep understanding of disease biology and attribute sciences, including epitope-paratope binding recognition. Mass spectrometry (MS) has become a critical technology platform in epitope mapping. MS-based approaches utilize chemical labeling to assess the changes of solvent accessibilities during binding interactions, and the labeling can be either reversible or irreversible. Reversible labeling is represented by hydrogen/deuterium exchange (HDX), which probes the changes via exchange between backbone amide hydrogen and deuterium in the solvent. Irreversible labeling targets amino acid residue side chains and involves chemical based labeling such as glycine ethyl ester labeling, radical based labeling such as fast photochemical oxidation of proteins (FPOP), and chemical cross-linking. All these methods have been developed extensively for characterization of binding interface within an immunocomplex. This review covers the fundamentals of these different MS-based methods and highlights recent case studies to illustrate unique capabilities of MS-based approaches in epitope mapping of protein therapeutics.

Sulfate-accelerated photochemical oxidation of arsenopyrite in acidic systems under oxic conditions: Formation and function of schwertmannite.

The weathering of arsenopyrite is closely related to the generation of acid mine drainage (AMD) and arsenic (As) pollution. Solar radiation can accelerate arsenopyrite oxidation, but little is known about the further effect of SO42- on the photochemical process. Here, the photooxidation of arsenopyrite was investigated in the presence of SO42- in simulated AMD environments, and the effects of SO42- concentration, pH and dissolved oxygen on arsenopyrite oxidation were studied as well. SO42- could accelerate the photooxidation of arsenopyrite and As(III) through complexation between nascent schwertmannite and As(III). Fe(II) released from arsenopyrite was oxidized to form schwertmannite in the presence of SO42-, and the photooxidation of arsenopyrite occurred through the ligand-to-metal charge-transfer process in schwertmannite-As(III) complex along with the formation of reactive oxygen species in the presence of O2. The photooxidation rate of arsenopyrite first rose and then fell with increasing SO42- concentration. In the pH range of 2.0-4.0, the photooxidation rate of arsenopyrite progressively increased in the presence of SO42-. This study reveals how SO42- promotes the photooxidation of arsenopyrite and As release in the AMD environment, and improves the understanding of the transformation and migration of As in mining areas.

Advances in mass spectrometry-based footprinting of membrane proteins.

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins (MPs) are determined regularly. These advances have been driven by over 15 years of technological improvements, first in macromolecular crystallography, and recently in cryo-electron microscopy. Obtaining information about MP higher order structure and interactions is also a frontier, important but challenging owing to their unique properties and the need to choose suitable detergents/lipids for their study. The development of mass spectrometry (MS), both instruments and methodology in the past 10 years, has also advanced it as a complementary method to study MP structure and interactions. In this review, we discuss advances in MS-based footprinting for MPs and highlight recent methodologies that offer new promise for MP study by chemical footprinting and mass spectrometry.

Epitope mapping of anti-drug antibodies to a clinical candidate bispecific antibody.

Anti-drug antibodies (ADA) can limit the efficacy and safety of therapeutic antibodies. However, determining the exact nature of ADA interactions with the target drug via epitope mapping is challenging due to the polyclonal nature of the IgG response. Here, we demonstrate successful proof-of-concept for the application of hydroxyl radical footprinting (HRF)-mass spectrometry for epitope mapping of ADAs obtained from goats that were administered a knob-into-hole bispecific antibody (BsAb1). Subsequently, we performed epitope mapping of ADAs obtained from cynomolgus (cyno) monkeys that were administered BsAb1 as we described in a recently published paper. Herein, we provide the first data to demonstrate the feasibility of using HRF for ADA epitope mapping, and show that both goat and cyno-derived ADAs specifically target the complementary-determining regions in both arms of BsAb1, suggesting that the ADA epitopes on BsAb1 may be species-independent.