A review of sample collection and analytical methods for detecting per- and polyfluoroalkyl substances in indoor and outdoor air.

Per- and polyfluoroalkyl substances (PFAS) are a unique class of chemicals synthesized to aid in industrial processes, fire-fighting products, and to benefit consumer products such as clothing, cosmetics, textiles, carpets, and coatings. The widespread use of PFAS and their strong carbon-fluorine bonds has led to their ubiquitous presence throughout the world. Airborne transport of PFAS throughout the atmosphere has also contributed to environmental pollution. Due to the potential environmental and human exposure concerns of some PFAS, research has extensively focused on water, soil, and organismal detection, but the presence of PFAS in the air has become an area of growing concern. Methods to measure polar PFAS in various matrices have been established, while the investigation of polar and nonpolar PFAS in air is still in its early development. This literature review aims to present the last two decades of research characterizing PFAS in outdoor and indoor air, focusing on active and passive air sampling and analytical methods. The PFAS classes targeted and detected in air samples include fluorotelomer alcohols (FTOHs), perfluoroalkane sulfonamides (FASAs), perfluoroalkane sulfonamido ethanols (FASEs), perfluorinated carboxylic acids (PFCAs), and perfluorinated sulfonic acids (PFSAs). Although the manufacturing of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) has been largely phased out, these two PFAS are still often detected in air samples. Additionally, recent estimates indicate that there are thousands of PFAS that are likely present in the air that are not currently monitored in air methods. Advances in air sampling methods are needed to fully characterize the atmospheric transport of PFAS.

Low temperature destruction of gas-phase per- and polyfluoroalkyl substances using an alumina-based catalyst.

Per- and polyfluoroalkyl substances (PFAS) pose a major health and environmental problem. Methods are needed to ensure that PFAS are not released into the environment during their use or disposal. Alumina-based catalysts have been used for the abatement of small perfluorocarbons, e.g. tetrafluoromethane and perfluoropropane, emitted during the silicon etching process. Here, an alumina-based catalyst was tested to determine if these catalysts may facilitate the destruction of gas-phase PFAS. The catalyst was challenged with two nonionic surfactants with eight fluorinated carbons, 8:2 fluorotelomer alcohol and N-Ethyl-N-(2-hydroxyethyl)perfluorooctylsulfonamide. The catalyst helped decrease the temperatures needed for the destruction of the parent PFAS relative to a thermal-only treatment. Temperatures of 200°C were sufficient to destroy the parent PFAS using the catalyst, although a significant number of fluorinated products of incomplete destruction (PIDs) were observed. The PIDs were no longer observed by about 500°C with catalyst treatment. Alumina-based catalysts are a promising PFAS pollution control technology that could eliminate both perfluorocarbons and longer chain PFAS from gas streams.Implications: The release of per- and polyfluoroalkyl substances (PFAS) into the atmosphere can cause problems for human health and the environment. It is critical to reduce and eliminate PFAS emissions from potential sources, such as manufacturers, destruction technologies, and fluoropolymer processing and application sites. Here, an alumina-based catalyst was used to eliminate the emissions of two gas-phase PFAS with eight fully fluorinated carbons. No PFAS were observed in the emissions when the catalyst was at 500°C, lowering the energy requirements for PFAS destruction. This shows that alumina-based catalysts are a promising area for research for PFAS pollution controls and the elimination of PFAS emissions into the atmosphere.

Characterization of PFAS air emissions from thermal application of fluoropolymer dispersions on fabrics.

During thermal processes utilized in affixing fluoropolymer coatings dispersion to fibers and fabrics, coating components are vaporized. It is suspected that per- and polyfluoroalkyl substances (PFAS) from the dispersions may undergo chemical transformations at the temperatures used, leading to additional emitted PFAS thermal byproducts. It is important to characterize these emissions to support evaluation of the resulting environmental and health impacts. In this study, a bench-scale system was built to simulate this industrial process via thermal application of dispersions to fiberglass utilizing relevant temperatures and residence times in sequential drying, baking, and sintering steps. Experiments were performed with two commercially available dispersions and a simple model mixture containing a single PFAS (6:2 fluorotelomer alcohol [6:2 FTOH]). Vapor-phase emissions were sampled and characterized by several off-line and real-time mass spectrometry techniques for targeted and nontargeted PFAS. Results indicate that multiple PFAS thermal transformation products and multiple nonhalogenated organic species were emitted from the exit of the high temperature third (sintering) furnace when 6:2 FTOH was the only PFAS present in the aqueous mixture. This finding supports the hypothesis that temperatures typical of these industrial furnaces may also induce chemical transformations within the fluorinated air emissions. Experiments using the two commercial fluoropolymer dispersions indicate air emissions of part-per-million by volume (ppmv) concentrations of heptafluoropropyl-1,2,2,2-tetrafluoroethyl ether (Fluoroether E1), as well as other PFAS at operationally relevant temperatures. We suspect that E1 is a direct thermal decomposition product (via decarboxylation) of 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (commonly referred to as HFPO-DA) present in the dispersions. Other thermal decomposition products, including the monomer, tetrafluoroethene, may originate from the PFAS used to stabilize the dispersion or from the polymer particles in suspension. This study represents the first researcher-built coating application simulator to report nontargeted PFAS emission characterization, real-time analyses, and the quantification of 30 volatile target PFAS.Implications: Thermal processes used to affix fluoropolymers to fabrics are believed to be a source of PFAS air emissions. These coating operations are used by many large and small manufacturers and typically do not currently require any air emissions control. This research designed and constructed a bench-scale system that simulates these processes and used several off-line and advanced real-time mass spectroscopy techniques to characterize PFAS air emissions from two commercial fluoropolymer dispersions. Further, as the compositions of commercial dispersions are largely unknown, a model three-component solution containing a single PFAS was used to characterize emissions of multiple PFAS thermal transformation products at operationally relevant conditions. This research shows that fluoropolymer fabric coating facilities can be sources of complex mixtures of PFAS air emissions that include volatile and semivolatile PFAS present in the dispersions, as well as PFAS byproducts formed by the thermal transformation of fluorocarbon and hydrocarbon species present in these dispersions.

Low Temperature Thermal Treatment of Gas-Phase Fluorotelomer Alcohols by Calcium Oxide.

Given the extent to which per- and polyfluoroalkyl substances (PFAS) are used in commercial and industrial applications, the need to evaluate treatment options that reduce environmental emissions and human and ecological exposures of PFAS is becoming more necessary. One specific chemical class of PFAS, fluorotelomer alcohols (FTOHs), have vapor pressures such that a significant fraction is expected to be present in the gas-phase even at ambient temperatures. FTOHs are used in a variety of PFAS applications, including synthesis and material coatings. Using two complementary mass spectrometric methods, the use of calcium oxide (CaO) was examined as a low temperature and potentially low-cost thermal treatment media for removal and destruction of four gas-phase FTOHs of varying molecular weights. This was accomplished by assessing the removal/destruction efficiency of the FTOHs and the formation of fluorinated byproducts as a function of treatment temperature (200 - 800 °C) in the presence of CaO compared to thermal-only destruction. During the treatment process, there is evidence that other PFAS compounds are produced at low temperatures (200 - 600 °C) as the primary FTOH partially degrades. At temperatures above 600 °C, thermal treatment with CaO prevented the formation or removed nearly all these secondary products.

Using the US EPA CompTox Chemicals Dashboard to interpret targeted and non-targeted GC-MS analyses from human breath and other biological media.

The U.S. EPA CompTox Chemicals Dashboard is a freely available web-based application providing access to chemistry, toxicity, and exposure data for ∼900 000 chemicals. Data, search functionality, and prediction models within the Dashboard can help identify chemicals found in environmental analyses and human biomonitoring. It was designed to deliver data generated to support computational toxicology to reduce chemical testing on animals and provide access to new approach methodologies including prediction models. The inclusion of mass and formula-based searches, together with relevant ranking approaches, allows for the identification and prioritization of exogenous (environmental) chemicals from high resolution mass spectrometry in need of further evaluation. The Dashboard includes chemicals that can be detected by liquid chromatography, gas chromatography-mass spectrometry (GC-MS) and direct-MS analyses, and chemical lists have been added that highlight breath-borne volatile and semi-volatile organic compounds. The Dashboard can be searched using various chemical identifiers (e.g. chemical synonyms, CASRN and InChIKeys), chemical formula, MS-ready formulae monoisotopic mass, consumer product categories and assays/genes associated with high-throughput screening data. An integrated search at a chemical level performs searches against PubMed to identify relevant published literature. This article describes specific procedures using the Dashboard as a first-stop tool for exploring both targeted and non-targeted results from GC-MS analyses of chemicals found in breath, exhaled breath condensate, and associated aerosols.

Exhaled breath condensate biomarkers in critically ill, mechanically ventilated patients.

Pneumonia is a significant risk for critically ill, mechanically ventilated (CIMV) patients. Diagnosis of pneumonia generally requires a combination of clinician-guided diagnoses and clinical scoring systems. Exhaled breath condensate (EBC) can be safely collected non-invasively from CIMV patients. Hundreds of biomarkers in EBC are associated with acute disease states, including pneumonia. We evaluated cytokines in EBC from CIMV patients and hypothesized that these biomarkers would correlate with disease severity in pneumonia, sepsis, and death. EBC IL-2 levels were associated with chest radiograph severity scores (odds ratio = 1.68; 95% confidence interval = 1.09-2.60; P = 0.02). EBC TNF-α levels were also associated with pneumonia (odds ratio = 3.20; 95% confidence interval = 1.19-8.65; P = 0.02). The techniques and results from this study may be useful for all mechanically ventilated patients.

Dataset of polycyclic aromatic hydrocarbon recoveries from a selection of sorbent tubes for thermal desorption-gas chromatography/mass spectrometry analysis.

This dataset contains raw area counts and percent recoveries of polycyclic aromatic hydrocarbon (PAH) standards desorbed from selected sorbent tubes and analyzed using thermal desorption-gas chromatography/mass spectrometry (TD-GC/MS). The results of this study were published in the article "Recovery and reactivity of polycyclic aromatic hydrocarbons collected on selected sorbent tubes and analyzed by thermal desorption-gas chromatography/mass spectrometry" in Journal of Chromatography A [1]. The sorbent tubes studied include stainless steel Carbograph 2TD/1TD, glass quartz wool-Carbograph 2TD, inert-coated stainless steel Carbograph 2TD, glass and stainless steel Tenax TA, PAH (chemical weapons), and glass and stainless steel XRO-440 sorbent tubes. Tables listing the experimental conditions, TD methods, and types of sorbent tubes are included in the manuscript. Data for experiments, including the investigation of incomplete desorption of PAHs from Carbograph 2TD/1TD and XRO-440 sorbent tubes, the comparison of PAH recoveries from three different TD methods, the analysis of PAH breakthrough from sorbent tubes, the investigation of the effect of heat on PAH percent recovery from sorbent tubes, and the formation of reaction products during PAH loading and desorption are included in Appendix A. These data can be used to guide sorbent tube selection for PAH analyses in future studies.

Firefighters' absorption of PAHs and VOCs during controlled residential fires by job assignment and fire attack tactic.

To better understand the absorption of combustion byproducts during firefighting, we performed biological monitoring (breath and urine) on firefighters who responded to controlled residential fires and examined the results by job assignment and fire attack tactic. Urine was analyzed for metabolites of polycyclic aromatic hydrocarbons (PAHs) and breath was analyzed for volatile organic compounds (VOCs) including benzene. Median concentrations of PAH metabolites in urine increased from pre-firefighting to 3-h post firefighting for all job assignments. This change was greatest for firefighters assigned to attack and search with 2.3, 5.6, 3.9, and 1.4-fold median increases in pyrene, phenanthrene, naphthalene, and fluorene metabolites. Median exhaled breath concentrations of benzene increased 2-fold for attack and search firefighters (p < 0.01) and 1.4-fold for outside vent firefighters (p = 0.02). Compared to interior attack, transitional attack resulted in 50% less uptake of pyrene (p = 0.09), 36% less uptake phenanthrene (p = 0.052), and 20% less uptake of fluorene (p < 0.01). Dermal absorption likely contributed to firefighters' exposures in this study. Firefighters' exposures will vary by job assignment and can be reduced by employing a transitional fire attack when feasible.

How do cancer-sniffing dogs sort biological samples? Exploring case-control samples with non-targeted LC-Orbitrap, GC-MS, and immunochemistry methods.

Early identification of disease onset is regarded as an important factor for successful medical intervention. However, cancer and other long-term latency diseases are rare and may take years to manifest clinically. As such, there are no gold standards with which to immediately validate proposed preclinical screening methodologies. There is evidence that dogs can sort samples reproducibly into yes/no categories based on case-control training, but the basis of their decisions is unknown. Because dogs are sniffing air, the distinguishing chemicals must be either in the gas-phase or attached to aerosols and/or airborne particles. Recent biomonitoring research has shown how to extract and analyze semi- and non-volatile compounds from human breath in exhaled condensates and aerosols. Further research has shown that exhaled aerosols can be directly collected on standard hospital-style olefin polypropylene masks and that these masks can be used as a simple sampling scheme for canine screening. In this article, detailed liquid chromatography-high resolution mass spectrometry (LC-HR-MS) with Orbitrap instrumentation and gas chromatography-mass spectrometry (GC-MS) analyses were performed on two sets of masks sorted by consensus of a four-dog cohort as either cancer or control. Specifically, after sorting by the dogs, sample masks were cut into multiple sections and extracted for LC-MS and GC-MS non-targeted analyses. Extracts were also analyzed for human cytokines, confirming the presence of human aerosol content above levels in blank masks. In preliminary evaluations, 345 and 44 high quality chemical features were detected by LC-MS and GC-MS analyses, respectively. These features were used to develop provisional orthogonal projection to latent structures-discriminant analysis (OPLS-DA) models to determine if the samples classified as cancer (case) or non-cancer (control) by the dogs could be separated into the same groups using analytical instrumentation. While the OPLS-DA model for the LC-HR-MS data was able to separate the two groups with statistical significance, although weak explanatory power, the GC-MS model was not found to be significant. These results suggest that the dogs may rely on the less volatile compounds from breath aerosol that were analyzed by LC-HR-MS than the more volatile compounds observed by GC-MS to sort mask samples into groups. These results provide justification for more expansive studies in the future that aim to characterize specific chemical features, and the role(s) of these features in maintaining homeostatic biological processes.

Recovery and reactivity of polycyclic aromatic hydrocarbons collected on selected sorbent tubes and analyzed by thermal desorption-gas chromatography/mass spectrometry.

This article describes the optimization of methodology for extending the measurement of volatile organic compounds (VOCs) to increasingly heavier polycyclic aromatic hydrocarbons (PAHs) with a detailed focus on recent sorbent tube technology. Although PAHs have lower volatility than compounds such as benzene, toluene, ethylbenzene and xylenes, these semi-volatile compounds can be detected in air and breath samples. For this work, PAHs were captured on sorbent tubes and subsequently analyzed using automated thermal desorption gas chromatography - mass spectrometry (ATD-GC/MS). While many different sorbent tubes are commercially available, optimization for airborne PAH sampling using sorbent tubes has not been previously considered. Herein, several commercially available sorbent tubes, including Carbograph 2 TD/1TD, Tenax TA, XRO-440, and inert-coated PAH tubes are compared to determine the relative recovery for eight PAHs commonly found in the environment. Certain types of sorbent materials were found to be better suited for PAH recovery during thermal desorption, and PAH reaction products were observed on several types of sorbent tubes, including graphitized carbon black sorbents with stainless steel tube materials. As such, selection of sorbent tube media should be carefully considered prior to embarking on a PAH study.

Targeted GC-MS analysis of firefighters' exhaled breath: Exploring biomarker response at the individual level.

Biomarker measurements can provide unambiguous evidence of environmental exposures as well as the resultant biological responses. Firefighters have a high rate of occupational cancer incidence, which has been proposed to be linked in part to their increased environmental exposure to byproducts of combustion and contaminants produced during fire responses. In this article, the uptake and elimination of targeted volatile organic compounds were investigated by collecting the exhaled breath of firefighters on sorbent tubes before and after controlled structure burns and analyzing samples using automated thermal desorption-gas chromatography (ATD-GC/MS). Volatile organic compounds exposure was assessed by grouping the data according to firefighting job positions as well as visualizing the data at the level of the individual firefighter to determine which individuals had expected exposure responses. When data were assessed at the group level, benzene concentrations were found to be elevated post-exposure in both fire attack, victim search, and outside ventilation firefighting positions. However, the results of the data analysis at the individual level indicate that certain firefighters may be more susceptible to post-exposure volatile organic compounds increases than others, and this should be considered when assessing the effectiveness of firefighting protective gear. Although this work focuses on firefighting activity, the results can be translated to potential human health and ecological effects from building and forest fires.

Detection and analysis of endogenous polar volatile organic compounds (PVOCs) in urine for human exposome research.

Background: The human exposome, defined as '…everything that is not the genome', comprises all chemicals in the body interacting with life processes. The exposome drives genes x environment (GxE) interactions that can cause long-term latency and chronic diseases. The exposome constantly changes in response to external exposures and internal metabolism. Different types of compounds are found in different biological media. Objective: Measure polar volatile organic compounds (PVOCs) excreted in urine to document endogenous metabolites and exogenous compounds from environmental exposures. Methods: Use headspace collection and sorbent tube thermal desorption coupled with bench-top gas chromatography-mass spectrometry (GC-MS) for targeted and non-targeted approaches. Identify and categorize PVOCs that may distinguish among healthy and affected individuals. Results: Method is successfully demonstrated to tabulate a series of 28 PVOCs detected in human urine across 120 samples from 28 human subjects. Median concentrations range from below detect to 165 ng/mL. Certain PVOCs have potential health implications. Conclusions: Headspace collection with sorbent tubes is an effective method for documenting PVOCs in urine that are otherwise difficult to measure. This methodology can provide probative information regarding biochemical processes and adverse outcome pathways (AOPs) for toxicity testing.

Beyond monoisotopic accurate mass spectrometry: ancillary techniques for identifying unknown features in non-targeted discovery analysis.

High-resolution mass spectrometry (HR-MS) is an important tool for performing non-targeted analysis for investigating complex organic mixtures in human or environmental media. This perspective demonstrates HR-MS compound identification strategies using atom counting, isotope ratios, and fragmentation pattern analysis based on 'exact' or 'accurate' mass, which allows analytical distinction among mass fragments with the same integer mass, but with different atomic constituents of the original molecules. Herein, HR-MS technology is shown to narrow down the identity of unknown compounds for specific examples, and ultimately inform future analyses when these compounds reoccur. Although HR-MS is important for all biological media, this is particularly critical for new methods and instrumentation invoking exhaled breath condensate, particles, and aerosols. In contrast to standard breath gas-phase analyses where 1 mass unit (Da) resolution is generally sufficient, the condensed phase breath media are particularly vulnerable to errors in compound identification because the larger organic non-volatile molecules can form identical integer mass fragments from different atomic constituents which then require high-resolution mass analyses to tell them apart.

Human biomarker interpretation: the importance of intra-class correlation coefficients (ICC) and their calculations based on mixed models, ANOVA, and variance estimates.

Human biomonitoring is the foundation of environmental toxicology, community public health evaluation, preclinical health effects assessments, pharmacological drug development and testing, and medical diagnostics. Within this framework, the intra-class correlation coefficient (ICC) serves as an important tool for gaining insight into human variability and responses and for developing risk-based assessments in the face of sparse or highly complex measurement data. The analytical procedures that provide data for clinical and public health efforts are continually evolving to expand our knowledge base of the many thousands of environmental and biomarker chemicals that define human systems biology. These chemicals range from the smallest molecules from energy metabolism (i.e., the metabolome), through larger molecules including enzymes, proteins, RNA, DNA, and adducts. In additiona, the human body contains exogenous environmental chemicals and contributions from the microbiome from gastrointestinal, pulmonary, urogenital, naso-pharyngeal, and skin sources. This complex mixture of biomarker chemicals from environmental, human, and microbiotic sources comprise the human exposome and generally accessed through sampling of blood, breath, and urine. One of the most difficult problems in biomarker assessment is assigning probative value to any given set of measurements as there are generally insufficient data to distinguish among sources of chemicals such as environmental, microbiotic, or human metabolism and also deciding which measurements are remarkable from those that are within normal human variability. The implementation of longitudinal (repeat) measurement strategies has provided new statistical approaches for interpreting such complexities, and use of descriptive statistics based upon intra-class correlation coefficients (ICC) has become a powerful tool in these efforts. This review has two parts; the first focuses on the history of repeat measures of human biomarkers starting with occupational toxicology of the early 1950s through modern applications in interpretation of the human exposome and metabolic adverse outcome pathways (AOPs). The second part reviews different methods for calculating the ICC and explores the strategies and applications in light of different data structures.

New breath related topics: sample collection for exhaled breath condensate and aerosol, development of real-time medical alerts, measurement of artificial atmospheres, and analysis of legalized cannabis product.

Exhaled breath technology is expanding beyond conventional gas-phase analysis, and conversely, methodology from other disciplines is finding applications in breath research. Recently, the authors attended conferences that incorporated new technologies into 'breath related' applications. The first was the International Submarine Air Monitoring and Air Purification (SAMAP) held in Uncasville Connecticut, November 2017, and the second was the Pittcon Conference and Exposition (Pittcon) held in Orlando, Florida, February 2018. Herein, we report some of the new topics and ideas encountered, ranging from very specific submarine related respiration issues revolving around carbon dioxide and oxygen from SAMAP to the eclectic topics of analytical chemistry from Pittcon.

Evolution of clinical and environmental health applications of exhaled breath research: Review of methods and instrumentation for gas-phase, condensate, and aerosols.

Human breath, along with urine and blood, has long been one of the three major biological media for assessing human health and environmental exposure. In fact, the detection of odor on human breath, as described by Hippocrates in 400 BC, is considered the first analytical health assessment tool. Although less common in comparison to contemporary bio-fluids analyses, breath has become an attractive diagnostic medium as sampling is non-invasive, unlimited in timing and volume, and does not require clinical personnel. Exhaled breath, exhaled breath condensate (EBC), and exhaled breath aerosol (EBA) are different types of breath matrices used to assess human health and disease state. Over the past 20 years, breath research has made many advances in assessing health state, overcoming many of its initial challenges related to sampling and analysis. The wide variety of sampling techniques and collection devices that have been developed for these media are discussed herein. The different types of sensors and mass spectrometry instruments currently available for breath analysis are evaluated as well as emerging breath research topics, such as cytokines, security and airport surveillance, cellular respiration, and canine olfaction.

Review: Endogenously Produced Volatiles for In Vitro Toxicity Testing Using Cell Lines.

Due to the ∼86,000 chemicals registered under the Toxic Substances Control Act and increasing ethical concerns regarding animal testing, it is not economically or technically feasible to screen every registered chemical for toxicity using animal-based toxicity assays. To address this challenge, regulatory agencies are investigating high-throughput screening in vitro methods to increase speed of toxicity testing, while reducing the overall cost. One approach for rapid toxicity testing currently being investigated is monitoring of volatile emissions produced by cell lines in culture. Such a metabolomics approach would measure gaseous emissions from a cell line and determine if such gaseous metabolites are altered upon exposure to a xenobiotic. Herein, we describe the history and rationale of monitoring endogenously produced volatiles for identification of pathologic conditions, as well as emerging applications in toxicity testing for such an approach.

Blood-borne biomarkers and bioindicators for linking exposure to health effects in environmental health science.

Environmental health science aims to link environmental pollution sources to adverse health outcomes to develop effective exposure intervention strategies that reduce long-term disease risks. Over the past few decades, the public health community recognized that health risk is driven by interaction between the human genome and external environment. Now that the human genetic code has been sequenced, establishing this "G × E" (gene-environment) interaction requires a similar effort to decode the human exposome, which is the accumulation of an individual's environmental exposures and metabolic responses throughout the person's lifetime. The exposome is composed of endogenous and exogenous chemicals, many of which are measurable as biomarkers in blood, breath, and urine. Exposure to pollutants is assessed by analyzing biofluids for the pollutant itself or its metabolic products. New methods are being developed to use a subset of biomarkers, termed bioindicators, to demonstrate biological changes indicative of future adverse health effects. Typically, environmental biomarkers are assessed using noninvasive (excreted) media, such as breath and urine. Blood is often avoided for biomonitoring due to practical reasons such as medical personnel, infectious waste, or clinical setting, despite the fact that blood represents the central compartment that interacts with every living cell and is the most relevant biofluid for certain applications and analyses. The aims of this study were to (1) review the current use of blood samples in environmental health research, (2) briefly contrast blood with other biological media, and (3) propose additional applications for blood analysis in human exposure research.

Thermodynamic Analysis of the Geldanamycin-Hsp90 Interaction in a Whole Cell Lysate Using a Mass Spectrometry-Based Proteomics Approach.

Geldanamycin is a natural product with well-established and potent anti-cancer activities. Heat shock protein 90 (Hsp90) is the known target of geldanamycin, which directly binds to Hsp90's N-terminal ATP binding domain and inhibits Hsp90's ATPase activity. The affinity of geldanamycin for Hsp90 has been measured in multiple studies. However, there have been large discrepancies between the reported dissociation constants (i.e., Kd values), which have ranged from low nanomolar to micromolar. Here the stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to measure the binding affinity of geldanamycin to unpurified Hsp90 in an MCF-7 cell lysate. The Kd values determined here were dependent on how long geldanamycin was equilibrated with the lysate prior to SPROX analysis. The Kd values determined using equilibration times of 0.5 and 24 h were 1 and 0.03 µM, respectively. These Kd values, which are similar to those previously reported in a geldanamycin-Hsp90 binding study that involved the use of a fluorescently labeled geldanamycin analogue, establish that the slow-tight binding behavior previously observed for the fluorescently labeled geldanamycin analogue is not an artifact of the fluorescent label, but rather an inherent property of the geldanamycin-Hsp90 binding interaction. The slow-tight binding property of this complex may be related to time-dependent conformational changes in Hsp90 and/or to time-dependent chemical changes in geldanamycin, both of which have been previously proposed to explain the slow-tight binding behavior of the geldanamycin-Hsp90 complex. Graphical Abstract ᅟ.