Analysis types and quantification methods applied in UHPLC-MS metabolomics research: a tutorial.

BACKGROUND: Different types of analytical methods, with different characteristics, are applied in metabolomics and lipidomics research and include untargeted, targeted and semi-targeted methods. Ultra High Performance Liquid Chromatography-Mass Spectrometry is one of the most frequently applied measurement instruments in metabolomics because of its ability to detect a large number of water-soluble and lipid metabolites over a wide range of concentrations in short analysis times. Methods applied for the detection and quantification of metabolites differ and can either report a (normalised) peak area or an absolute concentration. AIM OF REVIEW: In this tutorial we aim to (1) define similarities and differences between different analytical approaches applied in metabolomics and (2) define how amounts or absolute concentrations of endogenous metabolites can be determined together with the advantages and limitations of each approach in relation to the accuracy and precision when concentrations are reported. KEY SCIENTIFIC CONCEPTS OF REVIEW: The pre-analysis knowledge of metabolites to be targeted, the requirement for (normalised) peak responses or absolute concentrations to be reported and the number of metabolites to be reported define whether an untargeted, targeted or semi-targeted method is applied. Fully untargeted methods can only provide (normalised) peak responses and fold changes which can be reported even when the structural identity of the metabolite is not known. Targeted methods, where the analytes are known prior to the analysis, can also report fold changes. Semi-targeted methods apply a mix of characteristics of both untargeted and targeted assays. For the reporting of absolute concentrations of metabolites, the analytes are not only predefined but optimized analytical methods should be developed and validated for each analyte so that the accuracy and precision of concentration data collected for biological samples can be reported as fit for purpose and be reviewed by the scientific community.

Reference materials for MS-based untargeted metabolomics and lipidomics: a review by the metabolomics quality assurance and quality control consortium (mQACC).

INTRODUCTION: The metabolomics quality assurance and quality control consortium (mQACC) is enabling the identification, development, prioritization, and promotion of suitable reference materials (RMs) to be used in quality assurance (QA) and quality control (QC) for untargeted metabolomics research. OBJECTIVES: This review aims to highlight current RMs, and methodologies used within untargeted metabolomics and lipidomics communities to ensure standardization of results obtained from data analysis, interpretation and cross-study, and cross-laboratory comparisons. The essence of the aims is also applicable to other 'omics areas that generate high dimensional data. RESULTS: The potential for game-changing biochemical discoveries through mass spectrometry-based (MS) untargeted metabolomics and lipidomics are predicated on the evolution of more confident qualitative (and eventually quantitative) results from research laboratories. RMs are thus critical QC tools to be able to assure standardization, comparability, repeatability and reproducibility for untargeted data analysis, interpretation, to compare data within and across studies and across multiple laboratories. Standard operating procedures (SOPs) that promote, describe and exemplify the use of RMs will also improve QC for the metabolomics and lipidomics communities. CONCLUSIONS: The application of RMs described in this review may significantly improve data quality to support metabolomics and lipidomics research. The continued development and deployment of new RMs, together with interlaboratory studies and educational outreach and training, will further promote sound QA practices in the community.

Cross-Laboratory Standardization of Preclinical Lipidomics Using Differential Mobility Spectrometry and Multiple Reaction Monitoring.

Modern biomarker and translational research as well as personalized health care studies rely heavily on powerful omics' technologies, including metabolomics and lipidomics. However, to translate metabolomics and lipidomics discoveries into a high-throughput clinical setting, standardization is of utmost importance. Here, we compared and benchmarked a quantitative lipidomics platform. The employed Lipidyzer platform is based on lipid class separation by means of differential mobility spectrometry with subsequent multiple reaction monitoring. Quantitation is achieved by the use of 54 deuterated internal standards and an automated informatics approach. We investigated the platform performance across nine laboratories using NIST SRM 1950-Metabolites in Frozen Human Plasma, and three NIST Candidate Reference Materials 8231-Frozen Human Plasma Suite for Metabolomics (high triglyceride, diabetic, and African-American plasma). In addition, we comparatively analyzed 59 plasma samples from individuals with familial hypercholesterolemia from a clinical cohort study. We provide evidence that the more practical methyl-tert-butyl ether extraction outperforms the classic Bligh and Dyer approach and compare our results with two previously published ring trials. In summary, we present standardized lipidomics protocols, allowing for the highly reproducible analysis of several hundred human plasma lipids, and present detailed molecular information for potentially disease relevant and ethnicity-related materials.

Workshop report: Toward the development of a human whole stool reference material for metabolomic and metagenomic gut microbiome measurements.

INTRODUCTION: To date, there has been little effort to develop standards for metabolome-based gut microbiome measurements despite the significant efforts toward standard development for DNA-based microbiome measurements. OBJECTIVES: The National Institute of Standards and Technology (NIST), The BioCollective (TBC), and the North America Branch of the International Life Sciences Institute (ILSI North America) are collaborating to extend NIST's efforts to develop a Human Whole Stool Reference Material for the purpose of method harmonization and eventual quality control. METHODS: The reference material will be rationally designed for adequate quality assurance and quality control (QA/QC) for underlying measurements in the study of the impact of diet and nutrition on functional aspects of the host gut microbiome and relationships of those functions to health. To identify which metabolites deserve priority in their value assignment, NIST, TBC, and ILSI North America jointly conducted a workshop on September 12, 2019 at the NIST campus in Gaithersburg, Maryland. The objective of the workshop was to identify metabolites for which evidence indicates relevance to health and disease and to decide on the appropriate course of action to develop a fit-for-purpose reference material. RESULTS: This document represents the consensus opinions of workshop participants and co-authors of this manuscript, and provides additional supporting information. In addition to developing general criteria for metabolite selection and a preliminary list of proposed metabolites, this paper describes some of the strengths and limitations of this initiative given the current state of microbiome research. CONCLUSIONS: Given the rapidly evolving nature of gut microbiome science and the current state of knowledge, an RM (as opposed to a CRM) measured for multiple metabolites is appropriate at this stage. As the science evolves, the RM can evolve to match the needs of the research community. Ultimately, the stool RM may exist in sequential versions. Beneficial to this evolution will be a clear line of communication between NIST and the stakeholder community to ensure alignment with current scientific understanding and community needs.

Dissemination and analysis of the quality assurance (QA) and quality control (QC) practices of LC-MS based untargeted metabolomics practitioners.

INTRODUCTION: The metabolomics quality assurance and quality control consortium (mQACC) evolved from the recognized need for a community-wide consensus on improving and systematizing quality assurance (QA) and quality control (QC) practices for untargeted metabolomics. OBJECTIVES: In this work, we sought to identify and share the common and divergent QA and QC practices amongst mQACC members and collaborators who use liquid chromatography-mass spectrometry (LC-MS) in untargeted metabolomics. METHODS: All authors voluntarily participated in this collaborative research project by providing the details of and insights into the QA and QC practices used in their laboratories. This sharing was enabled via a six-page questionnaire composed of over 120 questions and comment fields which was developed as part of this work and has proved the basis for ongoing mQACC outreach. RESULTS: For QA, many laboratories reported documenting maintenance, calibration and tuning (82%); having established data storage and archival processes (71%); depositing data in public repositories (55%); having standard operating procedures (SOPs) in place for all laboratory processes (68%) and training staff on laboratory processes (55%). For QC, universal practices included using system suitability procedures (100%) and using a robust system of identification (Metabolomics Standards Initiative level 1 identification standards) for at least some of the detected compounds. Most laboratories used QC samples (>86%); used internal standards (91%); used a designated analytical acquisition template with randomized experimental samples (91%); and manually reviewed peak integration following data acquisition (86%). A minority of laboratories included technical replicates of experimental samples in their workflows (36%). CONCLUSIONS: Although the 23 contributors were researchers with diverse and international backgrounds from academia, industry and government, they are not necessarily representative of the worldwide pool of practitioners due to the recruitment method for participants and its voluntary nature. However, both questionnaire and the findings presented here have already informed and led other data gathering efforts by mQACC at conferences and other outreach activities and will continue to evolve in order to guide discussions for recommendations of best practices within the community and to establish internationally agreed upon reporting standards. We very much welcome further feedback from readers of this article.

Nontargeted lipidomics of novel human plasma reference materials: hypertriglyceridemic, diabetic, and African-American.

The unavailability of appropriate quality assurance/quality control materials in many lipidomics applications poses a significant challenge for lipidomics research. It is recommended that samples with certified values and/or consensus estimates, such as NIST SRM 1950-Metabolites in Frozen Human Plasma, be implemented in routine analyses to enable community-wide comparisons of lipidomics results and analytical workflows. Herein, we applied a nontargeted lipidomics method for the analysis of a new human plasma reference material suite developed by NIST (hypertriglyceridemic, diabetic, and African-American plasma pools), in addition to SRM 1950. We identified specific lipidomics fingerprints associated with each sample type, including lauric acid-containing lipids and elevated triacylglycerol levels in hypertriglyceridemic plasma, palmitoleic acid-containing lipids in diabetic plasma, and oxidized fatty acid-containing phospholipids in African-American plasma. This work highlights the importance of developing and profiling application-specific reference materials, while establishing reference data that may be used for system suitability and/or quality control metrics.Graphical abstract.

Detection of pathogenic Escherichia coli on potentially contaminated beef carcasses using cassette PCR and conventional PCR.

BACKGROUND: Over a one year period, swabs of 820 beef carcasses were tested for the presence of Shiga toxin-producing Escherichia coli by performing Polymerase Chain Reaction (PCR) in a novel technology termed "cassette PCR", in comparison to conventional liquid PCR. Cassette PCR is inexpensive and ready-to-use. The operator need only add the sample and press "go". Cassette PCR can simultaneously test multiple samples for multiple targets. Carcass swab samples were first tested for the presence of STEC genes (O157, eae, stx1 and stx2). Samples were considered to be pathogenic if positive for eae plus stx1 and/or stx2. For samples scored as pathogenic, further testing screened for 6 additional high frequency O-antigens (O26, O45, O103, O111, O121, and O145). RESULTS: Of the 820 samples, 41% were pathogenic and 30% were O157 positive. Of these, 19% of samples were positive for O157 and carried potentially pathogenic E. coli (eae plus stx1 and/or stx2). Of all samples identified as carrying pathogenic E. coli, 18.9, 38.8, 41.4, 0, 36.1, and 4.1% respectively were positive for O26, O45, O103, O111, O121, and O145. To validate cassette PCR testing, conventional PCR using STEC primers was performed on each of the 820 samples. Only 148 of 3280 cassette PCR tests were discordant with conventional PCR results. However, further fractional testing showed that 110 of these 148 PCRs reflected low numbers of E. coli in the enrichment broth and could be explained as due to Poisson limiting dilution of the template, affecting both cassette PCR and conventional PCR. Of the remaining 38 discordant tests, 27 initial capillary PCRs and 10 initial conventional tests were nominally discordant between cassette and conventional PCR, perhaps reflecting human/technical error on both sides of the comparison. CONCLUSIONS: Contaminated beef carcass swabs were often complex, likely harboring more than one strain of pathogenic E. coli. Cassette PCR had 98.8% concordance with parallel conventional PCR for detection of STEC genes. This indicates that cassette PCR is highly reliable for detecting multiple pathogens in beef carcass swabs from processing plants.

Lipidomics for wildlife disease etiology and biomarker discovery: a case study of pansteatitis outbreak in South Africa.

INTRODUCTION: Lipidomics is an emerging field with great promise for biomarker and mechanistic studies due to lipids diverse biological roles. Clinical research applying lipidomics is drastically increasing, with research methods and tools developed for clinical applications equally promising for wildlife studies. OBJECTIVES: Limited research to date has applied lipidomics, especially of the intact lipidome, to wildlife studies. Therefore, we examine the application of lipidomics for in situ studies on Mozambique tilapia (Oreochromis mossambicus) in Loskop Dam, South Africa. Wide-scale mortality events of aquatic life associated with an environmentally-derived inflammatory disease, pansteatitis, have occurred in this area. METHODS: The lipidome of adipose tissue (n = 31) and plasma (n = 51) from tilapia collected from Loskop Dam were characterized using state of the art liquid chromatography coupled to high-resolution tandem mass spectrometry. RESULTS: Lipid profiles reflected pansteatitis severity and were significantly different between diseased and healthy individuals. Over 13 classes of lipids associated with inflammation, cell death, and/or oxidative damage were upregulated in pansteatitis-affected adipose tissue, including ether-lipids, short-chained triglyceride oxidation products, sphingolipids, and acylcarnitines. Ceramides showed a 1000-fold increase in the most affected adipose tissues and were sensitive to disease severity. In plasma, triglycerides were found to be downregulated in pansteatitis-affected tilapia. CONCLUSION: Intact lipidomics provided useful mechanistic data and possible biomarkers of pansteatitis. Lipids pointed to upregulated inflammatory pathways, and ceramides serve as promising biomarker candidates for pansteatitis. As comprehensive coverage of the lipidome aids in the elucidation of possible disease mechanisms, application of lipidomics could be applied to the understanding of other environmentally-derived inflammatory conditions, such as those caused by obesogens.

Alloy-Free Band Gap Tuning across the Visible Spectrum.

We present evidence, from theory and experiment, that ZnSnN_{2} and MgSnN_{2} can be used to match the band gap of InGaN without alloying-by exploiting cation disorder in a controlled fashion. We base this on the determination of S, the long-range order parameter of the cation sublattice, for a series of epitaxial thin films of ZnSnN_{2} and MgSnN_{2} using three different techniques: x-ray diffraction, Raman spectroscopy, and in situ electron diffraction. We observe a linear relationship between S^{2} and the optical band gap of both ZnSnN_{2} (1.12-1.98 eV) and MgSnN_{2} (1.87-3.43 eV). The results clearly demonstrate the correlation between controlled heterovalent cation ordering and the optical band gap, which applies to a broad group of emerging ternary heterovalent compounds and has implications for similar trends in other material properties besides the band gap.

NIST lipidomics workflow questionnaire: an assessment of community-wide methodologies and perspectives.

INTRODUCTION: Efforts to harmonize lipidomic methodologies have been limited within the community. Here, we aimed to capitalize on the recent National Institute of Standards and Technology lipidomics interlaboratory comparison exercise by implementing a questionnaire that assessed current methodologies, quantitation strategies, standard operating procedures (SOPs), and quality control activities employed by the lipidomics community. OBJECTIVES: Lipidomics is a rapidly developing field with diverse applications. At present, there are no community-vetted methods to assess measurement comparability or data quality. Thus, a major impetus of this questionnaire was to profile current efforts, highlight areas of need, and establish future objectives in an effort to harmonize lipidomics workflows. METHODS: The 54-question survey inquired about laboratory demographics, lipidomic methodologies and SOPs, analytical platforms, quantitation, reference materials, quality control procedures, and opinions regarding challenges existing within the community. RESULTS: A total of 125 laboratories participated in the questionnaire. A broad overview of results highlighted a wide methodological diversity within current lipidomic workflows. The impact of this diversity on lipid measurement and quantitation is currently unknown and needs to be explored further. While some laboratories do incorporate SOPs and quality control activities, these concepts have not been fully embraced by the community. The top five perceived challenges within the lipidomics community were a lack of standardization amongst methods/protocols, lack of lipid standards, software/data handling and quantification, and over-reporting/false positives. CONCLUSION: The questionnaire provided an overview of current lipidomics methodologies and further promoted the need for community-accepted guidelines and protocols. The questionnaire also served as a platform to help determine and prioritize metrological issues to be investigated.

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

LipidQC: Method Validation Tool for Visual Comparison to SRM 1950 Using NIST Interlaboratory Comparison Exercise Lipid Consensus Mean Estimate Values.

As advances in analytical separation techniques, mass spectrometry instrumentation, and data processing platforms continue to spur growth in the lipidomics field, more structurally unique lipid species are detected and annotated. The lipidomics community is in need of benchmark reference values to assess the validity of various lipidomics workflows in providing accurate quantitative measurements across the diverse lipidome. LipidQC addresses the harmonization challenge in lipid quantitation by providing a semiautomated process, independent of analytical platform, for visual comparison of experimental results of National Institute of Standards and Technology Standard Reference Material (SRM) 1950, "Metabolites in Frozen Human Plasma", against benchmark consensus mean concentrations derived from the NIST Lipidomics Interlaboratory Comparison Exercise.

Metabolomics and proteomics reveal impacts of chemically mediated competition on marine plankton.

Competition is a major force structuring marine planktonic communities. The release of compounds that inhibit competitors, a process known as allelopathy, may play a role in the maintenance of large blooms of the red-tide dinoflagellate Karenia brevis, which produces potent neurotoxins that negatively impact coastal marine ecosystems. K. brevis is variably allelopathic to multiple competitors, typically causing sublethal suppression of growth. We used metabolomic and proteomic analyses to investigate the role of chemically mediated ecological interactions between K. brevis and two diatom competitors, Asterionellopsis glacialis and Thalassiosira pseudonana. The impact of K. brevis allelopathy on competitor physiology was reflected in the metabolomes and expressed proteomes of both diatoms, although the diatom that co-occurs with K. brevis blooms (A. glacialis) exhibited more robust metabolism in response to K. brevis. The observed partial resistance of A. glacialis to allelopathy may be a result of its frequent exposure to K. brevis blooms in the Gulf of Mexico. For the more sensitive diatom, T. pseudonana, which may not have had opportunity to evolve resistance to K. brevis, allelopathy disrupted energy metabolism and impeded cellular protection mechanisms including altered cell membrane components, inhibited osmoregulation, and increased oxidative stress. Allelopathic compounds appear to target multiple physiological pathways in sensitive competitors, demonstrating that chemical cues in the plankton have the potential to alter large-scale ecosystem processes including primary production and nutrient cycling.

Metabolomic serum profiling detects early-stage high-grade serous ovarian cancer in a mouse model.

Ovarian cancer is a deadly disease killing more than any other gynecologic cancer. Nonspecific symptoms, combined with a lack of early detection methods, contribute to late diagnosis and low five-year survival rates. High-grade serous carcinoma (HGSC) is the most common and deadliest subtype that results in 90% of ovarian cancer deaths. To investigate metabolic patterns for early detection of this deadly ovarian cancer, Dicer-Pten double knockout (DKO) mice that phenocopy many of the features of metastatic HGSC observed in women were studied. Using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS), serum samples from 14 early-stage tumor (ET) DKO mice and 11 controls were analyzed in depth to screen for metabolic signatures capable of differentiating early-stage HGSC from controls. Iterative multivariate classification selected 18 metabolites that, when considered as a panel, yielded 100% accuracy, sensitivity, and specificity for classification. Altered metabolic pathways reflected in that panel included those of fatty acids, bile acids, glycerophospholipids, peptides, and some dietary phytochemicals. These alterations revealed impacts to cellular energy storage and membrane stability, as well as changes in defenses against oxidative stress, shedding new light on the metabolic alterations associated with early ovarian cancer stages.

Schlieren visualization of fluid dynamics effects in direct analysis in real time mass spectrometry.

RATIONALE: The success of ambient analysis using plasma-based ion sources depends heavily on fluid dynamics and mass transport efficiency in the sample region. To help characterize the influence of these determining factors, visualization of the gas flow profile for a Direct Analysis in Real Time (DART) ion source at the mass spectrometer atmospheric pressure (AP) interface was performed using the Schlieren technique. METHODS: The DART helium flow pattern was imaged in model systems incorporating different interface designs, i.e. skimmer or capillary inlet, and for sampling strategies using several types of traditional DART sample probes including a glass capillary, swab, and drug tablet. Notably, Schlieren experiments were conducted on instruments equipped with the gas-ion separator tube (GIST) adapter and Vapur® pump, and on setups featuring the transmission mode (TM) DART module used in standard practice. RESULTS: DART sources were seen to expel a collimated, highly laminar helium stream across interface distances up to ~8 cm. The helium stream was robust to the influence of gas temperature (50-500 °C) and flow rate (≤3.5 L min(-1) ), but considerable DART gas deflection or full disruption was observed in each sampling scenario. The severity of the flow disturbance depended on probe size and placement, the GIST/Vapur® settings, or counter-current gas movements present at the interface. CONCLUSIONS: The real-time Schlieren visualizations introduced in this work provide new insight on the fluid dynamics within the DART-MS sample gap while also helping to identify those experimental parameters requiring optimization for improved transmission.

Feasibility of detecting prostate cancer by ultraperformance liquid chromatography-mass spectrometry serum metabolomics.

Prostate cancer (PCa) is the second leading cause of cancer-related mortality in men. The prevalent diagnosis method is based on the serum prostate-specific antigen (PSA) screening test, which suffers from low specificity, overdiagnosis, and overtreatment. In this work, untargeted metabolomic profiling of age-matched serum samples from prostate cancer patients and healthy individuals was performed using ultraperformance liquid chromatography coupled to high-resolution tandem mass spectrometry (UPLC-MS/MS) and machine learning methods. A metabolite-based in vitro diagnostic multivariate index assay (IVDMIA) was developed to predict the presence of PCa in serum samples with high classification sensitivity, specificity, and accuracy. A panel of 40 metabolic spectral features was found to be differential with 92.1% sensitivity, 94.3% specificity, and 93.0% accuracy. The performance of the IVDMIA was higher than the prevalent PSA test. Within the discriminant panel, 31 metabolites were identified by MS and MS/MS, with 10 further confirmed chromatographically by standards. Numerous discriminant metabolites were mapped in the steroid hormone biosynthesis pathway. The identification of fatty acids, amino acids, lysophospholipids, and bile acids provided further insights into the metabolic alterations associated with the disease. With additional work, the results presented here show great potential toward implementation in clinical settings.

Transmission mode direct analysis in real time mass spectrometry for fast untargeted metabolic fingerprinting.

RATIONALE: Untargeted metabolic fingerprinting is a discovery tool to better understand biochemical processes involved in detecting and characterizing disease states and responses to environmental stressors. Although current mass spectrometric (MS) methods are very powerful, there is a clear need for more rapid, high-throughput MS approaches for metabolomics studies. METHODS: A rapid metabolic fingerprinting method for human blood sera that utilizes a new transmission mode direct analysis in real time (TM-DART) sampling technique coupled with quadrupole time of flight mass spectrometry (QTOFMS) is presented. In this approach, the sample is deposited directly on a stainless steel mesh that is held in the ionization region by a custom-built module. As a result, the DART plasma gas stream interacts with the sample in a flow-through fashion, which maximizes the interaction between the sample and ionizing species and minimizes variance in sample positioning. RESULTS: The optimization of TM-DART parameters directly affecting metabolite desorption and ionization, such as sample position and ionizing gas desorption temperature, was critical in achieving high sensitivity and detecting a broad mass range of metabolites. Ramping the ionizing gas desorption temperature further enhanced analysis by adding a simple separation dimension to this ambient approach. In terms of reproducibility, TM-DART compared favorably with traditional probe mode (PM-) DART analysis, with coefficients of variation as low as 16%. The longer-lasting TM-DART signals enabled the acquisition of metabolite full scan and product ion accurate mass spectra in a single experiment, resulting in greater confidence in metabolite identification. CONCLUSIONS: TM-DART MS proved to be a powerful analytical technique for rapid metabolome analysis of human blood sera.

Effects of mercury deposition and coniferous forests on the mercury contamination of fish in the South Central United States.

Mercury (Hg) is a toxic metal that is found in aquatic food webs and is hazardous to human and wildlife health. We examined the relationship between Hg deposition, land coverage by coniferous and deciduous forests, and average Hg concentrations in largemouth bass (Micropterus salmoides)-equivalent fish (LMBE) in 14 ecoregions located within all or part of six states in the South Central U.S. In 11 ecoregions, the average Hg concentrations in 35.6-cm total length LMBE were above 300 ng/g, the threshold concentration of Hg recommended by the U.S. Environmental Protection Agency for the issuance of fish consumption advisories. Percent land coverage by coniferous forests within ecoregions had a significant linear relationship with average Hg concentrations in LMBE while percent land coverage by deciduous forests did not. Eighty percent of the variance in average Hg concentrations in LMBE between ecoregions could be accounted for by estimated Hg deposition after adjusting for the effects of coniferous forests. Here we show for the first time that fish from ecoregions with high atmospheric Hg pollution and coniferous forest coverage pose a significant hazard to human health. Our study suggests that models that use Hg deposition to predict Hg concentrations in fish could be improved by including the effects of coniferous forests on Hg deposition.