Progress 07/01/21 to 06/30/24
Outputs
Target Audience:RESEARCH Since its installation, the triple quadrupole mass spectrometer has assisted in 30 research projects across 12 research groups in the past two years. These projects include the elucidation of chemicals involved in plant-microbe signaling to enhance the nutrient uptake efficiency of crop plants, monitoring biomarkers in blood serum in cattle to monitor feeding efficiency and toxicosis, tracing the biotransformation of pesticides in plants to inform environment-friendly pesticide application practices, elucidating cellular pathways that facilitate the 'staygreen' characteristics in crop plants, elucidating the pigment chemistry of petals that attract pollinators and protect the reproductive phase from abiotic stressors, measuring allergens across peanut lines. The project results have been presented at 16 national meetings and are part of thirteen research publications. Beyond the targeted research projects, the instrument is being utilized to generate a mass spectral database of 2224 plant secondary compounds, including flavonoids, lignans, and phenylpropanoids, that would be available to the broader research community. TEACHING: Over the duration of this project more than 40 graduate students, 5 postdoctoral scholars and three technical staff have been trained on various aspects of sample analysis on the mass spectrometer. The instrument and the associated technique are incorporated into a graduate-level course. Changes/Problems:The project suffered an 8-month delay in implementation during 2022-2023 due to a weather-relatedflooding event resulting in lab shutdown. However, the project executions were accelerated after the instrument came online, which helped to partly overcome the disruption. What opportunities for training and professional development has the project provided?Graduate Course: The instrument and the associated technique are incorporated into a graduate-level course. Through lecture, students are introduced to the working principle of instrumentation, and the diverse array of mass spectrometric experiments that could be performed using this platform. The students are trained one-on-one in operating the instrument for the associated laboratory class. Further, they participate as a groupto develop methods for various analyses of the instrument. In the final stage of the course, students use the instrumentation independently to complete their class project, which is often focused on a research topic that is in line with their dissertation research. Over the duration of this project more than 40 graduate students, 5 postdoctoral scholars and three technical staff have been trained on various aspects of sample preparation analysis on the mass spectrometer. Apart from the graduate course, four postdoctoral associates and 12 undergraduate students have been trained in various aspects of the analysis. How have the results been disseminated to communities of interest?Over two years, the instrument has facilitated analysis across more than 30 research projects, across broader disciplines including Plant Sciences, Biology, Animal Sciences, Environmental Sciences, Genetics and Biochemistry, and has served 12 research groups within Clemson University, researchers at UC Davis, Uni. Arkansas, USDA-ARS. These studies have resulted in 14 publications in leading journals, and more than 16 presentations at national and regional meetings. Several studies are currently being completed, with five manuscripts at various stages of preparation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The higher sensitivity of analysis of the new triple-quadrupole mass spectrometer opened diverse research possibilities in agricultural and environmental sciences that were otherwise out of reach of research groups at Clemson University. These studies have resulted in 13 publications in leading journals, and more than 16 presentations at national and regional meetings. Several studies are currently being completed, with five manuscripts at various stages of preparation. Apart from facilitating sensitive, quantitative analysis across various research projects, the instrument also enabled several technology development activities; some notable ones are summarized below. Targeted screening of 108 phytohormones and their precursors in plant tissues. An instrument method was developed to screen 108 phytohormones, and was utilized in a study aimed at imparting drought resilience in corn by inoculating the rhizobiome from a congeneric, ruderal grass, Andropogon virginicus. With the extensive screening of phytohormones, we were able to show that one of the key mechanisms through which the rhizobia imparted stress resilience in corn is through the production of auxins that resulted in significantly higher branching of roots, and salicylic acid that resulted in stomatal closure to conserve water. Validating analytical method for lignin characterization. The instrument was part of the platform that tested the validity of traditional lignin quantification methods. Across ecological studies, lignin abundance has been characterized by several proximate and lignin-specific methods, without much understanding of their comparability. We validated the lignin method by comparing five analytical methods of estimating lignin abundance and composition in fine roots across 34 phylogenetically diverse tree species. Our results show a drastic difference in the estimated lignin contents between lignin-specific methods and a proximate acid-insoluble-fraction method routinely used to measure lignin abundance in ecological/agricultural studies. Using the isolated lignin standard, lignin-specific techniques estimated a lignin content of 2 to 10 % (w/w) in roots, whereas AIF-defined lignin contents were c. 5-10 fold higher, in the range of 21-50%. This study provides a refined framework for quantifying lignin in plant biomass and soil organic matter, which is relevant to bioenergy research, animal nutrition and soil carbon sequestration. Development of a sensitive method for analyzing biomarkers for skeletal muscle degradation in cattle. Methyl histidines (MH) are biomarkers for skeletal muscle degradation and could be used to monitor protein mobilization in dairy cows. The currently published methodology for monitoring 1-MH and 3-MH is extremely labor-intensive, time-consuming and lacks sensitivity. A new sensitive method was developed for the rapid and sensitive analysis of MH in plasma samples of dairy cows. The method involves rapid derivatization of MH with alkyl chloroformate and the subsequent analysis of the triple quadrupole with multiple reaction monitoring. Compared to the literature-reported method, the optimized method is: a) 1000 times more sensitive, with a limit of quantification of 2nM, b) requires 200 times less plasma, with robust detection at a sample volume of 5 microliters, c) is high throughput, with a processing time of 3 min per sample, compared to 2 days in the traditional method, d) robust with an internal standard recovery of >75%, e) the derivative is stable for at least 48 hrs. at room temperature. The method is currently being utilized to assess nutritional requirements in dairy cattle. Targeted metabolomics research pipeline: The higher sensitivity and the versatility offered by the instrumentation enabled several targeted metabolomics studies across several disciplines. The instrumentation enabled direct analysis of plant root exudates without any sample preparations and still could profile signaling molecules mediating plant-microbe interactions across 100 sorghum accessions. Compared to the traditional root exudate profiling, this approach increases the throughput of analysis by 10-fold, along with a four-fold increase in sensitivity. Targeted methods were also developed to elucidate the chemical landscape of plant-mycorrhizal symbiotic associations. These analyses revealed the changes in the physiology of the symbiont and the modulation of plant defense strategies that ensue mycorrhizal infection, which is further utilized to improve the phosphorus uptake of crops. Discerning the biological function of biomolecules through parallel data capture.In plants, flavonoids mitigate abiotic stress caused by factors such as ultraviolet (UV) light and drought, and they also play a critical role in plant-organismal interactions. Flavonoids in petals are vital for pollen viability and for attracting pollinators and thus are critical for crop yield. Flavonoids are one of the three classes of plant secondary metabolites and contain more than 5000 compounds, but authentic standards of only a few hundred flavonoids are commercially available. Even though mass spectrometric approaches provide sensitive quantitation of the flavonoids, this data doesn't relate to the biological functions of these compounds- e.g. uv-protection. To address the disconnect between the quantitation and biological function, we developed an analytical pipeline that would simultaneously measure the flavonoids with a diode array UV detector (to capture the absorption spectra of the flavonoids in the plant extract) and tandem mass spectrometer (for trace analysis and to distinguish between isomers). Mass spectral MRM library: Analysis of plant secondary metabolites is challenging due to the lack of authentic standards to optimize the instrument parameters for robust and sensitive analyses. To address this challenge and to serve the needs of the larger research community, we are optimizing instrument parameters for the analysis of 2224 plant secondary metabolites from authentic standards. These include 1252 flavonoids, 281 phenylpropanoids, 288 coumarins, 381 lignans, and 22 carotenoids. We use Mass Frontier and rely on a retention index based on N-alkylpyridinium-3-sulfonates as an additional dimension of comparison. These methods are transferable across triple quadrupole platforms, and any lab can analyze these compounds based on the optimized parameters without having to have authentic standards. Acquisition of Mass spec imaging platform: In complex systems, the biological function, rather than being driven solely by the quantity of compounds present in a cell/tissue/environmental matrices, is significantly influenced by the spatial organization of these compounds within the respective matrices. Thus, along with the quantity of the biomolecule, their spatial organization within the tissue matrix is critical for discerning the overall biological functions. Mass spectrometry imaging (MSI) is a robust tool that facilitates mapping thousands of molecules within the three-dimensional sample matrix. Focused on this vision and competitive funding from Clemson University, the PI acquired a Desorption electrospray ionization (DESI) sampling/ionization interface manufactured by Waters Cooperation (DESI™ XS) for the triple quadrupole platforms to perform MSI. The DESI™ XS has not yet been paired with the Waters TQ-XS, the top-of-the-line mass spectrometer currently available in terms of sensitivity and robustness. Waters Cooperation committed to working with the PI to bring this pairing to fruition. This effort will involve the Waters engineers being on-site at Clemson, and configuring and optimizing DESI™ XS to fit the data acquisition needs of the Clemson researchers. This collaborative partnership with Waters will provide unprecedented early access to this instrument platform for Clemson researchers to pursue MSI experiments in their respective fields.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Sandhu P, Leonard E, Nandula V, Tharayil N. 2023. Global Metabolome of Palmer Amaranth (Amaranthus palmeri) Populations Highlights the Specificity and Inducibility of Phytochemical Responses to Abiotic Stress. Journal of Agricultural and Food Chemistry. 71(7):3518-3530. DOI: 10.1021/acs.jafc.2c07162
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Koski M, Leonard E, Tharayil N. 2024. Foliar Flavonoids Across an Elevation Gradient: Plasticity in Response to Uv, and Links with Floral Pigmentation Patterning. [Preprint]. DOI: 10.2139/ssrn.4837607.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Havers M, Corkidi L, Leonard E, Palmer C, Tharayil N, Bethke J. 2024. Dynamic Behavior of Systemic Insecticide Residues in Snapdragons. HortScience. 59(6):806-816. DOI: 10.21273/HORTSCI17725-24
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Xia M, McCormack M, Suseela V, Kennedy P, Tharayil N. 2024. Formations of mycorrhizal symbiosis alter the phenolic heteropolymers in roots and leaves of four temperate woody species. New Phytologist. DOI: 10.1111/nph.19731
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2024
Citation:
Jatana B, Kitchens C, Ray C, Gerard P., Tharayi N. Chemical forms of nitrogen fertilization influence the content and composition of aroma volatiles and phytonutrients in strawberry fruits. Journal of Agricultural and Food Chemistry. In Revision.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Kumar R, Tharayil N. Copy number variation facilitated metabolic adaptation introduce robust physiological response of Palmer Amaranth to glyphosate. Plant Physiology
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Sandhu P, Kumar R., Tharayil N. Multi-omics analysis of glyphosate-induced response in Palmer amaranth (Amaranthus palmeri) highlights induced upregulation of stress response systems in herbicide-resistant biotypes.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Xia M, Singh D, Suseela V, Tharayil N Persistent effects of plant litter sources shaping molecular fingerprints of dissolved organic matter from decomposing litter concurrent with emergence of universal molecular structures.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Kaur G, Kumar R, Leonard E, Tharayil N. Stress Resilience in WeedsThrough Physiological Priming- A Case Study Using Palmer Amaranth," Weed Science Society ofAmerica, San Antonio Texas, San Antonio, TX, United States. (January 2024
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Tamura M, Tharayil N. A hybrid analytical approach for characterizing plant and microbial components in the extractable fraction of soil organic matter. Ecological Society of America Annual Meeting. Long Beach California. August 4-8, 2024
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Xia M, Suseela V, Tharayil N. Contrasting production and chemical signatures of dissolved organic matter from the decomposition of leaf litter and fine roots. Ecological Society of America Annual Meeting. Long Beach California. August 4-8, 2024
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Singh D, Xia M, Suseela, V Tharayil N. The optical characteristics of dissolved organic matter vary with litter identity and decomposition stage. Ecological Society of America Annual Meeting. Long Beach California. August 4-8, 2024
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Zhang Z, Suseela V, Tharayil N. Uptake of nitrogen from the protein-tannin complex by ectomycorrhizal fungi is independent of saprotrophic association. Functional Ecology
- Type:
Journal Articles
Status:
Submitted
Year Published:
2024
Citation:
Edayilam N, McKenna AM, Chen H, Powell BA, Tharayil N. lMolecular characteristic of dissolved organic matter is linked to the identity of plants, litter decomposition stage and differentially influences metal mobilization from minerals. Environmental Science Technology.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Tamura M, Tharayil N. Building Partnerships for Climate-Smart Commodities in South Carolina" AnnualMeeting, "Deciphering the chemical composition of the extractable fraction of soil carbon: Methodoptimization for soil biomarkers," Clemson University, Clemson, SC, United States. (May 2024).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Singh D, Xia M, Tharayil N.Building Partnerships for Climate-Smart Commodities in South Carolina" AnnualMeeting, "Deciphering the role of dissolved organic carbon in soil carbon storage in agricultural systems,"Clemson, SC, United States. (May 2024)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Tharayil N. , Appukuttan Suseela, V. , Clemson PostdoctoralSymposium 2024, "Proteomic and metabolomic landscape of the arbuscular mycorrhizal symbiosis inmaize roots," CU-VPR, Clemson, SC, United States
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2024
Citation:
Kumar R, Tharayil N. Metabolomics for deciphering cellular responses of gene dosage variation. Metabolomics Association of North America Conference. Florida October 2024.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2024
Citation:
Kaur G, Kumar R, Leonard E, Tharayil N. Metabolomics as a complementary conduit to elucidate the mechanisms of priming-mediated stress-memory in plants. Metabolomics Association of North America Conference. Florida October 2024.
|
Progress 07/01/22 to 06/30/23
Outputs
Target Audience:Since its installation, the mass spectrometer has served ten research projects across five research groups spanning agriculture, plant biology, animal sciences, and physiology. Apart from research groups at Clemson, the instrument met the analytical needs of researchers at UC Davis and USDA-ARS. These projects include the biotransformation of pesticides in plants, elucidating the pigment chemistry in petals, defining the metabolomic landscape of plant-mycorrhizal symbiosis,and monitoring biomarkers for skeletal muscle degradation in cattle. The results from these studies were published in three peer-reviewed journals and seven presentations at national conferences. Changes/Problems:Due to a weather-related flooding event that occurred in Dec 2022, the lab was closed for two months. Though the lab was partly functional after that, the instruments were not operational for another six months due to the dust generated as the repairs were underway. So, in total, we lost 8 months during 2022-23, which significantly affected the training and usage of the instrument. What opportunities for training and professional development has the project provided?Across different projects, 15 graduate students were trained on the theory and practical application of the instrument and associated techniques. The students were offered hands-on trainingand the opportunity to do independent research projects using the instrumentation. Six undergraduate students were trained on various aspects of sample preparation for the instrument analysis. How have the results been disseminated to communities of interest?The results from these studies were published in three peer-reviewed journals and seven presentations at national conferences. The instrumentation was incorporated into the graduate curriculum, and training opportunities were provided. The uniqueness of the instrument was showcased to sixnew faculty hires during their job interviews. The instrument served ten research projects across five research groups spanning agriculture, plant biology, animal sciences, and physiology. Apart from research groups at Clemson, the instrument met the analytical needs of researchers at UC Davis and USDA-ARS. What do you plan to do during the next reporting period to accomplish the goals?Along with completingthe trainingand quantitative analysis, we willfocus more on the technical contribution to the field by developing mass spectral libraries and acquiring imaging functionality for this instrumentation.
Impacts
What was accomplished under these goals?
The outcome of the key studies enabled by the instrumentation are summarized below: 1) Quantifying lignin abundance and composition in fine roots across temperate trees: a comparison of analytical methods. Across ecological studies, lignin abundance has been characterized using both proximate and lignin-specific method. Although the proximate acid-insoluble fraction (AIF) has been used to infer root lignin and decomposition, we showed that AIF-defined lignin content was disconnected from the lignin abundance estimated by techniques. In this reporting period, using the isolated lignin standard, lignin-specific techniques estimated a lignin content of 2 to 10 % (w/w) in roots, whereas AIF-defined lignin contents were c. 5-10 fold higher, in the range of 21-50%. The interspecific variation of AIF lignin was also found to be largely unrelated to that determined using lignin-specific techniques. Furthermore, we showed that high-throughput pyrolysis-gas chromatography-mass spectrometry, when combined with quantitative modeling, accurately predicted lignin content and composition, highlighting its feasibility for quicker assessment of lignin in roots. This study demonstrates that AIF should be interpreted separately from lignin in fine roots as its abundance is unrelated to the content of lignin polymers and provides the basis for informed decision-making with respect to lignin methodology in ecology. This study has been published in New Phytologist during this reporting period (doi: 10.1111/nph.18515). 2) Common and lifestyle-specific traits of mycorrhiza-associated metabolite alterations in plant roots reflects strategies of root-mycorrhizal interactions. Mycorrhizas are widespread belowground symbioses formed between plant roots and soil fungi. This plant-fungus partnership impacts terrestrial ecosystems by mediating plant performance and biogeochemical processes. The influence of mycorrhizas on plant and ecosystem functioning is ultimately driven by the biological processes that regulate plant-mycorrhizal interactions. Although convergent patterns in morphological and genetic traits of mycorrhizas have been well-documented and reflect key selection forces that shape the biology of mycorrhizas, generalizable traits of mycorrhizal-associated chemical changes in roots, which are more intimately linked to plant and ecosystem functioning, remained unexplored. Therefore, we compared mycorrhizal-associated plant metabolome alterations across multiple plant-mycorrhizal combinations forming either arbuscular mycorrhizas or ectomycorrhizas. We have described the greenhouse experiment and laboratory analysis in the previous report period. In addition, we added in silico class-level compound annotation, in order to maximize the coverage of annotation of mass features and provide a relatively unbiased way to assess the changes in root metabolomes in response to mycorrhizal colonization. Our result showed that mycorrhizas induced significant alterations in the root metabolome. Comparing across multiple plant-mycorrhizal combinations, our data revealed metabolite changes unique to mycorrhizal lifestyle and those common across plant-mycorrhizal associations irrespective of lifestyles. Roots colonized by arbuscular mycorrhizal and ectomycorrhizal fungi accumulated different sets of carbohydrates, reflecting unique carbon allocation strategies for mycorrhizas. Particularly, arbuscular mycorrhizal roots accumulated cyclic polyols inaccessible for their fungal partners, suggesting tight regulation by plants in carbon partitioning. By contrast, such accumulation was not observed in ectomycorrhizal-colonized roots. Mycorrhizas also altered specialized metabolism in roots, featuring frequent increases of flavan-3-ols but decreases in flavanols irrespective of mycorrhizal lifestyles, suggesting tactical reconfiguration of specialized metabolites to both facilitate and contain symbiosis. These characteristic metabolite alterations were largely root-specific and did not mirror in leaves. The study has been published in the Journal of EcologyDOI: 10.1111/1365-2745.14049. 3) The mechanism of rhizobiome-mediated drought resilience in crop plants. Climate change-induced drought stress is poised to threaten agricultural productivity and food security worldwide. Although plant adaptations to drought stress are often driven by physiological acclimation, recent research has revealed that beneficial microbial community that inhabits the root-rhizosphere (rhizobiome) play a significant role in imparting abiotic stress resilience in ruderal plants that thrive in marginal lands. However, despite numerous attempts, rhizobiome-mediated plant stress adaptations rarely translate from laboratory and greenhouse settings to the field conditions. This inconsistency in the outcome is mainly due to the complexity of plant-rhizobiome interactions, and due to a lack of understanding of the mechanisms of the drought resilience facilitated by rhizobiome. Using the triple quadrupole platform, we investigated the alterations in phytohormones in plants in the presence of rhizobiome. A novel approach was developed that simultaneously screened the plant extract for 108 phytohormones. Our results show that the drought resilience by rhizobiome was partly imparted by changes in phytohormone profile, especially the upregulation of auxins that resulted in fine root branching, thus as the efficient foraging of soil for water and by upregulation of salicylic acid, which stimulated stomatal closure, thus conserving the water.The study has been published in The Plant JournalI: 10.1111/tpj.15775.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Xia M, Suseela V, McCormack M, Kennedy P, Tharayil N. 2023. Common and lifestyle specific traits of mycorrhizal root metabolome reflect ecological strategies of plant-mycorrhizal interactions. Journal of Ecology. 111(3):601-616. DOI:10.1111/1365-2745.14049
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Kumar R. Tharayil N. Time-course metabolome variation in genetically diverse inbred lines identify pathways underlying staygreen trait in maize. 65th Annual Maize Genetics Meeting. St. Louis MO. March 16, 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Xia M, Suseela V, Tharayil N. Compound-specific chemistry in plant roots as a functional trait to understand plantmycorrhizal interactions and the associated ecological consequence. 8th International Symposium on Physiological Processes in Roots of Woody Plants.Penn State University
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Sandhu P, Tharayil N. An Integrated Multi-Omics Approach to Capture Stress-Induced Cellular Physiology in Plants: A Case Study using Glyphosate-Resistance in Palmer Amaranth (Amaranthus palmeri). Weed Science Society of America Annual Meeting.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Sandhu P, Tharayil N. Mapping the cellular physiology of glyphosate resistance in Palmer amaranth using global metabolomic approaches. Metabolomics Association of North America conference. October 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Jatana B, Kitchens C, Ray C, Gerard P, Tharayil N. Global Metabolomics reveal Nitrogen Fertilization as a Robust tool to Enhance Phytonutrient Content in Strawberry Fruits. Metabolomics Association of North America conference. October2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Kaur S, Tharayil N, Suseela v. Global effects of arbuscular mycorrhizal and ectomycorrhizal associations on root metabolomes feature alterations on carbon partitioning and flavonoid profiles. Metabolomics Association of North America conference. October 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Sandhu P, Tharayil N. Exploring dose-response in glyphosate-resistant and -susceptible Palmeri amaranth by UHPLCHRMS based untargeted metabolomics. Metabolomics Association of North America conference. October 2021.
|
Progress 07/01/21 to 06/30/22
Outputs
Target Audience:Research facilitated by the instrument aquisition was presented at national scientific meetings and department seminars. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctoral scholar and one lab technician was trained on various aspect of instrument evaluation and the genereration of the associated report. Twelve grad students were trained on the basic aspect of the instrument operation. How have the results been disseminated to communities of interest?The instrument was included as part of the graduate level course offered by the PI. The results obtained from instrument analysis were presented at meetings and seminars. What do you plan to do during the next reporting period to accomplish the goals?Facilitate the analysis of diverse research project on the new instrumentation. Actively pursue funding opportunities for the purchase of an imaging interface for the new mass spectrometer to enable targeted imaging mass spectrometry at Clemson University.
Impacts
What was accomplished under these goals?
A blinded sample analysis was conducted to identify the instrument with highest sensitivity and repeatability of analysis in complex biological matrices. INTRODUCTION: The purpose of the blinded sample analysis is to evaluate the performance of various ultra-high pressure liquid chromatography (UHPLC)-triple quadrupole (QqQ) mass spectrometers for their suitability to enhance the research infrastructure at of Multi-User Analytical Lab (MUAL) at Clemson University. This procurement aims to increase the sensitivity and robustness of targeted small molecule analysis that is currently done using a Shimadzu 8040 triple quadrupole mass spectrometer. A blind analysis was set up to evaluate the capability of UHPLC-QqQ instruments manufactured by different vendors. Irrespective of the cost of the instrument, all participating vendors were instructed to use the triple-quadrupole instrument of the highest sensitivity of this analysis. Since the instrument will be primarily used for quantitation of small molecules in diverse biological/environmental matrices, the blind analysis was formulated to capture the following pertinent instrument/analyses parameters: 1) Sensitivity: Sensitivity could be broadly defined as the magnitude of the signal produced by the analyte in the MS detector compared to the background noise. 1.1) Instrument detection limit (IDL): IDL is a measure of instrument sensitivity and is amenable to statistical validation, and repeatability of measurement is taken into account. IDL (ppb) = [tα × RSD × the concentration of injected standard (ppb)]/100 1.2) Signal-to-noise ratio (SNR): SNR is the traditional measure of sensitivity but could be biased by the post-data processing methods that are vendor-specific. 2) Matrix effect Matrix effect could be broadly described as the difference in signal intensity produced by the same analyte concentration in a clean solvent matrix compared to when present in a complex sample matrix. The matrix can result in an enhancement or suppression of the analyte signal depending on whether it facilitates or inhibits the ionization of the analyte. 3) Precision Precision could be broadly defined as the repeatability of measurement and is calculated as the relative standard deviation (RSD) of at least six replicate analyses of the same sample. 4) Ion transmission and fragmentations Measurement of the ion flux in the mass spectrometer and propensity of in-source fragmentation. ANALYSIS SETUP Matrix selection: The instrument response of ten compounds was tested in two matrices: i) clean acetonitrile matrix (simpler matrix) ii) extracted pollen matrix. This represents a relatively complex matrix with significant interferences from lipids and pigments. The pollen extract was prepared and partly cleaned using amodified QuEChERS method Analyte Selection: The following analytes ( Cortisol, Azithromycin, 13C4,15N-Thiamethoxam, 13C4-Clothianidin, D4-Imidacloprid, D5-Flupyradifurone, D3-Cyantraniliprole, Carbamazepine, Sulfamethoxazole, Ergotamine, Erythromycin, 13C5-Dinotefuran, Quercetrin, Isoquercitrin) were chosen based on their differential polarity that would result in varied retention times, the propensity of ionization at ESI that would test the instrument sensitivity, and the compounds that were routinely encountered in the samples submitted to MUAL (e.g., antibiotics, pesticides, plant metabolites etc.). There were four concentrations of the spike (high [20 ppb], medium [1 ppb], low [0.1 ppb], and ultra-low [20 ppt]) to evaluate the sensitivity of the instruments.Cortisol and azithromycin had the lowest instrument sensitivity based on the initial screening done at MUAL, and hence were spiked at higher concentrations relative to the other eight compounds. The spikes were done in both clean acetonitrile and extracted pollen matrix. EXECUTION and DATA ANALYSIS: The stability of the selected compounds in the matrix was tested for a two-week time period at MUAL before finalizing the compounds and concentrations for the spiking. Specific information was provided to the vendors as part of the bid process to execute this blind analysis (Appendix 1). Each spike concentration was injected eight times (ultra-low was injected three times) to enable the statistical analysis of the results. All data were collected with a specified LC method with predefined solvent gradient and injection volume in positive ionization mode. Including the two matrices, four concentrations, associated blanks and 3 to 8 replicate injections there were total of 69 analyses, and the order of execution of the analysis was specified. Method details and the requested order of analysis are in Appendix-1. The identity of the samples (acetonitrile/pollen) and the concentration of the analyte in each sample was withheld from the vendors to facilitate blind analysis. The unprocessed instrument data files of the 69 analyses were submitted by the vendor to MUAL. The raw data from all vendors were processed using Skyline by MUAL staff to derive the parameters that are being evaluated. DISCUSSION: SENSITIVITY: Within the sensitivity parameter, Vendors A, B & C maintained a lower instrument detection limit (IDL), mostly in <200 per trillion (ppt) concentration. Analyte A2 had a higher IDL across all vendors since it formed doubly charged ions, and the precursors in MRMs were different for different vendors (Fig. 1). With respect to SNR, vendor-A was superior in medium-spike concentration across all analytes, except for A2 (Fig. 2). The sensitivity of vendor-A was more evident in the analysis of ultra-low spike concentration (Fig. 3), where vendor-A had acceptable SNR (>3) for seven out of the 10 compounds, of which five analytes had acceptable RSD (Fig. 7). Vendor-B had three analytes with acceptable SNR and RSD, whereas none of the other two instruments had any analyte with SNR of >3 (Fig. 3). The signal-to-noise ratio of the analyte spike (medium-concentration) in pollen matrix was robust only for vendor-A (9 out of 10 compounds); all the other vendors lost at least five compounds due to poor SNR in the pollen matrix. MATRIX EFFECT: Significant matrix effect was observed across all instruments for all analytes (Fig. 6). For all analytes, the matrix effect resulted in suppression of analyte ions, except for A2 where significant ion enhancement was observed. PRECISION: Precision of analysis was matrix dependent with a lower RSD in acetonitrile matrix, and a higher RSD in pollen extracts for the medium-concentration spike. Out of the ten analytes, vendor-A had a better precision of measurement for nine analytes irrespective of the sample matrix, and in all cases, this was <10% in both matrices (Fig. 7 and Fig. 8). ION TRANSMISSION AND FRAGMENTATIONS: The ion intensities of all the MRMs and the Q1 scan showed significantly higher ion transmission and higher SNR for vendor-A, which is a desirable characteristic (Fig. 9). For Q1 scan, ion intensities were 100 times higher than the other two vendor instruments. As assessed by the presence of flavonoid aglycone (quercetin) compared to their glycosylated forms (quercetin and isoquercitrin), the amount of in-source fragmentation was 30-50% higher for vendor-A compared to vendor-B. Higher insource fragmentation is less desirable, but its negative effect on sensitivity could be compensated for higher ion intensities. CONCLUSION: The four vendors that participated in the bid combinedly holds more than 75% of the triple quadrupole market and used their elite instrument for this analysis. Thus, this blind analysis compared the top performing instruments currently available on the market. The instrument from vendor-A was the most robust performing across all the four parameters tested. This instrument was purchased, installed and users were trained.
Publications
- Type:
Other
Status:
Other
Year Published:
2022
Citation:
Huynh, K, Leonard, EL, Tharayil, N. 2021. Evaluation Report of Blind Sample analysis. Associated with the bid process for the procurement of an Ultra-
High Performance Liquid Chromatography (UHPLC) Triple
Quadrupole (QqQ) Mass Spectrometer. Submitted to Clemosn University
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Zhang, Z., Jatana, B., Campbell, B., Gill, J., Vidya, S., Tharayil, N. 2022. Cross inoculation of rhizobiome from a congeneric ruderal plant imparts drought tolerance in maize (Zea mays) through changes in root morphology and proteome. Plant Journal. https://doi.org/10.1111/tpj.15775
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Xia, M., Valverde-Barrantes, O., Suseela, V., Blackwood, C., Tharayil, N.2022.Characterizing natural variability of lignin in tree roots: a comparison of analytical methods and assessment of high-throughput techniques for large-scale screening. New Phytologist (https://doi.org/10.1111/nph.18515)
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
Xia, M., Valverde-Barrantes, O., Suseela, V., Blackwood, C., Tharayil, N. 2022. Common and lifestyle-specific traits of mycorrhizal-associated metabolome alterations in plant roots reflects strategies of plant-mycorrhizal interactions. BioRxiv https://doi.org/10.1101/2022.01.27.478019 (In press-Journal of Ecology).
|