Metabolic phenotyping of maize stem extracts using DIESI-MS
1st. National Workshop on Fodder Maize“Metabolic phenotyping of maize stem extracts using DIESI-MS” MC Martín García Flores Supervisor: Dr. Axel Tiessen Lab Metabolomics & Molecular Physiology Irapuato, Gto., January 23, 2013
Outline• Introduction – Forage maize – Genotype-Phenotype dilemma• Experimental strategy and Methods – Workflow Metabolomics – Harvesting and sample prep – Mass spectrometry (DIESI-MS)• Control experiments• Results – Spectral comparisons, heatmaps
Forage production 2012 Year Forage production 1600000 1400000 1200000 1000000Ton 800000 600000 400000 200000 0 QUERETARO AGUASCALIENTES PUEBLA MEXICO GUANAJUATO ZACATECAS CHIHUAHUA DURANGO COAHUILA JALISCO State http://www.siap.gob.mx/ 22/01/2013
Average Yield 2012 year. Yield maize 70 62 61 60 51 48 50 44 40 40 40 35Ton/ha 31 30 30 20 10 0 MEXICO GUERRERO HIDALGO DURANGO QUERETARO COAHUILA OAXACA GUANAJUATO AGUASCALIENTES BAJA CALIFORNIA SUR State http://www.siap.gob.mx/ 22/01/2013
Phenotype• Measurable characteristics• Agronomic traits• Morphological traits• Physiological traits• Biochemical traits• Depends on both Gen,Env, and Gen x Env
Phenotype dilemmaGenotype DNA Genes Epigenetic Transcription modifications Signal RNA transduction Environment Translation Agronomy Proteins Photosynthesis ActivityPhenotype Growth Development Partitioning Metabolites Biomass Physiology Grain Yield Tiessen, 2009
Metabolomics• Characterization, identification and quantification of all metabolites • Functional genomics (function of genes) • Physiology • Phenotyping, Breeding • QTL analysis • Metabolic Engineering
ThroughputAnalytic methods:• HPLC-DAD (20 sa/day)• GC-FID (30 sa/day)• GC-EI-MS (18 sa/day) High through-put. Sample pre-treatment.• DIESI-MS (80 sa/day) Omitted fractioning and separation. Reduction of analysis time. Minimizes influence of sample manipulation. Brings down costs.• Enzymatic and colorimetric assays (384 sa/day)
Typical workflow in Metabolomics Koek et al., 2011
Biological questionsWhat is the function of the stem inthe maize plant?Which metabolites change underdifferent field conditions?Experimental design (Lattice)Texcoco: 11 Genotypes, 6 biological samples, 3 technicalsamples (Low Nitrogen and Normal Nitrogen)Tlaltizapan:14 Genotypes, 6 biological samples, 3technical samples (Water stress and Well watered)
Sampling Experimental field trial Maize stem sampling Physiological data recording Grounding and collection of extracted juice sample, 1 ml Freezing in 96-well microplates, dry ice for 30 s. and storaged in liquid “N” (-80 °C).
Sample preparationFilter 0.45 µm mesh PVDF; 10 µL sample and990 µL De-Ionizade H2O (1:100 dilution).Add 25 µL formic acid to 475 µL sample, mix 3min and Read: DIESI-MS. Defreezing in ice bath for 1 hr Centrifugate for 10 min, 4000 rpm, 4 °C. Supernatant filtration: 0.45µm mesh PVDF, activated carbon treatment, 100 µL aliquotes in 96-well microplates Freezing (-18 °C) before DIESI-MS García-Flores et al., 2012
Data acquisition DIESI Conditions Water micromass Q/Z spectrometer ES (+) source. Voltages: Capillary (Kv) 3.0 Temperatures. Cone (V) 60 source ·C 80 Extractor (V) 3 desolvation ·C 150 Rf lens (V) 0.5 Gas flow. Desolvation (L/hr) 250 Cone (L/hr) 50 Syringe. Pump flow (µl/min) 10 Analyzer. LM resolution 15 HM resolution 15 Ion energy 0.5 Multiplier 650 García-Flores et al., 2012
Direct infusion electron spray ionization mass spectrometry (DIESI-MS). Waters micromass (Z/Q). Mass spectrometer (m/z).
Evaluating the physiological state of maize (Zea mays L.) plants by direct-injection electrospray mass spectrometry (DIESI-MS).Martín García Flores; Sheila Juárez Colunga; Josaphat Miguel Montero Vargas; Janet Ana Isabel López Arciniéga; Alicia Chagoya; Axel Tiessen and Robert Winkler. Molecular Biosystems García-Flores et al., 2012
ConclusionsDIESI-MS has high throughput. It is the cheapest andfastest MS strategy. We have successfully set up themethod at CINVESTAV.We can detect >200 of different ms peaks. We canmeasure more that 80 samples per day.Some peaks vary acording to Gen and Env effectsBiochemical phenotyping of maize stem fluids enablesthe rapid evaluation of the physiological state of plants.It also allows to discriminate between genotypes (breeding)Metabolic heatmaps are useful for MS datarepresentation
PerspectivesThe method will be applied at large scale forinvestigating the metabolic stress response of variousZea mays L. genotypes.We hope we can detect biomarkers for selectionDerived metabolic markers can complement the DNAbased markers for breeding.
Acknowledgments• CIMMYT MasAgro-IMIC – Dr. Marc Rojas (IMIC) – Dr. Felix San Vicente (CIMMYT)• CONACYT Grants• Dr. Axel and Dr. Robert• Laboratory team: Mayela, Andrés, Adrián, Erandi, Sheila, Obed, Iván, Julio, Viviana, Daniel, etc