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Taller Nacional de Maiz Forrajero, 1er; Guanajuato (Mexico); 23 enero 2013
Citation preview
Supervisor: Dr. Axel TiessenLab Metabolomics & Molecular Physiology
Irapuato, Gto., January 23, 2013
1st. National Workshop on Fodder Maize
MC Martín García Flores
“Metabolic phenotyping of maize stem extracts
using DIESI-MS”
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
http://www.siap.gob.mx/ 22/01/2013
2012 Year Forage production
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
JAL
ISC
O
ME
XIC
O
DU
RA
NG
O
AG
UA
SCA
LIE
NT
ES
QU
ER
ET
AR
O
CO
AH
UIL
A
CH
IHU
AH
UA
GU
AN
AJU
AT
O
PU
EB
LA
ZA
CA
TE
CA
S
State
To
n
Average Yield
http://www.siap.gob.mx/ 22/01/2013
2012 year. Yield maize
40
303135
4044
4851
6162
0
10
20
30
40
50
60
70
QU
ERET
ARO
BAJA
CAL
IFO
RN
IASU
R
AGU
ASC
ALIE
NTE
S
GU
ANAJ
UAT
O
HID
ALG
O
MEX
ICO
GU
ERR
ERO
OAX
ACA
DU
RAN
GO
CO
AHU
ILA
State
Ton/
ha
Phenotype dilemma
Genotype
Phenotype
Environment
DNA
BiomassGrain Yield
Agronomy
Tiessen, 2009
Genotype
• Genetic information• DNA (Nuclear. Mit, Chl)
• Sequence (Base order)
• Unique identity• Variety, Species, Genus
Epi-genotypeHeredable modificationsMethylationHistone-AcetylationmicroRNAs, etcProtein-states, etc
Phenotype
• Measurable characteristics• Agronomic traits• Morphological traits• Physiological traits• Biochemical traits• Depends on both Gen,Env, and Gen x Env
Phenotype dilemma
Genes
Proteins
Metabolites
Genotype
Phenotype
Environment
RNA
DNA
Activity
BiomassGrain Yield
Translation
Transcription
GrowthDevelopmentPartitioning
Epigenetic modifications
Photosynthesis
Signal transduction
Physiology
Agronomy
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)
• DIESI-MS (80 sa/day)
• Enzymatic and colorimetric assays (384 sa/day)
High through-put.Sample pre-treatment.Omitted fractioning and separation.Reduction of analysis time.Minimizes influence of sample manipulation.Brings down costs.
Typical workflow in Metabolomics
Koek et al., 2011
Biological questionsWhat is the function of the stem in the maize plant?
Which metabolites change under different field conditions?
Experimental design (Lattice)
Texcoco: 11 Genotypes, 6 biological samples, 3 technical samples (Low Nitrogen and Normal Nitrogen)
Tlaltizapan:14 Genotypes, 6 biological samples, 3 technical samples (Water stress and Well watered)
Genetic materialCIMMYT; experimental fields
Texcoco: Low Nitrogen Tlaltizapan: Water Stress
Entry Pedigree
1 H-55
6 HID-15
8 CMT 099001 (CML457/CHWE235)//CHWE231
4 P1684
*9 CMT 099003 (CML457/CHWL147)//CHWE229 (NN)
7 (CML457/CML459)//IML-6
5 ASPROS-823
2 H-57
11 TESTIGO LOCAL#!
10 CMT 099027 (CML457/CHWE235)//CHWE233
*3 CV-702 (LN)
Entry Pedigree
*8 DTPYC9-F143-5-4-1-2-B*5/CML312SR (WW and WS)
20 DTPWC9-F24-2-3-1-1-B*5/CML312SR
26 CML323-BB/CML312SR
69 LaPostaSeqC7-F78-2-1-1-1-B*4/CML312SR
72 CML486-BB/CML312SR
87 CML311/MBRC3BcF95-2-2-1-B*7/CML312SR
89 LaPostaSeqC7-F96-1-2-1-3-B*4/CML312SR
104[[KILIMAST94A]-30/MSV-03-1-10-B-1-BB-1xP84c1F27-4-1-6-B-5-B]F8-3-2-2-
1xG16SeqC1F47-2-1-2-1-B*5-xP84c1F26-2-2-6-B-3-B]-3-1-B/CML395]-1-1-BB/CML312SR
125CML311xCML311/CML311xMBRC1BcF94-3-1-1-B*4-1-1-
B*7/CML312SR
132CLQ-RCWQ83=(CML146xCML150)-B-32-1-2-B-1-B*4/
CML312SR
143 CL02450Q/CML451QLocalCheckQPM2
147LaPostaSeqC7-F64-2-6-2-2-BB/
CML495=Check2IntermediateMaturity
91 DTPYC9-F69-3-5-1-1-B*4/CML312SR
99 LaPostaSeqC7-F64-2-6-2-2-BB/CML312SR
Sampling
Grounding and collection of extracted juice sample, 1 ml
Experimental field trial
Maize stem sampling
Physiological data recording
Freezing in 96-well microplates, dry ice for 30 s. and storaged in liquid “N”
(-80 °C).
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
Sample preparation
Filter 0.45 µm mesh PVDF; 10 µL sample and 990 µL De-Ionizade H2O (1:100 dilution). Add 25 µL formic acid to 475 µL sample, mix 3 min and Read: DIESI-MS.
Data acquisition
García-Flores et al., 2012
DIESI ConditionsWater 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) 250Cone (L/hr) 50
Syringe.Pump flow (µl/min) 10
Analyzer.LM resolution 15HM resolution 15Ion energy 0.5Multiplier 650
Direct infusion electron spray ionization mass spectrometry (DIESI-MS).
Waters micromass (Z/Q).
Mass spectrometer (m/z).
Mass Spectrometer components
Warwick, 2007
Electrospray ionization
Martín, 2012
Capillary column, small quantities of sample,continuos flow, vacuum interlock. Electro-ionization.
Data processing
TOPPAS Software
Data acquisition and spectrum analysis
Input files
Output files
Output files
Output files
Output files
File Convert
er
Spectra Merger
NoiseFiltersgolay
TextExporter
File Convert
er
Peak Picker
wavelet
Winkler, 2011
Data processing
Average of 6 spectra
Control experiments
Default machine peaks
Top Peak: (m/z) 43.26, Intensity: 217896000
Top Peak: (m/z) 29.62, Intensity: 8164352
Solvent Water
CLQ-RCWQ83=(CML146xCML150)-B-32-1-2-B-1-B*4/CML312SR
Tlaltizapan
Drought stress (SS)
Deinonized Water
Formic acid
DilutionsGreen: 1:100
Red: 1:1000
Blue: 1:10000
Peak discrimination
Aminoacid (M.W.)+H Arginine 175.01
Asparagine 133.00
Glutamine 147.06
Proline 116.06
Serine 106.04
Tryptophan 205.08
Threonine 120.05
Tyrosine 182.07
Leucine 132.17
Metabolites of interest
Lysine
Mass positive: 147.10
C6H14N2O2
Fragments
m/z intensity
147.14 6 600 192
Lysine
LysineLysine
REP 1
REP 2 REP 3
Valine
Valine: (m/z) 118.08
Solvent Peak: (m/z) 43.32
AAs:Most common signals
m/z Histidine Isoleucine Lysine Methionine Valine Serine Threonine Cysteine Tryptophan Phenylalanine
39.451 6.89E+06 5.09E+06 7.55E+06 1.47E+07 4.76E+06 7.01E+06 5.74E+06 7.44E+06 2.10E+06 5.97E+06
47.4482 3.93E+08 3.97E+08 4.83E+08 4.05E+08 4.32E+08 4.92E+08 4.45E+08 4.20E+08 3.74E+08 3.88E+08
48.5103 8.85E+06 6.08E+06 2.22E+07 6.85E+06 1.68E+07 2.18E+07 1.74E+07 1.42E+07 6.08E+06 6.05E+06
59.3195 8.44E+07 3.48E+07 4.29E+07 3.59E+07 3.75E+07 4.59E+07 3.74E+07 3.05E+07 3.13E+07 2.80E+0661.3814 3.13E+07 1.77E+07 2.18E+07 2.23E+07 2.00E+07 2.45E+07 2.12E+07 1.80E+07 1.59E+07 1.59E+0769.1918 6.18E+06 4.37E+06 1.39E+07 1.04E+07 8.09E+06 1.59E+07 8.23E+06 8.84E+06 3.50E+06 4.97E+06
75.1904 3.86E+07 2.27E+08 4.37E+07 1.77E+08 5.75E+07 6.10E+07 1.75E+08 1.50E+08 2.18E+08 1.68E+0889.0626 1.53E+07 1.05E+07 1.11E+07 1.15E+07 1.06E+07 1.29E+07 1.20E+07 1.01E+07 9.94E+06 9.92E+0689.9376 1.52E+06 7.38E+05 1.09E+06 1.63E+06 8.09E+05 1.04E+06 9.51E+05 8.96E+05 6.84E+05 6.83E+0593.0619 5.30E+08 5.98E+08 6.49E+08 6.03E+08 6.14E+08 6.82E+08 6.42E+08 6.27E+08 5.85E+08 5.86E+0894.3742 2.53E+07 3.12E+07 3.86E+07 3.52E+07 3.25E+07 4.27E+07 3.97E+07 3.53E+07 3.07E+07 3.00E+0797.1238 4.59E+06 3.34E+07 4.33E+06 4.98E+07 7.11E+06 8.88E+06 2.97E+07 3.40E+07 2.90E+07 2.83E+07
101.1232 5.28E+07 4.13E+07 3.48E+07 4.55E+07 3.90E+07 4.56E+07 4.55E+07 3.84E+07 3.92E+07 3.71E+07102.873 4.02E+06 2.57E+06 2.70E+06 3.09E+06 2.58E+06 3.09E+06 3.43E+06 2.54E+06 2.73E+06 2.44E+06
105.0602 1.93E+07 1.16E+07 1.10E+07 1.31E+07 1.08E+07 1.04E+07 1.30E+07 1.18E+07 1.15E+07 1.07E+07107.2475 1.19E+07 8.35E+06 9.39E+06 9.74E+06 8.45E+06 1.10E+07 9.67E+06 8.48E+06 8.01E+06 7.38E+06117.1841 6.74E+07 5.23E+07 4.31E+07 6.18E+07 4.40E+07 5.64E+07 5.94E+07 4.86E+07 5.14E+07 4.79E+07121.0588 1.07E+07 1.35E+08 1.03E+07 1.13E+08 2.21E+07 2.02E+07 1.02E+08 9.64E+07 1.40E+08 1.05E+08131.9958 1.70E+06 6.48E+07 1.10E+06 2.60E+06 9.43E+05 1.07E+06 1.02E+06 9.49E+05 8.62E+05 0.00E+00171.0599 1.05E+07 8.64E+06 1.83E+07 1.29E+07 6.67E+06 1.72E+07 1.29E+07 1.22E+07 9.88E+06 7.95E+06191.9376 1.33E+07 2.13E+07 1.50E+07 1.66E+07 2.75E+07 2.59E+07 2.31E+07 1.96E+07 1.82E+07 2.01E+07238.0731 7.07E+06 1.62E+07 1.01E+07 1.35E+07 1.93E+07 1.88E+07 1.86E+07 1.78E+07 1.47E+07 1.64E+07
HigherLower
AAs most common signals Plot
39.4
5
47.4
4
48.5
1
59.3
1
61.3
8
69.1
9
75.1
9
89.0
6
89.9
3
93.0
6
94.3
7
97.1
2
101.
12
102.
87
105.
06
107.
24
117.
18
121.
05
131.
99
171.
05
191.
93
238.
07
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
Intensity
m/z
AAs
AAs Surface Plot
0.00E+00-2.00E+08 2.00E+08-4.00E+08 4.00E+08-6.00E+08 6.00E+08-8.00E+08
PheTryCysThrSerValMetLysIsoHis
C1C2C3C4C5C6C7C8C9C10
0.00E+00
1.00E+08
2.00E+08
3.00E+08
4.00E+08
5.00E+08
6.00E+08
7.00E+08
Int
m/z
AAs
AAs Surface Plot
0.00E+00-1.00E+08 1.00E+08-2.00E+08 2.00E+08-3.00E+083.00E+08-4.00E+08 4.00E+08-5.00E+08 5.00E+08-6.00E+086.00E+08-7.00E+08
PheTryCysThrSerValMetLysIsoHis
Excel 2003
AA’s fragments Summary AA Ionize Intensity FragmentsHistidine Low 56 452 120.08, 121.09, 123.06
Glutamic acid Low 2 749 184 112.88, 114.09, 130.90
Aspartic acid Low 3 822 336 99.03, 101.03, 117.03
Isoleucine Standard 13 760 512 98.09, 99.09, 115.98
Arginine Low 3 046 144 139.97, 140.98, 157.97
Lysine Standard 6 600 192 112.19, 113.16, 130.16
Alanine High-quality 134 176 768 75.50, 87.43, 89.50
Methionine Low 3 736 320 115.91, 116.92, 132.87
Glutamine High-quality 94 728 192 128.83, 129.87, 147.06
Valine Standard 11 805 696 101.07, 116.03, 117.06
Serine High-quality 63 971 328 102.99, 103.99, 104.99
Threonine High-quality 64 405 504 102.96, 117.94, 118.85
Asparagine Low 6 648 576 111.88, 112.88, 115.85
Tyrosine Low 2 569 472 148.92, 162.94, 164.88
Cysteine Standard 19 729 408 101.98, 102.96, 104.90
Proline Standard 13 458 432 100.20, 115.11, 232.15
Glycine High-quality 103 301 120 75.43, 151.06, 226.17
Tryptophan Low 897 216 170.11, 172.09, 188.06
Phenylalanine Low 4 782 336 130.90, 146.92, 162.94
Leucine High-quality 3 208 960 100.17, 117.32, 131.40
Results
García-Flores et al., 2012
Results DIESI-MS spectrum
Comparison
¿Which MS peaks vary according to the environmental conditions?
Comparison of spectra
CLQ-RCWQ83=(CML146xCML150)-B-32-1-2-B-1-B*4/CML312SR
Tlaltizapan
Control (WW)
Drought stress (SS)
CLQ-RCWQ83=(CML146xCML150)-B-32-1-2-B-1-B*4/CML312SR
Tlaltizapan
Control (WW)
Drought stress (SS)
Control-Low nitrogen spectra
Control (NN)
CV-702 Batan
Low Nitrogen
CV-702 HL Batan
Control (NN)
Low Nitrogen
Can we use the MS info for classifying samples?
Statistical analysis with R
ANOVA analysisHerarchical clusteringHeatmap bicluster
Metabolic HeatMap
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 and fastest MS strategy. We have successfully set up the method at CINVESTAV.
We can detect >200 of different ms peaks. We can measure more that 80 samples per day.
Some peaks vary acording to Gen and Env effects
Biochemical phenotyping of maize stem fluids enables the rapid evaluation of the physiological state of plants.
It also allows to discriminate between genotypes ( breeding)
Metabolic heatmaps are useful for MS data representation
Perspectives
The method will be applied at large scale for investigating the metabolic stress response of various Zea mays L. genotypes.
We hope we can detect biomarkers for selection
Derived metabolic markers can complement the DNA based 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
LaboratoryMetabolomics & Molecular Physiology
Questions?Many thanks for
being here.
CINVESTAV IRAPUATO
Plant Biotechnology
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