1 Plant Biology Division Current and Future Directions of LC/MS Current and Future Directions of LC/MS Based Metabolomics of the Model legume Based Metabolomics of the Model legume Medicago truncatula Medicago truncatula Mohamed A. Farag, Corey D. Broeckling, David V. Huhman, Ewa Urbanczyk-Wochniak, Zhentian Lei, Bonnie S. Watson, Satish Nagaraj, and Lloyd W. Sumner Marina Naoumkina, Bettina Deavours, and Richard A. Dixon Plant Biology Division Biochemistry is a Dynamic Multi Biochemistry is a Dynamic Multi- level level Reaction Network Reaction Network mRNA υ 1 Active Protein υ 3 υ 4 υ 2 Polypeptde Protein degradation υ 10 Metabolite B Metabolite A Protein Sorting υ 8 υ 5 υ 7 Protein-Protein Interaction υ 9 υ 6 Transcriptome Proteome Metabolome genome PTMs
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1
Plant Biology Division
Current and Future Directions of LC/MS Current and Future Directions of LC/MS Based Metabolomics of the Model legume Based Metabolomics of the Model legume
Medicago truncatulaMedicago truncatula
Mohamed A. Farag, Corey D. Broeckling, David V. Huhman, Ewa Urbanczyk-Wochniak, Zhentian Lei, Bonnie S. Watson, Satish
Nagaraj, and Lloyd W. Sumner Marina Naoumkina, Bettina Deavours, and Richard A. Dixon
Plant Biology Division
Biochemistry is a Dynamic MultiBiochemistry is a Dynamic Multi--level level Reaction NetworkReaction Network
mRNAυ1 Active
Protein
υ3 υ4υ2Polypeptde
Proteindegradation
υ10 Metabolite BMetabolite A
Protein Sorting
υ8υ5
υ7
Protein-Protein Interaction
υ9υ6
Transcriptome
Proteome
Metabolome
genome
PTMs
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Plant Biology Division
Proteome Metabolome
Alter Gene Expression
Integrated Functional Genomics & Systems BiologyIntegrated Functional Genomics & Systems BiologyTranscriptomeGenome
An integrated approach to functional genomics and An integrated approach to functional genomics and bioinformatics in a model legumebioinformatics in a model legume
• Sample collection at 21 time points post elicitation
• Triplicate controls & elicited• 640 total samples• Generate bioinformatic tools to
determine causal relationships between genes, proteins, and natural products
• Isoflavonoid pathway serves as internal control and validation
HPLC/PDA/MS/MS is a Highly SpecificHPLC/PDA/MS/MS is a Highly SpecificMultidimensional Metabolic Profiling ToolMultidimensional Metabolic Profiling Tool
•HPLC retention time•On-line photodiode array spectra for
compounds with chromophores•On-line mass selective detection•On-line tandem and MSn capabilities
Basic peroxidase cDNA isolated from soybean hypocotyls infected with Phytophthora sojae f.sp. glycines
Identified in elicited cell culture and fungal infected plant
TC 56738, pI=8.9
TC 50982& 50994
Gene Discovery -Peroxidases elicited with Yeast
0-48hr Control 0-48hr Elicited
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Plant Biology Division
OH
O
OH
HOO
O
HO
OHperoxidaseFunctional analysis of peroxidases in E. coli
Isoliquiritigenin (IL) Hispidol (H)
5 10 15 20 25 30 35 Time [min]
TC56738
H
nm240 260 280 300 320 340 360 380
DAD1, 32.542 (212 mAU,Bln) of MF000026.D
TC50994
nm250 275 300 325 350 375 400 425
DAD1, 33.944 (227 mAU,Apx) of MF000025.D
IL
IL
pET-28a vector
H
H
TC50982
IL
161.6
269.3
32.3min
100 120 140 160 180 200 220 240 260 280
253.3-MS, 33.8min (#1210)
100 120 140 160 180 200 220 240 260 m/z
5 10 15 20 25 30 35 Time [min]
5 10 15 20 25 30 35 40 Time [min]
5 10 15 20 25 30 35 Time [min]
325 nm
50x
45x
OH
O
OO
M+
M+390 nm
Quinol vinyl ether
40x
Plant Biology Division
Need for Increased Metabolomics Depth of Coverage Need for Increased Metabolomics Depth of Coverage
● Current metabolome estimates Bacteria ~ 600 to 900 metabolites Single plant species ~ 15,000 metabolites Plant Kingdom ~200,000 due to secondary metabolism
● Currently profiling approx 600 components by GC/MS + 150 by LC/MS = 750 total
● Therefore, there is a dramatic need for more comprehensive metabolome coverage
● Increased depth of coverage can be achieved by increased resolution in both the chromatography and mass domains
Bino et al, Trends in Plant Science, 9(9): 418-425.
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Plant Biology Division
Separation Efficiencies & Peak CapacitiesSeparation Efficiencies & Peak Capacities● Separation efficiency – unitless measure of band
broadening per unit time
● Peak capacity - maximum number of theoretical peaks resolvable by the chromatographic system
( ) p
RRR
dLR
wtttN =
−=⎟⎟
⎠
⎞⎜⎜⎝
⎛=⎟
⎟⎠
⎞⎜⎜⎝
⎛=⎟⎟
⎠
⎞⎜⎜⎝
⎛= 2
22
½
'2
b
'2'
11654.5
W16
ασ
1ln4 1
2 +⎟⎟⎠
⎞⎜⎜⎝
⎛=
tt
RNn
Plant Biology Division
Need for Increased Resolving Power (M/Need for Increased Resolving Power (M/∆∆M) M)
Increased Comprehensive Coverage using Ultra Increased Comprehensive Coverage using Ultra performance liquid chromatography (UPLC) performance liquid chromatography (UPLC) QtofMSQtofMS
↑ resolution/plate #↑ Peak capacity↑ metabolomics
potential
UPLC-QtofMS base-peak ion chromatograms obtained for the combined methanol extracts from soybean and Medicago truncatula (cv Jemalong A17). Separations were achieved using a Waters Acquity UPLC 2.1 x 100 mm, BEH C18 column with 1.7 µm particles, a flow of 600 µl/min, and a linear gradient of 0.1%acetic acid:acetonitrile (5:95 to 30:70 over 30min). Mass spectra were collected on a Waters QTOFMS Premier.
Plant Biology Division
500,000UPLC (2.1 x 100 mm @ 600 µL/min)
225,000Capillary HPLC (300 µm x 150 mm @ 4 µL/min)
87,000Analytical HPLC (4.6 x 250mm @ 0.8mL/min)
Ave Efficiency (plates per meter)
HPLC system
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Plant Biology Division
O
O
OCH2OH
HOHO O
O
CH2OH
CO
O
O CH2OH
OH
CH2OH
HOHO OH
COOH
CO
HO CH2OH
OH
HO CH2OH
O
O
HO
COOH
HOHO O
O CH2OH
OHOHHO
O CH2OH
OHOHHO
O
OOH
HO OH
OCH2OH
HO
HO O
O
OH
HOOH
CH2OH
COOH
HO
HO
CH2OH
COOH
HO COOH
COOH
HO
HO
Glycosyltransferases
P450s
E
β-Amyrin
HO
A B
C D
HederageninBayogenin Medicagenic acid Soyasapogenol ESoyasapogenol B
3-Glc, 28Glc-Medicagenic acid3-Glc-Ara, 28-Glc-Hederagenin Soyasaponin I
(3-Rha-Gal-GlcA-Soyasapogenol B)
β-Amyrin synthase (AS)
Squalene epoxidase (SE)
Squalene synthase (SS)
FPP
β-Amyrin
Mevalonic acid
2,3-Oxidosqualene
Squalene
Cycloartenol
Sesquiterpenes
12
3 45
67
8
9
10
1112
13
14
1516
17
18
19 20 21
22
2324
25 26
27
28
2930
HO
O
HO
O
PPO
Cycloartenol synthase (CAS)
Constructing a Constructing a TriterpeneTriterpene Saponin Pathway in Saponin Pathway in Medicago truncatula Medicago truncatula
Achnine et al, Plant J, Mar 2005; 41(6): 875-87. Suzuki et al., Plant J. 2002, 32(6):1033-48.Suzuki et al, Planta, Mar 2005; 220(5): 696-707. Huhman & Sumner, Phytochemistry, 2002, 59, 347-360. Huhman et al, J. Ag Food Chem., 2005, 53(6):1914-20.
Plant Biology Division
Ecotype Profiling•Approx. 120 Ecotypes
(natural mutants, ie genotypicalvariants)
•Elucidate biochemical basis of genotypes/phenotypes using
metabolite profiling
•Establish relationships between genotypes
•Mine ecotypes for saponin enrichment or deficiencies
3-Glc-28-Glc-Medicagenic AcidRha-Hex-Hex-Soyasapogenol E
Medicagenic Acid
3-Glc-Glc-28-Ara-Rha-Xyl-Api-Medicagenic Acid
Rha-Hex-?
3-Ara-Glc-Ara-Hederagenin
Bayogenin
3-Glc-Glc-28-Ara-Rha-Xyl-Medicagenic Acid
Pent-Hex-Hederagenin
Rha-Hex-?
Rha-Glc-Med or Hex-GlcA-Hed
Hex-Hederagenin
Hex-Hex-Hex-Bayogenin
Rha-Hex-Hex-Bayogenin
GlcA-? Multiple Aglycone in t
GlcA?
Gal-GlcA-Soyasapogenol B
Rha-Ara-GlcA-Soyasapogenol B
3-Rha-Xyl-GlcA-Soyasapogenol E
Soyasapogenol E
Hex-HexA-Soy E
Glc-Gal-GlcA-Soyasapognol B
Ara-GlcA-Soyasapogenol B
Rha-Gal-GlcA-Soyasapogenol B (
PCA Loadings of Elite CultivarsPCA Loadings of Elite Cultivars
Plant Biology Division
3pM_Soy1_13pM_Soy1_10
3pM_Soy1_2
3pM_Soy1_3
3pM_Soy1_43pM_Soy1_53pM_Soy1_63pM_Soy1_7
3pM_Soy1_83pM_Soy1_8
3pM_Soy1_9
BORUNG_2 86
BORUNG_3 29
CALIPH_1 50
CRE_005_1 161
CRE_005_3 187
CRE_006_1 35CRE_006_2 13CRE_006_3 24
CRE_007_1 57
CRE_007_2 71
CRE_007_3 26
CRE_009_1 15CRE_009_2 64
DZA_033_1 33DZA_033_2 91DZA_033_3 40DZA_045_1 99
DZA_045_3 16
DZA_046_1 174
DZA_046_2 219
DZA_046_3 159
DZA_055_1 294DZA_055_2 276
DZA_055_3 260
DZA_058_1 141
DZA_058_2 156
DZA_058_3 175
DZA_059_1 220DZA_059_2 142
DZA_061_1 51
DZA_061_2 90
DZA_061_3 42
DZA_202_2 172DZA_202_3 130
DZA_210_1 176DZA_210_2 214DZA_210_3 213
DZA_219_1 157
DZA_219_2 166DZA_219_3 165
DZA_221_1 125DZA_221_2 211DZA_221_3 112
DZA_230_1 177
DZA_230_2 109
DZA_230_3 124
DZA_231_1 210DZA_231_2 136
DZA_231_3 127
DZA_233_1 222
DZA_233_2 181
DZA_233_3 201
DZA_236_2 120
DZA_236_3 198
DZA_241_1 89DZA_241_2 100DZA_241_3 54
DZA_242_1 268DZA_242_2 169
DZA_242_3 306
DZA_243_1 290DZA_243_2 310
DZA_243_3 162
DZA_244_1 194
DZA_244_2 289
DZA_244_3 315
DZA_246_1 228DZA_246_2 151
DZA_246_3 274
DZA_309_1 245DZA_309_2 262DZA_309_3 267
DZA_312_1 45DZA_312_2 62DZA_312_3 107
DZA_327_1 202
DZA_327_2 167
DZA_327_3 208
ESP_050_1 22ESP_050_3 84
ESP_080_2 248
ESP_080_3 314
ESP_098B_1 158ESP_098B_2 186
ESP_098B_3 173
ESP_101_1 301
ESP_101_2 265
ESP_101_3 285
ESP_105_1 139ESP_105_2 148ESP_105_3 193
ESP_140_1 316
ESP_140_2 229
ESP_155_1 269ESP_155_2 283ESP_155_3 232
ESP_158_1 302ESP_158_2 287
ESP_158_3 266
ESP_161_1 261
ESP_161_2 275ESP_161_3 298
ESP_163_3 247
ESP_170_1 231
ESP_170_2 234ESP_170_3 226
ESP_171_2 20
ESP_172_2 97
F11_007_1 70F11_007_2 81F11_007_3 56
F11_008_1 80F11_008_2 32F11_008_3 88
F20_031_2 44
F20_031_3 48F20_047_1 28F20_047_2 38F20_047_3 36
F20_058_1 12F20_058_2 41F20_058_3 75
F20 081 1 279
F20_086_1 291
F20_086_2 191
F20_086_3 300F20_089_1 150
F20_089_2 131F20_089_3 113
GRC_020_1 278
GRC_020_2 184
GRC_020_3 249
GRC_033_1 138
GRC_033_2 117
GRC_033_3 118
GRC_037_1 21GRC_037_2 61GRC_037_3 37
GRC_040_1 149
GRC_040_2 207
GRC_040_3 216
GRC_043_1B 238GRC_043_2B 303GRC_043_3B 160
GRC_063_1 215GRC_063_2 153
GRC_063_3 126
GRC_064_1 284
GRC_064_2 230
GRC_064_3 288
GRC_093_2 76GRC_093_3 14
GRC_098_1 140GRC_098_2 185GRC_098_3 199
PARABINGA_1 9PARABINGA_2 43
PARABINGA_3 10
PARAGGIO_1 46PARAGGIO_2 103
PARAGGIO_3 87
PRT_179_1 5PRT_179_2 30PRT_179_3 93
SEPHI_1 25SEPHI_2 69SEPHI_3 19
A17_1 49
A17_2 101A17_3 67
CALIPH_2 79
CALIPH_3 53
CRE_009_3 98
CYPRUS_1 55
CYPRUS_2 58
CYPRUS_3 7
DZA_045_2 23
_ _
DZA_059_3 188
DZA_105_1 218
DZA_105_2 135DZA_105_3 225
DZA_202_1 221
DZA_213_1 189DZA_213_2 111DZA_213_3 171
DZA_323_1 217DZA_323_2 212DZA_323_3 116
ESP_031_1 110ESP_031_2 182ESP_031_3 121
ESP_039_1 190ESP_039_2 163ESP_039_3 224
ESP_040_1 251
ESP_040_2 308
ESP_040_3 270
ESP_041_1 206ESP_041_2 152ESP_041_3 195
ESP_043_1 317
ESP_043_2 237
ESP_043_3 273
ESP_045_1 286
ESP_045_2 227ESP_045_3 236
ESP_048_1 281
ESP_048_2 277ESP_048_3 296
ESP_050_2 2
ESP_074_1 313ESP_074_2 299ESP_074_3 253
ESP_096_1 257ESP_096_2 132ESP_096_3 146
ESP_098A_1 168ESP_098A_2 223ESP_098A_3 143
ESP_100_1 115ESP_100_2 133ESP_100_3 154
ESP_103_1 264ESP_103_2 254
ESP_103_3 256
ESP_104_1a 271
ESP_104_2 137ESP_104_1 129
ESP_140_3 240ESP_156_1 242ESP_156_2 252
ESP_156_3 307
ESP_159_1 105ESP_159_2 108ESP_159_3 72
ESP_160_1 196ESP_160_2 204ESP_160_3 164
ESP_162_1 123ESP_162_2 147ESP_162_3 178
ESP_163_1 258ESP_163_2 255
ESP_165_2 292ESP_165_3 243
ESP_169_1 293ESP_169_2 304
ESP_169_3 241
ESP_171_1 85ESP_171_3 78
ESP_172_1 18ESP_172_3 39
ESP_173_1 155ESP_173_2 280ESP_173_3 179
ESP_175_1 17
ESP_175_2 104
ESP_175_3 8
F20_009_1 77F20_009_2 34F20_009_3 96
F20_015_1 233F20_015_2 250F20_015_3 239
F20_025_1 235F20_025_2 309
F20_025_3 259
F20_048_1 144F20_048_2 145F20_048_3 200
F20_061_1 183F20_061_2 114F20_061_3 170
F20_081_1 279
F20_081_2 209
F20_081_3 282
F20_087_1 246
F20_087_2 205
F20_087_3 297
GRC_052_1 203GRC_052_2 192
GRC_052_3 122
GRC_065_1 1GRC_065_2 60GRC_065_3 27
HARBINGER_1 68HARBINGER_2 73HARBINGER_3 82
JEM_1 66JEM_2 83
JEM_3 11
MOGUL_1 59MOGUL_2 63MOGUL_3 3
PRT_176_1 74
PRT_176_2 65
PRT_176_3 4
PRT_177_1 128PRT_177_2 119PRT_177_3 134
PRT_178_1 6PRT_178_2 95
ESP_178_3 106PRT_180_1 295PRT_180_2 311
PRT_180_3 272
SALERNES_1 102SALERNES_2 52SALERNES_3 94
Hex
Hex
Rha
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in
Hex
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Rha
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Agl
ycon
e
Hex
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in
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-Hex
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erag
enin
3-G
lc-2
8-G
lc-M
edic
agen
ic A
cid
3-G
lc-G
lc-2
8-A
ra-R
ha-X
yl-A
pi-M
edic
agen
ic A
cid
3-G
lc-G
lc-2
8-A
ra-R
ha-X
yl-M
edic
agen
ic A
cid
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lcA
-28-
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-Xyl
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icag
enic
Aci
d
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8-G
lc-M
alon
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cid
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Plant Biology Division
Metabolomics, Proteomics, transcriptomics, and integrated functional genomics are rapidly maturing discovery sciences yielding new:
● Hypothesis: MeJA induces concurrent response in primary metabolism and saponin biosynthesis suggesting a significant repartitioning of carbon from primary to secondary metabolism.
● Gene Validation: Metabolic profiling data validates the presence and induction of a threonine aldolase in plants
● Mechanistic Insight: MeJA induces isoflavonoidaccumulation via a different mechanism than Yeast.
SummarySummary
Plant Biology Division
?Fishing??Fishing?
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Plant Biology Division
AcknowledgementsAcknowledgementsMetabolomics $$$ The Noble FoundationCorey D. Broeckling (Colorado State Univ.) $$$ NSF PGRP #DBI-0109732Mohamed Farag (Univ. Cairo) $$$ NSF 2010 MCB-0520283David V. Huhman $$$ NSF 2010 MCB-0520140Ewa Urbanczyk-Wochniak $$$ State of OklahomaAnthony L. Duran (Cargill) LECOJ. Tim Smith & Brad Williams (SEOSU)
Noble Bioinformatics Proteomics Patrick Zhao, Indira Reddy, & Yuanji Zhang Zhentian Lei Bonnie S. Watson Virginia Bioinformatics InstituteSatish Nagaraj Pedro Mendes
Bharat MehrotraSaponins & Cell CultureRichard Dixon, Lahoucine Achnine Genomics/EST/MicrorrayHideyuki Suzuki, Jack Blount Richard Dixon & Group
Marina Naoumkina, Bettina DeavoursGregory D. May & Group Bruce Roe & International Consortium NCGR/TIGR/VBI