Foodomics: food science in the post-genomic era Alejandro Cifuentes

Laboratory of Foodomics, CIAL National Research Council of Spain (CSIC) Madrid, Spain

CEICS Forum 2011, Tarragona CURRENT CHALLENGES IN FOOD SCIENCE

Production of new functional foods (with scientifically proved claims)

Food safety, quality and traceability (ideally as a whole) using approaches

Develop, produce and monitor new transgenics foods

Understand the effects of gene-food interaction on human health (Nutrigenomics)

Explain the different answers from individuals to food: personalized diet (Nutrigenetics) CURRENT CHALLENGES IN FOOD SCIENCE s ic Production of new functionalm foods o (scientifically proved claims)d o o Food safety, quality and traceability :F (ideally as a whole) susing omics r approachese w s Develop, producen and monitor new transgenicsa foods w e Understand the effectsn of gene-food interaction on human, health (Nutrigenomics) s d e Explain differente answers from individuals to food: personalizedn diet (Nutrigenetics) w e N Foodomics We have defined Foodomics for the first time in a SCI journal as: A discipline that studies the Food and Nutrition domains through the application of advanced omics technologies to improve consumer’s well-being, health, and confidence. (Cifuentes et al.; J. Chromatogr. A 1216 (2009) 7109; Electrophoresis 31 (2010) 205; Mass Spectrom. Rev. 2011, DOI 10.1002/mas).

The interest in Foodomics coincides with a clear shift in medicine and biosciences toward prevention of future diseases. Foodomics: A new omics for a new food era

FOODOMICS Goal To improve consumers well-being and confidence Food quality fulfilling legislation

Epidemiological Food safety studies Nutrigenomics Nutrigenetics

Development of: Nutraceuticals Functional foods GM foods New foods

Bioinformatics GM foods monitoring Toxicity assays In-vitro assays In-vivo assays Clinical trials Foodomics papers from our group

-A. Cifuentes “Food Analysis and Foodomics” J. Chromatogr. A 1216 (2009) 7109-7110.

-M. Herrero, V. García-Cañas, C. Simo, A. Cifuentes “Recent advances in the application of CE methods for food analysis and foodomics” Electrophoresis 31 (2010) 205-228

-C. Simó, E. Domínguez-Vega, M.L. Marina, M.C. García, G. Dinelli, A. Cifuentes “CE-TOF MS analysis of complex protein hydrolyzates from genetically modified soybeans. A tool for Foodomics” Electrophoresis 31 (2010) 1175–1183

-M. Herrero, C. Simó, V. García-Cañas, E. Ibáñez, A. Cifuentes “Foodomics: MS-based strategies in modern Food Science and Nutrition” Mass Spectrom. Rev. 2011, DOI 10.1002/mas (impact factor: 3.569) Special issue on: “Advanced Food Analysis and Foodomics”

Editor: Alejandro Cifuentes [email protected] (to be published in summer 2012) A book is now under preparation on:

“FOODOMICS: ADVANCED MASS SPECTROMETRY IN MODERN FOOD SCIENCE AND NUTRITION”

Editor: Alejandro Cifuentes [email protected] (to be published in autumn 2012) Foodomics

Genomics & Epigenomics

Metabolomics

Transcriptomics

Proteomics FOODOMICS: CURRENT CHALLENGES

Production of new functional foods (scientifically proved claims)

Food safety, quality and traceability (ideally as a whole) using omics approaches

Develop, produce and monitor new transgenics foods

Understand the effects of gene-food interaction on human health (Nutrigenomics)

Explain the different answers from individuals to food: personalized diet (Nutrigenetics) Transgenic maize (Bt corn) A new CryIA(b) gene (encodes for a Bacillus thuringiensis protoxin) is inserted by recombinant DNA techniques into the maize genome. The new protoxin acts as insecticide against lepidopters.

Transgenic soybean (RR soybean) A new CP4 EPSPS gene from Agrobacterium (that encodes for a CP4 5-enolpyruvylshikimate-3-phosphate sintase, CP4-EPSPS) is inserted by recombinant DNA techniques into the soy genome. The new CP4-EPSPS enzyme allows to the GM plant to resist the effect of the herbicide glyphosate.

Can the new inserted genes give rise to other unintended effects? The European Food Safety Agency (EFSA) recommends the development of profiling techniques to study these unexpected effects. Second Generation GMOs

THEIR SUCCESS WILL DEPEND (AMONG OTHER FACTORS) ON PROVIDING STRONG SCIENTIFIC EVIDENCES ON: -THEIR (HEALTH) BENEFITS FOR CONSUMERS Macronutrients: -THE EQUIVALENCE WITH THEIR NATURAL COUNTERPARTS Proteins -NO ENVIRONMENTAL RISKS Amino acid composition Functionality, e.g. bread dough Drought tolerance Carbohydrates Starch composition, inulin, monosaccharides Vegetable oils High-PUFA (e.g., oleic acid)

Micronutrients: Vitamins, anti-oxidants (Golden rice) Minerals (iron-fortified rice)

Miscellaneous: Hypoallergenic foods Drought tolerance Prolonged ripening Ideal Foodomics platform for GMO analysis

GMO detection / Traceability GMO labeling TRANSCRIPTOMICS SYSTEMS BIOLOGY Gene Protein Metabolite Data GMO biomarkers expression expression expression integration Unintended effects STATISTICS GMO METABOLITES PROTEINS DNA/mRNAs GMO safety Legal issues GMO removed or improved DNA analysis by CGE-LIF

Development of a novel analytical methodology, based on MLGA-CGE- LIF, to simultaneously detect multiple GMOs in a single reaction Multiplex ligation dependent genome amplification (MLGA)

Vector (universal sequence) III. Circular DNA enrichment Long oligonucleotide (exonuclease treatment) probe (Selector)

Target 3 II. Ligation of targets I. Digestion of gDNA with (Circularization) specific endonucleases Target 1 Target 2 Targets 5’ 5’ Fwd primer 5’ Target 3 Rev primer V. Detection of PCR products Target 2 Target 1

1 3 Target 1 IV. Linearization of targets and PCR 2 5’ 3’ Target 2 5’ 3’ Target 3 CGE-LIF 5’ 3’ Simultaneous detection of multiple GMOs with MLGA-CGE-LIF

mon863 selector CGE-LIF ga21 selector Fwd primer adh V2 selector 3.5 1% GA21, 1% MON863 & 1% MON810 maize mixture mon810 selector Rev primer Adh V2 selector (2.5 nM) vector MON863 selector (5 nM)

3.0 Mon810 selector (2.5 nM)

GA21 selector (5 nM) 2.5

Ligation Linearization 2.0 & & Enrichment PCR RFU 1.5 Mon810 (172 bp) mon863 (155 bp)

Digested 1.0 Ga21 (131 bp) Adh (199 bp) genomic Circular DNA DNA targets 0.5

0.0 MLGA is very flexible since the incorporation 0 2 4 6 8 10 12 14 16 18 20 22 24 of new additional selector probes to the ligation Minutes step requires minor adjustments of the selector Calculated LODs (S/N=3) concentration to detect all the DNA target with 0.2% GA21 maize LODs below 1% 0.3% MON863 maize 0.1% MON810 maize SHOTGUN PROTEOMICS by CE-TOF-MS: GM vs. wild soybean

SOYBEAN Protein content 40 %

Well-known difficulties in protein separation: ¾ Different physico-chemical properties (size, isoelectric point, hydrophobicity) within a wide range of concentrations. ¾ Difficult to separate complex mixtures of proteins. ¾ Challenging identification of (large) proteins.

SHOTGUN PROTEOMICS by CE-TOF-MS Analysis of peptides obtained after hydrolysis of complex protein mixtures

GM soybean PEPTIDE PROTEIN ENZYMATIC DATA ANALYSIS BY EXTRACTION HYDROLYSIS ANALYSIS Wild soybean CE-TOF-MS

Optimization required Intens. x105 Base peak electropherogram 4 3 2 1 0 0 5 1015202530(min)

Some (63) extracted ion electropherograms Relative intensity Relative

0 5 1015202530Time (min) CE-TOF MS ANALYSIS UNDER SELECTED CONDITIONS

F BPE Soy Protein Isolate 5.0 B C E

D

2.5

G H

A

0.0 0 5 10 15 20 25 30 Time (min) Intens. M+H Intens. x104 5 641.3511 Mass spectra x10 M+3H Mass spectra M+H 919.4486 5 (9.5 min, peak A) (25.7 min, peak G) 417.2570 0.8 4 Intens. x105 M+4H Mass spectra M+H 734.1155 0.6 M+2H 3 631.3779 2.5 (15.2 min, peak C) 1378.6605 M+H 0.4 2 685.3153 2.0

0.2 1 1.5 M+1H 0 1.0 658.3186 0.0 200 300 400 500 600 700 800 900 m/z 500 1000 1500m/z M+5H M+3H 0.5 587.4964 978.4777

0.0 400 600 800 1000 1200 1400 1600 m/z

Complexity of the Peptidic Map Automated interpretation OPTIMIZATION OF AUTOMATED INTERPRETATION

Use of deconvolution tool → Study of the peptides obtained in 5 consecutive injections

The same peptides were not ↑ cutoff in order to eliminate 5 % Cutoff found in all the injections unstable signals

The same peptides were found in all injections 15 % Cutoff (simultaneous analysis of more than 150 peptides)

Intens. Intens. M+H Mr (3) x104 641.3511 Mass spectra x105 Deconvoluted (9.5 min, peak A) 640.3 mass spectra M+H (9.5 min, peak A) 5 417.2570

1.0 Mr (1) 4 416.2

M+H 3 631.3779 Mr (2) 630.4 M+H 2 685.3153 0.5 Mr (4) 684.3

1

0 0.0

200 300 400 500 600 700 800 900 m/z 200 300 400 500 600 700 800 900 m/z SHOTGUN PROTEOMICS by CE-TOF-MS: GM vs. wild soybean

Intens. x105 BPE

7.5

6.0 Conventional

4.5

3.0

Transgenic 1.5

0.0 0 5 10 15 20 25 30 Time (min) SIMILAR PEPTIDE PROFILE

No differences were observed between the GM and wild soybean when a 15% abundance cutoff was used for the automatic deconvolution of the detected ions METABOLOMICS by FT-ICR-MS, PLE and CE-TOF-MS: GM vs. wild corn

Intens. 175.06122 257.11426 x108 301.03528

6

515.12475 339.09313

380.99850

276.98773 4

577.13423

477.06693 2 605.19248 12 tesla FT-ICR-MS at GSF/Munich Germany P. Schmitt Kopplin

0 150 200 250 300 350 400 450 500 550 600 m/z Intens.9 -MS x10 438.23902 4 Details on m/z 438.239 GMO 3 2 439.24247 1 440.24610 0 Mass resolution: >600.000 in full scan 2500 438.23873 Theoretical compound Mass accuracy: <0.1 ppm 2000 C H N O "Lunarine 1500 25 32 3 4 >10.000 signals/mass spectra 1000 439.24213 >300 elementary composition assignements 500 440.24498 0 (depending on the extraction conditions) 438.23899 2000 Theoretical compound 1500 C4H50N6O6S5 1000 440.23478 500 439.24044 0 438.0 438.5 439.0 439.5 440.0 440.5 m/z Although the high resolution and sensitivity provided by FT-MS allows the detection and identification of an impressive number of compounds, PLE and CE-TOF-MS can provide additional information useful to corroborate (or not) the metabolites identification. METABOLOMICS by FT-ICR-MS, PLE and CE-TOF-MS: GM vs. wild corn FT-ICR-MS of wild maize

Mass resolution: >600.000 in full scan; Mass accuracy: <0.1 ppm; Signals/mass spectra: > 10.000 Elementary composition assignements: >300 (depending on the extraction conditions) Extraction by PLE with:

Hexane

Methanol

Water PROCEDURE FOR THE TENTATIVE CHARACTERIZATION OF METABOLITES BASED ON FT-MS and CE-TOF-MS DATA

MOLECULAR ION DETERMINATION

ISOTOPIC PATTERN

MOLECULAR FORMULAE ASSIGNATION

SEARCH IN DATABASES

EXPECTED ELECTROPHORETIC MOBILITY

DATA ANALYSIS

SAMPLES CLASSIFICATION/BIOMARKERS DETECTION Partial least squares–discriminant analysis (PLS- DA)(Q2(cum)=0.52 and R2(Y)=0.99) with six different maize varieties analyzed by FT-MS.

Maize samples: A) PR33P66; B) PR33P66 Bt; C) Tietar; D) Tietar Bt; E) Aristis; and F) Aristis Bt.

The score scatter plot underlines a different pattern for the transgenic (they are represenrepresented in blue color) and wild lines (red color). The different properties of the discriminative masses (represented in blue and red in the loading plot) are investigated with MassTRIX. The model was built up with the data measured in negative mode.

Problem to be solved: Number of available samples Publicaciones de nuestro grupo sobre GMOs

-C. Simó, R. González, C. Barbas, A. Cifuentes Anal. Chem. 77 (2005) 7709-7716 ---Proteomics

-M. Herrero, E. Ibáñez, P.J. Martin-Alvarez, A. Cifuentes Anal. Chem. 79 (2007) 5071-5077---Metabolomics

-T. Levandi, C. Leon, M. Kaljurand, V. Garcia-Cañas, A. Cifuentes Anal. Chem. 80 (2008) 6329-6335 ---Metabolomics

- V. García-Cañas, M. Mondello, A. Cifuentes Electrophoresis 31 (2010) 2249–2259 ---Genomics

-C. Leon, I. Rodriguez, M. Lucio, V. Garcia-Cañas, P. Schmitt-Kopplin, A. Cifuentes J. Chromatogr. A 1216 (2009) 7314-7323---Metabolomics

-C. Simó, E. Domínguez-Vega, M.L. Marina, M.C. García, G. Dinelli, A. Cifuentes Electrophoresis 31 (2010) 1175–1183---Proteomics

-V. García-Cañas, C. Simó, C. León, E. Ibáñez, A. Cifuentes Mass Spectrom. Rev. 30 (2011) 396– 416 –Proteomics + Metabolomics Running Foodomics projects at our lab on bioactivity of new functional ingredients on:

Alzheimer Diabetic children Colon cancer Leukemia

Population study Clinical trial Human cell lines Human cell lines

Biological sample: Biological samples: Biological samples: Biological samples: Cebrospinal fluid Urine and Plasma DNA, RNA, DNA, RNA, (CSF) proteins and proteins and metabolites metabolites

In collaboration with In collaboration with GSF In collaboration with Karolinska Institute (Munich, Germany) Univ. Miguel Hernandez, Elche, Spain (Stockholm, Sweden) La Paz Hospital University of Granda, Granada, Spain (Madrid, Spain) Univ. CEU-SP (Madrid, Spain) GENERAL CONCLUSION

Foodomics is a suitable approach to solve new challenges in Food Science and Nutrition… 20th International Symposium on Electro- and Liquid-Phase Separation Techniques

6-9 October, 2013

CHAIRMAN: Alejandro Cifuentes (National Research Council of Spain, CSIC, Spain) CO-CHAIRMAN: Javier Hernández-Borges (University of La Laguna, Tenerife, Spain) Thank you!