G Model

PBA-7217; No. of Pages 15

Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis
journal homepage: www.elsevier.com/locate/jpba


Advances in Nutrigenomics research: Novel and future analytical approaches to investigate the biological activity of natural compounds and food functions
˜ V. García-Canas, C. Simó, C. León, A. Cifuentes ∗
Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, Madrid, Spain

a r t i c l e

i n f o

a b s t r a c t
In recent years, nutrition research has moved from classical epidemiology and physiology to molecular biology and genetics. Following this trend, Nutrigenomics has emerged as a novel and multidisciplinary research field in nutritional science that aims to elucidate how diet can influence human health. It is already well known that bioactive food compounds can interact with genes affecting transcription factors, protein expression and metabolite production. The study of these complex interactions requires the development of advanced analytical approaches combined with bioinformatics. Thus, to carry out these studies Transcriptomics, Proteomics and Metabolomics approaches are employed together with an adequate integration of the information that they provide. In this article, an overview of the current methodologies and a thorough revision of the advances in analytical technologies and their possibilities for future developments and applications in the field of Nutrigenomics is provided. © 2009 Elsevier B.V. All rights reserved.

Article history: Received 2 February 2009 Received in revised form 6 April 2009 Accepted 9 April 2009 Available online xxx Keywords: Nutrigenomics Transcriptomics Proteomics Metabolomics Systems Biology

Contents 1. 2. Introduction to Nutrigenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advances in Transcriptomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Gene expression microarray technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Sequencing-based technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Bioinformatics and Gene Ontology database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advances in Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Bottom-up approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Top-down approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Other Proteomic approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advances in Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Analytical techniques in Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Mass spectrometry (MS) approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Nuclear magnetic resonance (NMR) approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Data analysis in Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Systems Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future needs and developments in Nutrigenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00



5. 6.

1. Introduction to Nutrigenomics Diet is a key environmental factor affecting health and the incidence of many chronic diseases [1]. Nutrition research has

∗ Corresponding author. E-mail address: acifuentes@ifi.csic.es (A. Cifuentes). 0731-7085/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2009.04.019

traditionally explored the importance of this relation by classical approaches based on human intervention studies and the use of biomarkers. The effects of nutrient deficiencies, imbalance of macronutrients or toxic concentrations of certain food compounds on health have been the main topics in Nutrition research [2,3]. On the other hand, other bioactive food constituents such as certain polyphenols, vitamins, carotenoids and terpenoids have significant beneficial effects for health promotion and disease prevention by

˜ Please cite this article in press as: V. García-Canas, et al., J. Pharm. Biomed. Anal. (2009), doi:10.1016/j.jpba.2009.04.019

the expression of individual genes has been determined by quantification of mRNA with Northern blotting.G Model PBA-7217. involving not only genetic factors but also a number of behavioural and environmental factors such as exposure to certain food components [14]. possible molecular biomarkers.19. focuses on the study of the impact of specific nutrients and diets on health through the expression of genetic information by the integration of “omics” technologies such as Transcriptomics. (ii) the identity of genes that are involved in the previous stage to the onset of the disease. These disorders are complex and multifactorial in their origin. etc. the discovery of such biomarkers is not easy since diet–gene interactions are complex. Proteomics and Metabolomics [12. obesity (Nutrigenetics). many of which have specific biological activity. 2.) through diet. On the opposite. This classical technique has gradually been replaced by more sensitive techniques such as real-time PCR.g. there is a necessity for understanding the molecular mechanisms that describe homeostasis at biochemical. and might also be ideal for elucidating the effects of novel functional foods and nutraceuticals on global expression of genetic information and cell function without making assumptions about what to look for in terms of risk. and providing new means for discovering biomarkers for efficacy testing of bioactive functional food ingredients [1]. These early effect biomarkers should accurately reflect subtle changes in homeostasis and the efforts of the body to maintain it [16]. molecular and cellular mechanisms that underlies the beneficial or adverse effects of certain bioactive food components. Typically.9]. attached to a substrate. As a consequence. metabolic syndromes. the pre-disease state [3]. The mentioned profiles can be considered as ‘dietary signatures’ that Nutrigenomics will use to understand the cellular functions of nutrients and other active food components and how they affect homeostasis in specific tissues within the whole organism.5].019 . representing thousands of genes. Gene expression microarray technology During the last years. there is a need for molecular biomarkers that allow early detection of the onset of disease or. Owing to the lack of effective analytical approaches that take into account multiple aspects of the biological effects of food compounds. doi:10. The loss of homeostasis and altered biochemical composition of cells and tissues can be a primary cause in disease [15]. J. the analysis of changes in mRNA expression by nutrients and bioactive food constituents is often the first step to study the flow of molecular information from the genome to the proteome and metabolome and one of the main goals in Nutrigenomics research [8]. Biomed. of Pages 15 ARTICLE IN PRESS V. the production of metabolites [8. however. ˜ Please cite this article in press as: V. protein expression and metabolite production will be more useful than single markers. and therefore. This is an important limitation for their application in Nutrigenomics research since the analysis of a reduced number of genes may not provide insights about the causative relationship between the bioactive food constituent and its biological effect [19]. associated with a healthy status and how diet affects this homeostatic control [16]. As a result. Pharm. ideally. to determine health status and reflecting the functional response to a bioactive food component [8]. several steps may be regulated by nutrients and other bioactive compounds in food. and (iii) the effect of bioactive food constituents on crucial molecular pathways [8. cellular and organ levels. function and health. (2009). Anal. Also. gene expression microarray has become a leading analytical technology in Nutrigenomics research for the investigation of the interactions between nutrients and other bioactive food compounds and genes [15. the main characteristics of these -omics techniques and their use in Nutrigenomics are reviewed.11].1. Nutrigenomics makes use of an integrated analytical approach including the latest developments in high-throughput omics techniques for the comprehensive study of different aspects of this biological complexity. nutrients and other bioactive food constituents have been referred to as signals that are detected by cellular sensor systems and affect the expression of the genome at several levels (mRNA and proteins) and subsequently. Nutrigenomics. complete biomarker profiles of gene expression. This integrated analytical approach puts together Transcriptomics.. Thus.21]. More specifically. To do this. These aspects have motivated current trends in Nutrition research to study how diet affects the balance between health and disease by altering the expression of an individual’s genetic makeup (Nutrigenomics) or how the genetic variability among individuals can influence their predisposition to suffer specific illness related to diet as e.2009.13]. Nowadays. usually a glass slide. Proteomics and Metabolomics. Unlike the comparative simplicity of the single-gene disorders. Nutritional Genomics (Nutrigenomics and Nutrigenetics) has emerged as a new field that focuses on the study of the interaction between nutrition and human genome [10.18]. and the other group of techniques is based on DNA sequencing [20]. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n reducing the process of sustained inflammation that accompanies chronic disease [4. two conceptually different analytical approaches have emerged to allow quantitative and comprehensive analysis of changes in mRNA expression levels of hundreds or thousands of genes. Transcriptomics. bioactive food compounds also interact with each other. each combination making a relatively small contribution to overall homeostasis.1016/j. In the past decade. chronic diseases are likely the result of multiple genes and multiple variants of each gene interacting with multiple environmental factors. making even more difficult any effort to identify their biological activity [6]. However. One of the main interests in Nutrigenomics research relates to health and prevention of chronic diseases (such as e. One approach is based on microarray technology. the analysis of global gene expression may offer better opportunities to identify the effect of bioactive food constituents on metabolic pathways and homeostatic control and how this regulation is potentially altered in the development of certain chronic diseases [13]. cardiovascular diseases.04. owing to the extensive optimization and standardization. Most foods are composed of diverse constituents. This technique is based on specific nucleic acids hybridization and it can be used to measure the relative quantities of specific mRNAs in two or more samples for thousands of genes simultaneously. it is recognized that understanding the effect of diet on health requires the study of the mechanisms of nutrients and other bioactive food constituents at the molecular level. cancer. animals and cell cultures demonstrating that nutrients and other bioactive compounds in food can regulate gene expression in diverse ways [7]. protein expression and metabolite production. Proteomics and Metabolomics [17] has created extraordinary opportunities for increasing our understanding about (i) the biochemical.jpba. Both techniques. For years. This is supported by the increasingly growing number of studies in humans. García-Ca˜as et al. at predefined locations within a grid pattern. To attain this. The development of Genomics. The topic of this review. 2 No. the importance of the biological homeostasis maintenance for disease prevention has been underlined. et al. García-Canas. In line with this. In the following sections.g. little is known about their molecular functions and the biological processes involved. Consequently.. the availability of advanced analytical techniques will be essential for the investigation of complete biomarker profiles of gene expression. 2. Advances in Transcriptomics In the expression process of genomic information.. can only analyze gene expression for a limited number of candidate genes at a time. DNA microarrays are collections of oligonucleotides or probes. In addition to this.

In RT-PCR analysis. Redrawn from [53]. the mechanisms of dietary long chain polyunsaturated fatty acids in molecular function cancer and normal cells [48. and possible differences in isoforms detected by the two technologies [21].41]. sensitivity. Data acquisition and pre-processing are important posttechnical steps in microarray experiments. have been adopted by a number of scientific journals. Once hybridization is complete. quantitative radioactive in situ hybridization and verification at the level of the protein. including fold change and statistical significance determined by comparison statistics is necessary in order to identify candidate genes that are differentially expressed. In order to fully exploit the possibilities of DNA microarrays. Scanned image of a microarray composed of a total of 250 kinases and phosphatases showing the expression profile obtained from human prostate carcinoma LNCaP cells treated with 12 mM epigallocatechin gallate (EGCG) or water-only for 12 h. and determination of the biological meaning of both individual genes and group genes. mRNA is converted into cDNA and subsequently amplified in a PCR by specific primers in the presence of a fluorescent dye or internal probes. Differences in precision. García-Ca˜as et al. In a recent study. a careful study of the experimental design is required [23]. since they need to be constantly expressed between all samples and conditions in the experiment. Data analysis process can be divided into three main parts: identification of significantly regulated genes. Regardless of the platform used for the analysis. Also. the differentially expressed genes are then classified into discrete groups or clusters. J. in an effort to make MIAME-compliant data publicly available. Moreover.49] and the effects on transcriptome of a high. Biomed. labelled with a detectable marker (typically. often housekeeping genes. but variability arising throughout the measurement process can obscure the biological signals of interest. quantitative reverse transcriptase polymerase chain reaction (RT-PCR) [34–38]. there is a good correlation between microarray results and other more traditional methods. identification of global patterns of gene expression. through routine Western blotting [40]. To achieve this.or low-carbohydrate intake [50.G Model PBA-7217. strict quality criteria for array-PCR data analysis. The most common approaches adopted for normalization are total intensity normalization. Theoretically. this database accepts processed data files generated with the very recent RNA-Seq technologies that will be discussed later. aimed to improve the data sharing between platforms. (2009). 1).04. cells. immunohistochemical or immunocytochemical assays has also been used [31. indicating the ability of the technology to produce reliable results [33]. the Microarray Gene Expression Data (MGED) organization [26] has established Minimum Information About a Microarray Experiment (MIAME) guidelines for microarray data annotation and reporting. García-Canas. The expression level of the target gene is computed relative to the expression level of one or more reference genes. In addition. After filtering data. Gene expression microarrays are powerful.51]. Pharm. Early applications of microarray to Nutrigenomics were related to the effects of caloric restriction on aging [42–45]. fluorescent dye) and allowed to hybridize to the microarrays with individual DNA sequences hybridizing to their complementary gene-specific probes on the microarray. Anal. based on expression pattern. sophisticated bioinformatics tools such as unsupervised clustering. the fluorescent signal of derivatized-nucleic acids bound to any probe is a function of their concentration. filtering criteria. the ArrayExpress microarray database [27] has been created as a repository for Transcriptomics experimental data. the application of a 96-well plate PCRarray analysis for validation of microarray data suggested that most differences between the results obtained by two different technologies were attributed to saturation problems in the microarray. The fundamental goal of microarray expression profiling is to identify genes that are differentially expressed in the condition of interest [31]. The validation is generally performed by analyzing a selection of differentially expressed genes of interest in the samples using real-time. No. differences in labelling or detection efficiencies between the fluorescent dyes used. the typical experimental procedure is based on the same analytical steps: RNA is extracted from a source of interest (tissue.2009. The latter involves data normalization. of Pages 15 ARTICLE IN PRESS V. Furthermore. and systematic biases in the measured expression levels. Locally Weighted Scatterplot Smoothing (LOWESS) and the use of housekeeping genes [24. samples are washed and imaged using a confocal laser scanner (Fig. In order to alleviate in part cross-comparison problems among distinct microarray platforms. Most of the microarray studies published to date corroborate the gene expression data by an alternative sensitive technique.47].. This step allows the identification of groups of co-regulated genes. the annotations across different platforms are not represented by exactly the same gene sequence regions.jpba.019 . The relative fluorescence intensity for each gene is extracted and transformed to a numeric value [22]. et al. ArrayExpress Atlas [28] is a recent tool developed to allow the user to query for conditions-specific gene expression across multiple data sets within the increasingly growing ArrayExpress database [29]. or other materials). although other methods such as Northern blotting [39]. The molecular mechanisms of cer- ˜ Please cite this article in press as: V. First. 1. make comparisons between experimental platforms unreliable. Selecting proper housekeeping genes is one of the most critical aspects of the analysis. which consists of adjusting the individual hybridization intensities in order to remove variation derived from unequal quantities of starting RNA. Soon. The analysis of the vast amount of microarray data for extracting biologically meaningful information is perhaps the most challenging and daunting tasks [30].33]. or specificity between different microarray platforms. These guidelines.1016/j. principal component analysis and self-organizing maps are the most widely used [32. In general.25]. the technology was extended to study other interesting aspects in Nutrigenomics including the effects of dietary protein in the gene expression of cells [46. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n 3 Fig. doi:10.

Modulation of glucose sensitivity in humans Prevention of the oxidative DNA damage. et al.4 PBA-7217.019 No.04. Pharm. doi:10.1016/j. Bioactive compound/food ingredient Anthocyanins C3G and cyanidin Cy Astaxanthin Food/beverage Fruits.. García-Canas. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n Table 1 Applications of expression DNA microarrays in Nutrigenomics research. Anal. reduction of inflammatory response Chemopreventive agent in cancer Anti-proliferative action Anti-proliferative action Regulation camp signalling and cell differentiation Effects in cardiovascular disease Regulation of glycogen metabolism and protein biosynthesis Regulation of hepatic beta-oxidation and gluconeogenesis Regulation of oxidative stress and inflammation Chemopreventive agent in cancer Chemopreventive agent in cancer Chemopreventive agent in cancer Analytical methodology Affymetrix Human Genome microarray Affymetrix Mouse Expression microarray Ref. Biomed. vegetables.2009. red wine Fish. of Pages 15 V. algae Studied model Human adipocyte cells Mice Expected effect/target illness Regulation of adipocyte function Regulation of oxidative phosphorylation and oxidative stress Regulation of fat and glucose metabolism. J. García-Ca˜as et al. G Model ˜ Please cite this article in press as: V. (2009). [37] [52] ARTICLE IN PRESS Chlorella algae intake Diet Healthy men (blood cells) Custom diabetes-related microarray (Hitachi) [6] Epicatechin Cocoa Human colon adenocarcinoma Caco-2 cells Clontech Human Haematology microarray [36] Epigallocatechin-3 gallate Green tea Human bronchial epithelial 21BES cells Human prostate carcinoma LNCaP cells Human HT 29 colon carcinoma cells Postmenopausal women (peripheral lymphocytes) Rats Healthy men (blood cells) Mice Custom printed Human microarray cDNA microarray Affymetrix Human Genome microarray Human oligo microarrays Custom printed cDNA microarray Affymetrix Human Genome microarray Affymetrix Murine Genome microarray [35] [53] [40] [24] [54] [50] [48] Genistein High-cholesterol intake High-protein/high-carbohydrate intakes Long chain polyunsaturated fatty acids Soybean Diet Dairy-based breakfasts Fish oil Low-calorie intake Omega-3 fatty acid Quercetin Sulforaphane Diet Fish oil Fruits and vegetables Cruciferous vegetables Obese men (blood cells) Human colon adenocarcinoma Caco-2 cells CO115 colon-adenocarcinoma cells Mice Agilent Human Oligo microarray Clontech Human Atlas Glass Arrays Affymetrix Human Genome microarray Affymetrix Murine Genome microarray [55] [49] [56] [39] .jpba.

which with Sanger sequencing would not have been economically or logistically practical before [70. 2. a variety of high-throughput enrichment tools (e. to a reference genome or reference transcripts. J. Anal. usually derived from one end of an mRNA. many SAGE tags are shared by the transcripts from different genes. García-Ca˜as et al. In their work. A recent study of the effects of soy isoflavones. mapping RNA splice isoforms and discovery of small RNAs [78–80]. quantify the gene expression [72]. a complex RNA sample is converted to a library of cDNA with adaptors attached to one or both ends. the MPSS technology has been restricted to only a few specialized laboratories [64].jpba. Enrichment analysis would not be possible without appropriately structured databases such as Gene Ontology [83]. recent enrichment tools also integrate information extracted from other databases as for instance. the biological interpretation of such results is very challenging. for example.G Model PBA-7217. Following sequencing. to comprehensively map and quantify transcriptomes. sequenced SAGE library rarely exhibits saturating tag counts that would indicate complete representation of the cellular transcriptome [62]. Thus. is based on a global sequence census approach. with reference genomes available. Gene Ontology provide a systematic and controlled language. therefore. cloning and bacterial cell propagations that these techniques involve may result in a quantitative bias for different tags [64]. which are applied to genes and proteins are then. These short reads will represent a challenge for either mapping them to reference genome or de novo assembling without genomic reference [69]. Gene Ontology database and information from Dragon Estrogen Responsive Genes Database [24]. for the consistent description of attributes of genes and gene products. An additional limitation of these techniques is that owing to the short length. et al.019 . based on arginine-sensitive regulation that coordinates aspects related to nutrient availability in hepatic cells [38]. In the past few years. A recent work by Leong et al. Thus. Sequencing-based technologies In contrast to microarray technology. In addition to the aforementioned Gene Ontology database. GOminer. their potential is underlined by their adoption in studies of transcriptomes during physiological changes. Next-generation sequencing technologies apply distinct concepts and procedures aimed to increase sequencing throughput in a cost-effective and rapid manner. Once mapped. including LongSAGE [58]. More specifically. the co-functioning genes involved should have a higher (enriched) potential to be selected as a relevant group by high-throughput screening technologies. Unfortunately. 2.2. powerful bioinformatics tools are needed in order to align the resulting short reads (30–300 bp).71]. the many PCR amplifications. EASE. This approach increases the probability for researchers to identify the correct biological processes most pertinent to the biological mechanism under study [82]. cDNA molecules. which can complicate gene identification [63]. First. ProfCom. the development of “next-generation” sequencing methods. Despite the more statistical robustness and less stringent standardization and replication requirements than those used for microarrays. These bioinformatics resources systematically map the list of interesting (differentially expressed) genes to the associated biological annotation terms and then statistically examine the enrichment of gene members for each of the terms by comparing them to a control (or reference). Generally. the use of biological knowledge accumulated in public databases by means of bioinformatics. the use of Affymetrix microarray platform and stringent data analysis provided interesting insights into a homeostatic mechanism. Nevertheless. Onto-Express. (2009). Cap Analysis of Gene Expression (CAGE) [59]. depending on the sequencing platform used. of Pages 15 ARTICLE IN PRESS V. standard biological phrases.65]. most of these methods are based on expensive Sanger sequencing method and.3. doi:10.. A summary of some representative applications of microarray in Nutrigenomics field is given in Table 1. These novel technologies have had an enormous impact on research in a short time period. it uses a different strategy that does not involve propagation in bacteria neither Sanger sequencing [62]. short reads are sufficient to map their locations rather than sequencing the entire large genome. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n 5 tain bioactive food constituents have also been investigated by microarray technology. Bioinformatics and Gene Ontology database As mentioned. The new features of next-generation sequencers have stimulated the development of new techniques that have expanded their applications. Biomed. are subjected to massively parallel sequencing in short reads without bacterial cloning as a pre-requisite. That is. the principle behind these novel techniques for Transcriptomics. and for the comparative analysis between different disease states or conditions [66]. referred to as terms. also referred to as either “massive parallel” or “ultra-deep” sequencing. Over the last years. allows to systematically analyze large gene lists in an attempt to assemble a summary of the most enriched and significant biological aspects [81]. biological process and cellular component. the Kyoto Encyclopedia of Genes and ˜ Please cite this article in press as: V. In SAGE-based methods. has revealed potential mechanisms of action for genistein by the combined use of GoMiner as the enrichment tool.2009. based on gene expression microarray data. in three key biological domains that are shared by all organisms: molecular function. and it is likely to increase further in the future. linked or associated with other Gene Ontology terms by trained curators at genome databases [84]. restriction enzymes are used to obtain short tags of 14–21 bp. which have been termed RNA-Seq methods. Massively Parallel Signature Sequencing (MPSS) is a more sophisticated “clone-andcount” technique that also generates small tags of each mRNA species. Similarly to the concept adopted in SAGE and MPSS. however. No. with or without amplification (depending on the sequencing platform). These techniques include Serial Analysis of Gene Expression (SAGE) [57] and some of its variants. shows the potential of exploiting bioinformatics tools for extracting valuable biological information from microarray experiments. FatiGO. depending on the sequencing platform and method used. the sequence hits are counted to determine their density and distribution and then. García-Canas.g.. which are then concatenated. The principle behind enrichment analysis is that if a certain biological process is occurring in a given study. etc. Moreover. RNA-Seq is still a technology under active development that is being evaluated in multiple laboratories for RNA profiling [73–77] and other applications such as discovery of small RNAs. cloned and sequenced to determine the expression profiles of their corresponding mRNAs. Gene Identification Signature (GIS) [60]. The read lengths typically achievable by these technologies range from 30 to 300 bp. Pharm. high-throughput technologies in Transcriptomics usually generate large lists of differentially expressed genes as final output. Last advances in emulsion PCR and non-cloning based methods for DNA amplification.) have been developed since 2002 in order to assist microarray end user to understand the biological mechanisms behind the large set of regulated genes. is changing the way in which gene expression is studied [20. together with improved imaging instruments have facilitated the development of sequencers capable of reading up to tens of million of bases per run in a massively parallel fashion [67–69].04.1016/j. DAVID. or ontology. Although they have not been applied in Nutrigenomics yet. sequencing-based techniques consist of counting tags of DNA fragments to provide “digital” representation of gene expression levels using sequencing. the abundance of a particular mRNA species is estimated from the count of tags derived from one of its ends. the size of the sample tags should be increased in order to improve the precision and accuracy for detecting rare mRNAs [61]. However.

has brought about an important improvement. tissue or cell in a particular moment. the main drawback regarding this approach is that co-depletion of a certain fraction of the proteome with the abundant proteins can occur. sumoylation. it is dynamic and varies according to the cell type and functional state.019 . however. In addition. For this reason. including PTM in proteins (and peptides) is the methodology of choice when a specific population of the proteome is under study [98]. It is already known that dietary components can also modify the translation of RNA to proteins and the post-translational procedures [92]. being still in not enough concentration for subsequent analysis. one of the major sources of error in 2DE is gel-to-gel variation. Another strategy for proteome fractionation that is gaining more and more significance is the sequential or simultaneous immuno-affinity depletion of the most-abundant proteins [99]. The chemical tagging strategies. It has become the alternative in certain application. Cy3. nutrients or diets on the proteome. a compound found in processed garlic. which evidently will influence chemical properties and functions of proteins. PTMs are key to the control and modulation of many processes inside the cell. Pharm. 2DE is laborious. There are limited studies on the effect of specific natural compounds. DIGE methodology is based on the use of novel ultra high sensitive fluorescent dyes (typically. it has been observed that dietary components such as diallyl disulfide. glycosylation. Anal. In spite of the higher sample complexity due to the huge number of peptidic species obtained from the protein mixture hydrolysate. in Proteomics fractionation and subsequent concentration of the proteome is often needed [95. 2DE is the methodology that currently provides the highest protein species resolution capacity with low-instrumentation cost. the main problem of the mentioned strategies is that after fractionation or depletion. Thus. large-scale analyses of proteomes are accomplished by the combination of two-dimensional gel electrophoresis (2DE) followed by MS analysis [105]. acting by reducing the signal of high-abundance proteins while increasing the level of the low-abundance ones to bring their signal within the detection limit of the present-day analytical instruments [102]. in which the molecular masses of the peptides from the protein digest are compared with those simulated of already sequenced proteins. One of the main differences when working with proteins is that there is not an amplification methodology for proteins comparable to PCR. rapid and automatic identification of complex protein mixtures. 2DE has also some limitations to separate highly hydrophobic biomolecules or proteins with extreme isoelectric point or molecular weight values. J. Namely. cell types. and within the same cell depending on cell activity and state.2009. a 1010 dynamic range has been estimated in serum for protein concentration [94]. The selected analytical strategy for the detection of PTMs will depend on the type of modification: acetylation. of Pages 15 ARTICLE IN PRESS V. or (ii) by tandem MS in a sequence tag search in which a few peptidic sequences obtained from MS/MS analysis (using collisional activation or some other energy deposition process) can be used to search protein databases. combined with the everyday more powerful. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n Genomes (KEGG) pathways (metabolites) in order to improve the comprehensiveness of this type of studies [82]. et al. An essential part of shotgun Proteomics is the use of powerful and highly resolving separation methods prior to MS analysis. MS and databases of protein sequences are then use in different ways for protein identification.85–90]. These studies are summarized in Table 2. fractionation of the proteome can be done by differential centrifugation into different populations of organelles [97] and also attending solubility properties into different cellular compartments.96].1.104]. Although Proteomics has been scarcely applied to study the effect of nutrients on health. 3. easy to use and affordable mass spectrometers. García-Ca˜as et al. In this sense.. phosphorylation. 3. In other cases post-translational regulation of proteins by dietary components can involve the modification of their thiol groups [93]. in which gel variation is eliminated by loading different samples in the same gel [106]. After image analysis of the 2DE gels. has been shown to post-translationally modify proteins [92]. peptides are in general better separated and analyzed by MS than proteins. Biomed. Cy5 and Cy2) to label up to three different protein samples that will be separated in the same 2DE run. In general. two fundamental analytical strategies can be employed: the bottom-up and the top-down approach. Advances in Proteomics The proteome is the set of expressed proteins at a given time under defined conditions. there are hundreds of different types of post-translational modifications (PTMs). doi:10. the introduction of difference gel electrophoresis (DIGE). Thus. Because of the complexity of proteome the use and development of high-resolving separation techniques as well as highly accurate mass spectrometers is nowadays critical in Proteomics [103. Moreover. Currently.jpba. In the case of a Nutrigenomic study. Shotgun Proteomics is an advanced bottom-up approach in which a complex protein mixture is digested with endoproteinases of known specificity [119].04. being the number of review papers higher than research papers [91]. García-Canas. however. the remaining proteins (in case of depletion) or the proteins in the fractions of interest remain dilute.G Model PBA-7217. ˜ Please cite this article in press as: V. the importance of Proteomics as a tool to understand the effect of the diet on health has already been recognized by several authors [18. most of them being based in the bottom-up approach. loosing information about PTM. This separation step is followed by analysis of the isolated proteins (or peptides) by mass spectrometry (MS) via the so-called “soft ionization” techniques. depending strongly on staining and visualization techniques. In a first instance. Most recently. They differ among individuals. etc. Another important drawback in the proteome study is the huge dynamic concentration range of proteins in biological fluids or tissues. Physical and chemical diversity of proteins are also higher than nucleic acids. This situation causes many detection difficulties since a large number of proteins are below the level of sensitivity of the most advanced instruments. it has been described the use of a library of combinatorial ligands. providing a comprehensive. the proteome provides a “picture” of the impact of specific bioactive nutrients and diets in a certain organism. time-consuming and presents low sensitivity. although also LC-MS/MS has been applied with this purpose. The main limitation of the bottom-up approach is that information obtained is related to a fraction of the protein. following: (i) the peptide mass fingerprint (PMF) approach using MS data. ubiquitination. more than a single electrophoretic or chromatographic step is used to separate the thousands of proteins found in a biological sample. Bottom-up approach The bottom-up approach is the most widely used in Proteomics since it can apply conventional or modern methodologies. in order to reduce the complexity of the sample. more precisely in classical 2DEMS. such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). involving the modification of functional groups of amino acid residues. up to now the application of Proteomics in Nutrigenomics is still rather limited. the protein spots of interest are subsequently submitted to an ingel digestion step with a protease enzyme. However.1016/j. Chromatographic [100] and electrophoretic [101] strategies have also been applied to the pre-fractionation of the proteome. 6 No. Both methodologies differ on the separation requirements and the type of MS instrumentation. (2009). In a conventional approach.

The main advantages over the bottom-up approach are that higher sequence coverage is obtained.04. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n [107] [108] [109–113] [113] [114] [115] [116] [117] 7 Table 2 Applications of Proteomics in Nutrigenomics research. MALDI-TOF-MS 2DE. García-Canas. as for instance those based on hydrophilic interaction liquid chromatography (HILIC) [128]. García-Ca˜as et al. MALDI-TOF-MS 2DE. Bioactive compound/food ingredient Quercetin Fatty acids Genistein and daidzein isoflavones Isoflavones Monacolin K Volatile compounds Polyphenols No specific bioactive compound allowing the study of complicated biological samples while avoiding suppression ionization problems. Pharm.1016/j. of Pages 15 ARTICLE IN PRESS V.126] (Fig. ESI has been the interface of choice to ionize peptides eluted from LC or CE columns and to analyze them by MS: from the lower resolving power ion trap instruments able to generate MSn spectra. It has also been described the great potential of monolithic compared to particle-based columns due to monolithic can be used together with higher flows and gradients [127]. CE combined with MS has also come out as an effective strategy for proteomic studies. (2009). MALDI-Q-TOF-MS/MS 2DE. Liquid chromatography (LC) [120] and capillary electrophoresis (CE) [121. The vast majority is based on the combination of ion exchange chromatography and reversed-phase (RP) chromatography. as in the case of ultra performance LC systems (an LC system using sub-2 m packing columns combined with high operating pressures).122] in their different modes are the main methodologies applicable to the separation of complex peptide mixtures due to their high resolving power and their potential for full automation and high sampling rates. TOF-TOF and the recently introduced hybrid linear ion trap (LTQ)-Orbitrap (OrbitrapTM ) [130]. MALDI-TOF-MS. lower sample and reagent consumption than nLC and CE.2. J. Top-down approach A growing number of researchers are focusing on the use of topdown Proteomics. MALDI-TOF-MS 2DE.2009. To carry out shotgun Proteomics most efforts have been focused on the development of on-line combination of various chromatography and/or electrokinetic separation methods. as well as the use of instruments capable of delivering nanoliter/minute flow rates such us nano-scale liquid chromatography (nLC). Special attention is currently being paid to the use of CE in microchips since it provides much shorter separation times [124]. Although physico-chemical diversity of peptides makes them well suited to be separated by different liquid-based separation modes.019 Isoflavones Vitamin A – Dietary fish oil Soy extract Cereal bars Fungus Coffee bean Green tea Diets with different % of vegetable Soy foods – Food/beverage Human serum Rat plasma Vascular protection Changes in nutritional status DIGE. Anal. The technical problems generated by the non-atomospheric working conditions required by MALDI interface could be compensated for a lower interference with sample matrix and chromatographic eluents and an improvement on proteome coverage [131]. Other novel couplings have been described. et al. No. This is of special interest in a multidimensional chromatography setup where the separation and identification of a vast number of analytes in a short time is mandatory mainly in the second dimension. doi:10. it permits the study of 2DE. Biomed. The capabilities of multidimensional systems involving on-line combination of capillary isoelectric focusing (CIEF) with nano-RPLC have also been demonstrated to be effective in the analysis of proteins and peptides [129] creating new and encouraging perspectives in the discovery of biomarkers in Nutrigenomics studies. Some proteomic studies have also focused on the use of MALDI interface for chromatographic separations and MS. Due to the analytical characteristics of these micro-devices further high-throughput in shotgun Proteomics is expected in the near future.jpba. MALDI-TOF/TOF-MS 2DE. reversed phase (RP) and aqueous mobile phases are normally used for the chromatography of peptides. On the other hand. in which structure of proteins is studied through measurement of their intact mass followed by direct ion dissociation in the gas phase [132]. . MALDI-TOF-MS Colorectal cancer prevention Lipid and glucose metabolism study Atherosclerosis-preventive activities Atherosclerosis-preventive activities Chemopreventive agent in cancer Antioxidant and stress relaxation Anticancer activity Colorectal cancer prevention Human SW480 colon carcinoma cells Leiden transgenic mice Human endothelial cells Human peripheral blood mononuclear cells Caco-2 cells Rat brain Human lung adenocarcinoma A549 cells Mouse colon mucosal cells Analytical methodology Expected effect/target illness Studied model ˜ Please cite this article in press as: V. coupled with MS or tandem MS [125. Special attention has been put in miniaturization of columns through the reduction of column diameters and sorbent particles. since CE is a robust system that provides fast and high-resolution separations employing inexpensive capillaries [123]. 3. 2). LC-MS/MS SELDI-TOF [118] [143] Ref. to the more advanced hybrid instrumentation such us quadrupoletime of flight (Q-TOF). nLC-ESI-Q-TOF-MS/MS 2DE. as well as in multidimensional protein identification technology (MudPIT). a relatively new approach compared to bottomup.. MALDI-TOF-MS 2DE.G Model PBA-7217.

The microarray patterns generated can then be transformed into proteomic maps. they can be subjected to a limited proteolysis to produce large polypeptides that are then analyzed employing topdown Proteomic approaches. therefore. while consuming only a few microliters of sample. García-Ca˜as et al. (2009). suitable for routine topdown Proteomics as recently reported [134]. 8 No. resolving power. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n Fig. This alternative technique is based on the fragmentation of multiple charged protein cations due to interaction with low-energy electrons.(or nano) arrays: analytical and functional protein microarrays.G Model PBA-7217. Today. a variety of instruments have been used for top-down Proteomics.jpba. This has been called the middle-down approach. Alternative dissociation techniques such us electron capture dissociation (ECD) represent one of the most recent and significant advances in tandem MS [138]. enzymes and ligands) that bind one specific protein or group of proteins. There are two major classes of protein micro.. ESI-Q/TOF. This technology enables to speed up the process significantly with the capacity to run and analyze several hundreds samples in parallel per day.1016/j.019 . such us MALDI-TOF/TOF. due to the capabilities of CE for complex protein separation. Different SELDI chip types have different surfaces. Surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS).3. Redrawn from [126]. to surfaces with a specific biomolecular affinity (e. its high purchase. After import data files from the microarray scanning sources and normalization and quantification of protein microarrays. J. Both charge reduction dissociation techniques. Protein microarrays can be composed by recombinant protein molecules or antibodies immobilized in a high-density format on the surface of a substrate material. resulting in easier identification of the modification sites. These miniaturized arrays can be fabricated with an almost infinite number of antibodies (in the particular case of antibody microarrays) carrying the desired specificities and being capable of simultaneously profiling numerous low-abundant protein analytes in complex proteomes. However. can help to save time and research effort in studies about the effect of bioactive food components on the proteome. a protein isolate from a previous fractionation or purification step is directly infused to the high-resolution MS. antibodies. Other Proteomic approaches New Proteomic approaches are under development. maintaining the advantages of high sequence coverage and PTM information. which also uses charge reduction by electron transfer [139]. The efficacy of the SELDI-TOF technology has been proved in the analysis of plasma samples (previously fractionated by anion-exchange chromatography) by quickly identifying the proteins whose expres- ˜ Please cite this article in press as: V. In those cases in which the molecular mass of proteins exceeds the analytical power of the mass spectrometer. depending on the type of proteins to be analyzed. infrared multiphoton dissociation (IRMPD) [136]. Pharm. Fourier transform ion cyclotron resonance (FTICR) MS offers the highest mass resolution. PTM and it makes possible to discern between biomolecules with a high degree of sequence identity. rapid and high-throughput Proteomic approach to identify differentially expressed peptides and proteins and. A newer dissociation method similar to ECD is electron transfer dissociation (ETD). In general. mostly derived from the expensive superconducting magnet and liquid helium supply required. an emerging tool for protein profiling and biomarker discovery applicable to minute amounts of starting material [142] can also be used as a complementary. In this regard. being the antibody-based microarray the most common platform in proteomic studies [141]. Nevertheless. a statistical analysis has to be carried out. with a growing increase of works focused on the detection of protein biomarkers. or detailed molecular fingerprints. N is the sample fraction. Anal. Usually.g. receptors. Workflow of multidimensional separation-MS/MS-based shotgun Proteomics strategy. of Pages 15 ARTICLE IN PRESS V. ECD and ETD initially used in FTICR-MS and now extended to (LTQ)-Orbitrap instruments provide more extensive sequence fragments over the entire protein backbone preserving the PTM after cleavage. ESI-IT and the novel OrbitrapTM . One of the approaches that are gaining popularity is based on the array technology. CE coupling with high-resolution MS is one of the most promising methodology in top-down studies [140]. 2.04.. although ultraviolet photodissociation (UVPD) [135]. A key to the top-down approach is the capacity to fragment intact proteins inside the mass spectrometer. doi:10. low-energy multiple collision-induced dissociation (CID) is used. et al. revealing the composition of the proteome. range from chromatographic chemistries. improved ECD and ETC speed will be needed for future high-throughput CE-MS (and also LC-MS) applications in top-down Proteomics. Thus. Biomed. García-Canas. precludes its general use. and blackbody infrared radiative dissociation (BIRD) [137] can be found in literature. Top-down Proteomics approaches are usually limited to simple protein mixtures since very complex spectra are usually generated by multiple charged proteins. accuracy and sensitivity among present MS technologies [133]. 3.2009. and maintenance costs.

The most common metabolites are amino acids. even more complex than in other areas of research. small peptides or carbohydrates. metabolic fingerprinting does not aim to identify all metabolites. tissue. Redrawn from [143]. Advances in Metabolomics As defined by Trujillo et al. and soybased estrogen analogues in cancer [149]. so it is hard to understand the effects separately and to link directly metabolites to genes and proteins [152].720. the metabolome can be described as the full set of endogenous or exogenous low molecular weight metabolic entities of approximately <1000 Da (metabolites). Thus. according to some authors [12].G Model PBA-7217. The two most common analytical techniques used so far in Metabolomics are nuclear magnetic resonance (NMR) and MS.. and its association with health benefits. but to compare patterns of metabolites that change in response to the cellular environment [147]. (2009). Moreover. García-Canas. doi:10. There are three basic approaches used in Metabolomic research. 15. determination of the metabolic effects of environmental changes in the body or early disease detection [148]. metabolic profiling and metabolic fingerprinting. the biological activity of these compounds is an important topic of investigation in Nutrigenomics [150]. any change in metabolite concentration may describe better the biochemical state of a biological system than proteomic or transcriptomic variations. 4). Its importance not only lies on the information obtained about the molecular events involved in nutrition and how the body adapts through metabolic pathways to different nutrient fluxes. 4. organism or species). In general.1016/j. Bold line. making it easy to exceed the linear range of the analytical technique employed.jpba. most Metabolomics studies in mammals have used urine as the vehicle for investigation and. of stannols in cholesterol metabolism. dotted line. As no single technique can be expected to meet all these requirements. vitamin A-deficient Mr profile. SELDI-TOF mass spectra representing protein expression differences in plasma of vitamin A-deficient (n = 3) and vitamin A-sufficient (n = 3) rats at three different m/z: 10. there are several challenges that Metabolomics has to face in human nutrition. Metabolomics has diverse applications such as biomarker discovery.693. organ. 3). vitamins. lipids. however. It is one of the basic approaches to phenotyping as the study of metabolic profiles of a cell gives a more accurate description of a phenotype [146]. of Pages 15 ARTICLE IN PRESS V. Humans feed on other organisms. 3. but also on the identification of certain metabolites such as cholesterol or glucose as biomarkers for health or disease status [145]. the relative concentration of metabolites in the biofluids varies from millimolar level (or higher) to picomolar. Therefore. So far. Metabolic profiling is a non-targeted strategy that focuses on the study of a group of related metabolites or a specific metabolic pathway. Some examples are the potential benefits of flavones in heart diseases. The most relevant application of Metabolomics in Nutrigenomics is the possible health benefits provided by the ingest of functional compounds. many Metabolomics approaches can employ several analytical techniques [12.203 and 18. such as a specific biomarker or reaction product. metabolism is dynamic and depends on the different cellular environments and physiological situation. et al. García-Ca˜as et al. so the number of different metabolomes that conform our diet is significant enough to make Nutrigenomics a very complex discipline [151]. J. Meanwhile.2009. the ideal steps should be first to elucidate the nature and concentration of the searched metabolites and second to share the information in accessible databases [88] (Fig. To date. In Nutritional Metabolomics the complex interactions between gut microflora and host metabolism as well as other extrinsic factors such as food habits. Metabolomics has to deal with very different compounds of very diverse chemical and physical properties. diet and other lifestyle parameters give rise to high metabolic variability [153–156].019 . Anal. so new analytical techniques and methods are continuously being developed and will continue in the near future in order to achieve its goals. as a general result it can be stated that the influence of ˜ Please cite this article in press as: V.151]. Biomed. Metabolites are the real endpoints of gene expression and of any physiological regulatory processes. No. Pharm. in the field of Nutrigenomics blood and urine are the most likely candidates for sample choice [157]. which intend to determine a single chemical class of compounds (mRNA or proteins). In order to gain a detailed knowledge on human metabolism by means of Metabolomic tools. One of the advantages of Nutritional Metabolomics is that there are several accessible body fluids that contain possible biomarkers in human body.1. 4. This is certainly interesting in the nutrition field as can determine variations in different metabolic pathways due to the consumption of different compounds in the diet. Metabolomics is proving to be very useful for the analysis of metabolic patterns and changes in the metabolism derived from different situations in the cellular environment.04. The objective of Metabolomics within the frame of Nutrigenomics is to investigate the metabolic alterations produced by the effect of nutrients or bioactive food constituents in the different metabolic pathways. and the small pathway motifs that are present in a biological system (cell. the effects of phytochemicals in human health are the most studied. each with its own metabolome. Application of Metabolomics in Nutrigenomics (or Nutritional Metabolomics) is. In this regard. Analytical techniques in Metabolomics Unlike Transcriptomics or Proteomics. Because of the extensive consumption of polyphenols in the diet. Target analysis aims the quantitative measurement of selected analytes. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n 9 Fig. sion changed in response to vitamin A status from retinol-sufficient and retinol-deficient rats [143] (Fig. Therefore. target analysis. Metabolomics is an emerging technology. [144]. vitamin A-sufficient Mr profile.

diet in metabolism has been clearly established using these techniques. the most usual ionization techniques are the ones that use atmospheric pressure ionization. Thus. in urine and plasma has been developed [164]. These kinds of analyzers should lead the metabolomic studies in the near future. The most recent introduction into the field of separation techniques with enormous potential in Metabolomics research is the ultra performance liquid chromatography coupled to MS (UPLCTM MS) technology. J.2. amino acids and carbohydrate levels in plasma from five healthy pre-menopauseal women. 10 No. This method identified 11 phenolic acids in urine samples whose concentration increased after tea consumption. In another study. A summary of main Metabolomic applications in Nutrigenomics field is listed in Table 3. have provided an improvement in detection sensitivity that will increase even further the number of applications of NMR in metabolomic studies [173]. García-Ca˜as et al. Yin et al. thus suggesting a metabolic alteration due to soy consumption [170]. an improvement in renal function and an increase in the urinary excretion of the osmolyte trimethylamine-N oxide has been proven [171].1. As for Proteomics. but can also give interesting results in Metabolomics. the recent introduction of new instrumentation. have studied the relationship between the consumption of chamomile tea and some human biological responses. For example HPLC NMR has been used for the identification of hippuric acid. Apart of GC. have the advantages of mass accuracy given by a TOF analyzer combined with the possibility to fragment the ions and thus. UPLCTM -MS has been used in the determination of metabolic profiles in human urine [166]. Direct injection is performed for Metabolomics with high or ultra-highresolution mass analyzers as TOF-MS (mass accuracy < 10 ppm) or FTICR-MS which provides a mass accuracy < 1 ppm and detection limits lower than attomole or femtomole levels. However.G Model PBA-7217. liquid–liquid extraction. 3-(3-hydroxyphenyl) propionic acid. In another 1 H NMR study on the effect of soy isoflavones. García-Canas.04. and 3hydroxycinnamic acid in rat urine using reverse phase gradient HPLC coupled with 1 H NMR [172].019 . The aim of this LC-Q-TOF-MS strategy was to identify potential biomarkers and the metabolic pathways altered by an herbal preparation used to treat some diseases such as cardiovascular problems or cancer. et al.2009. provide information about the structure of the detected metabolites [161]. This study showed changes in the levels of isobutyrate when the mixture of juice and wine was taken but could not find any difference in the metabolic profile due to the intake of juice. Mass accuracy and resolution provided by MS instruments are usually not enough to undoubtedly separate and identify all metabolites in a sample by direct infusion. as it can work with a wide range of polarities [162]. Coupling of NMR with separation techniques is less extended than in MS. Metabolic profiling after dietary intervention with soy isoflavones has also been determined by 1 H NMR differences in lipoprotein. In this method. Nuclear magnetic resonance (NMR) approaches NMR is a high reproducibility technique that has demonstrated its great potential in Metabolomics studies [167]. A GC-MS method for the analysis of flavonoids. Wang et al. [160] which stated that different strains of Salinibacter ruber could be differentiated by some characteristic metabolites.1016/j. Hybrid analyzers. like 1 H-detection triple resonance cold probes.1 g/ml for most phenolic acids. GC-MS is so far the most common coupling for metabolomic studies. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n polyphenols. Biomed. derivatization and analysis by GCTOF-MS have been optimized. The lack of sensitivity of NMR. Mass spectrometry (MS) approaches MS can be used as a standalone technique or most commonly combined with a preliminary chromatographic separation technique. high performance chromatography (HPLC) with MS (HPLC-MS) or CE-MS [151]. Offline coupling with solid-phase extraction (SPE) or on-line coupling with HPLC are the most extended in metabolomic research. providing limits of detection below 0. doi:10. the effect of diet on urea cycle and purine metabolic pathways was demonstrated using both 1 H NMR and ion trap MS [158]. Another study involving the use of both 1 H NMR spectroscopy and high performance liquid chromatography (HPLC) coupled to a Q-TOF-MS reported that acute changes in human urinary metabolomic profiles occur after the consumption of dietary phytochemicals [159]. either gas chromatography (GC). other separation techniques have also been successfully coupled to MS instruments to carry out Nutritional Metabolomics. Anal.1. To overcome these limitations MS analyzers are usually coupled to separation techniques for metabolomic studies. Thus. This could be explained through the modulation of microbial gut metabolism produced by the polyphenols present in wine.. Pharm. in the range of g. A complete methodology for metabolite elucidation is provided by Chen et al. has been one of the main problems of this analytical technique. or different approaches such as dynamic nuclear polarization. Statistical differences in three different excreted metabolites between high-resolution 1 H NMR analyses of urine samples taken before and after chamomile tea consumption were found [168]. 5). Fig.1. a sub class of 4. the metabolic profiling approach using high resolution 1 H NMR spectroscopy has been applied to study metabolite changes in human faeces by the intake of grape juice and ethanol-free wine extracts [169] (see Fig.jpba. (2009). 4. Using this analytical technique. [163] in the metabolomic identification of biomarkers of individuals at risk of diabetes. [165] have recently developed a reliable metabolic profiling method for human serum analysis using a C-18 column coupled to a Q-TOF-MS analyzer. Metabolomics workflow in Nutrigenomics research. as for example Q-TOF. ˜ Please cite this article in press as: V. These characteristics make them ideal tools for metabolomic studies as was shown by Rosello-Mora et al. especially ESI interfaces. 4. of Pages 15 ARTICLE IN PRESS V. For example. The small particle size and the high pressure allow to reduce analysis time approximately ten times respect to conventional LC while increasing efficiency and maintaining resolution.

The aim of PLSDA is to discriminate the complete peak list and reduce it with the most relevant ones [151]. NMR profiles of faeces from different subjects using two different extraction methods (water and methanol).2009. their very different concentrations and the numerous targets with different affinities and specificities that they may have. As described above. individuals. Systems Biology approaches may encompass molecules. it has been necessary to develop strategies to convert the complex raw data obtained into useful information. García-Ca˜as et al. protein. However. tissue. among others. Data analysis in Metabolomics Due to the huge amount of data obtained in metabolomic studies. careful attention will need to be given to the methodologies used to identify these responses. from food complexity. For this matter.. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n 11 Fig. Anal. Pharm. of Pages 15 ARTICLE IN PRESS V. organs. [159] [164] H NMR and HPLC-ESI-Q-TOF GC-TOF-MS Polyphenols Polyphenols Isoflavones Isoflavones 1 H NMR H NMR H NMR H NMR [168] [169] [170] [171] 1 1 1 ˜ Please cite this article in press as: V. After data reduction a multivariate analysis is usually performed.G Model PBA-7217.177]. Metabolomic data reduction is normally accomplished using principal component analysis (PCA) or other related techniques.jpba. Each large data set contains sufficient noise to preclude the identification of multiple minor but relevant changes that could Table 3 Metabolomics applications in Nutrigenomics research. PCA is a tool for exploratory data analysis that determines correlation differences among sample sets. cells. J. the following challenge is to identify it and determine its biological significance [148]. in Nutrigenomic studies biologic responses to a bioactive food component may be subtle and. 5. For all these reasons. Once an unknown compound is determined. Systems Biology Nutrigenomics has to face important difficulties derived. Systems Biology exploits global data sets to derive useful information [181]. or even ecosystems and it is regarded as an integrative approach of all information at the different levels of genomic expression (mRNA. The most common is partial least square discriminant analysis (PLS-DA). ‘omics’ platforms need to be integrated in order to obtain optimal means to understand the influences of bioactive food components on the investigated system (e. Transcriptomics. proteins and metabolites. doi:10. Redrawn from [169]. In particular for Nutritional Metabolomics the most interesting biochemical databases are the ones operated by the European Nutrigenomics Organization [175] that allows to upload and edit scientific information. One of the most accessed databases available online is KEGG [174]. which provides information on more then 2180 metabolites from human. and the Human Metabolome Database [176. the large number of different nutrients and bioactive food compounds. Metabolomics has been considered in the NIH roadmap as an interesting tool for the overall initiative to carry out possible solutions to human metabolic diseases [88]. cell. which can be caused by either a biological difference or a methodological bias.04. García-Canas.. plasma and fecal samples Human urine samples Human fecal samples Human plasma samples Human urine samples Expected effect/target illness Acute effects in urinary metabolism Reduce risk of cardiovascular disease via study of the gut microbial impact in polyphenols Increased excretion of hippurate and glycine and depleted of creatinine Modulation of the gut microflora to prevent inflammatory bowel Changes in carbohydrate and energy metabolism Improved glomerular function or general kidney function Analytical methodology 1 Ref. Proteomics and Metabolomics represents powerful analytical platforms developed for the analysis of genes. et al.g. 5. Thus. Modern Systems Biology is the analysis of the relationships among the elements in a system in response to genetic or environmental perturbations. (2009).019 . there are different large-scale databases available on the web. It is usually used as a first step to have information about the quality of the data.1016/j. 4. metabolite). Unlike any reductionist approach that would take these techniques individually.2. organ) giving rise to the growing of a new area of biology called Systems Biology. therefore. Biomed. with the goal of understanding the system or the emergent properties on the system [178–180]. No. However. Bioactive compound/food ingredient Phytochemicals Flavonoids/phenolic compounds Food/beverage Low and standard phytochemical diet Red wine/red grape juice/tea Chamomile tea Wine/grape juice Soy enriched diets Soy enriched diets Studied model Human urine samples Human urine.

RNA-Seq currently faces several challenges including those concerning library construction issues. the library construction involves some manipulation stages and procedures that could originate some bias [66]. Fortunately. Contrary to microarray. it affects the ability of the technique to accurately detect differences in gene expression levels [41].. 6. most of them hybrid instruments in a combination of two or more analyzers. are improving current technologies and have catalyzed the development of new Proteomic approaches. however.188]. it is expected that relatively new protein microarray technology will play an important role in Proteomics in the near future due to the possibility to simultaneously analyze a large number of different proteins. used the context likelihood of relatedness (CLR) algorithm (gene network analysis) in combination with gene expression microarrays and Gene Ontology-based enrichment analysis to construct and filter gene connectivity maps of bacteria under antibiotic treatment [183]. improvements in the resolution of peptides are desirable to provide increased protein coverage.2009. Anal.1016/j.04. and designing a novel concept for dietary prevention and intervention of disease [16]. although much less employed than the methodologies used today in Proteomics. For that. In this sense it has been proved the robustness of MudPIT for the analysis of complex mixtures of peptides. as well as with label-free hybridization strategies are being investigated as cheaper and sensitive alternatives to current optical detection systems [184. The gene networks were further enriched with data derived from antibiotic growth high-throughput screening to provide insight into the pathway whereby the antibiotic under study triggers its bactericide action. On the other hand. García-Ca˜as et al. A second problem is related to the efficiency and specificity of the hybridization reaction that is subject to variability and cross-hybridization and thus. Also. low signal-to-noise ratios). In a recent work. it is expected that new innovations in proteomic technology will help proteomic profiling to become standard practice in the Nutrigenomic field. Although microarrays is currently the technique of choice for profiling RNA populations under different conditions.G Model PBA-7217. These probes offer high specificity and appear as good candidates for mismatch discrimination. This will aid in rapidly identifying new biomarkers for nutritional status and disease progression. both in terms of hardware and software. Future needs and developments in Nutrigenomics Despite the use of DNA microarrays is a powerful analytical approach. providing unique opportunities to establish interrelationship between dietary components and the development and/or progression of diseases. An adequate Systems Biology approach in Nutrigenomics should provide a holistic view of the molecular mechanisms underlying the beneficial or adverse effects of certain bioactive food components. MS is essential for the systematic investigation in Proteomics. Although Systems Biology has been scarcely applied in Nutrigenomic studies. or nanoparticle labels. their potential is underlined by their adoption by other disciplines. As can be seen in the low number of proteomic applications in Nutrigenomic studies. Kohanski et al. one of the main limitations of RNA-Seq stems from the short length of the sequence reads provided.194]. redox-active indicators. based on sequencing both ends of each interrogated DNA fragment.jpba. without prior knowledge. mapping the short reads to the reference transcriptome can be complicated. it should help in the discovery of key genes and proteins that function to regulate metabolic pathways and whose expression is affected by specific bioactive food compound. Some of these problems are associated with the high background noise that specially hinders the detection of low signals (i. ˜ Please cite this article in press as: V. Sequencing depth is another important aspect that affects sequence coverage (percentage of transcripts surveyed). by confining the information can provide a filter for “distracting” noise generated in each individual platform and minimize the data to be interpreted by focusing on only those endpoints common between the various experimental platforms [178.185]. RNA-Seq consists of absolute numbers of reads that provide accurate estimates of the relative abundance of given transcripts and it is exempt from cross-hybridization problems [72. In Proteomics. For instance. dynamic range of expression level in RNA-Seq depends on the sequencing depth total number of all the sequences reads or base pairs represented in the experiment) and it has been reported to be larger (up to five orders of magnitude) than the one provided by microarray scanners (few-hundred-fold) [189]. there are technical limitations that have to be addressed for optimal implementation. Still. In such case.. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n be unnoticed without adequate statistical tools since the researcher is focused on the changes that are really significant within the whole data set. Pharm. 2DE. In this sense. determines the number of expressed genes and rare spliced isoforms detected and is directly proportional to the sequencing costs [192]. In this regard. Despite these advantages. some interesting alternatives to typical linear probes have been proposed such as molecular beacon probes and peptide nucleic acids [185]. some advantages of RNA-Seq over existing technologies make this technique valuable for comprehensive transcriptome studies. spliced transcript isoforms [191] and sequence variations such as single nucleotide polymorphisms (SNPs) in the transcribed regions [66]. to identify critical factors affecting data quality. longer reads and paired-end reads sequencing. which rely on continuous signals.186]. J. Apart from the everyday more sophisticated sample treatments and separation techniques. conventional mass spectrometers are giving way to the more sophisticated and compact mass spectrometers. CE and MS have become the most used methodologies. New improvements will probably include the establishment of routine data analysis methods and increases in the numbers and lengths of sequence reads as well [193. Another important advantage of RNA-Seq relative to gene expression microarray is their ability to identify. Also.e. have been proposed to help alleviate the problem [72]. et al. There is an evident need of development of improved or alternative technologies to become the routine analysis for proteome research a reality. RNA-Seq has very low background signal because DNA sequences can be unambiguously mapped to unique regions of the genome. the MicroArray Quality Control (MAQC) Consortium establishes quality control criteria to ensure data quality. In addi- tion. RNA-Seq does not require cloning. Rapid advances in MS instrumentation. García-Canas. however. allowing detection of very low expressed mRNA. a Systems Biology approach has been applied to improve our knowledge about carbohydrate metabolism in yeast [178]. the cost is likely to keep falling.182]. and to optimize and standardize microarray procedures [187]. Next-generation methods in Transcriptomics will undoubtedly continue to technically improve in several ways within the next years. To achieve this. however. unlike hybridization-based approaches. provided that sequencing depth is sufficient [190]. Systems Biology. For large and complex transcriptomes. doi:10. First. (2009). of Pages 15 ARTICLE IN PRESS V. problems derived from element-to-element differences within a microarray and microarray-to-microarray differences need also to be solved [23. especially if an important fraction of sequence reads match multiple locations in the genome. LC.019 . bioinformatics and complete coverage at reasonable costs. 12 No. allowing next-generation sequencing methods demonstrate full potential for the study of transcriptomes and provide new applications and extensive use of these technologies in Nutrigenomics research. Biomed. appropriate statistical models have to be used in order to filter through the large data sets and highlight only those important changes. Novel approaches focused on the use of electrochemical transducers in combination with either enzymatic.

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