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PBA-7217; No. of Pages 15
ARTICLE IN PRESS
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
1. Introduction to Nutrigenomics Diet is a key environmental factor affecting health and the incidence of many chronic diseases . 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
Thus. cellular and organ levels. and (iii) the effect of bioactive food constituents on crucial molecular pathways [8. Gene expression microarray technology During the last years.jpba. 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 . In line with this.. 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. Typically. owing to the extensive optimization and standardization. involving not only genetic factors but also a number of behavioural and environmental factors such as exposure to certain food components . chronic diseases are likely the result of multiple genes and multiple variants of each gene interacting with multiple environmental factors. 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. complete biomarker proﬁles of gene expression. usually a glass slide. doi:10. Both techniques.g. This classical technique has gradually been replaced by more sensitive techniques such as real-time PCR. These early effect biomarkers should accurately reﬂect subtle changes in homeostasis and the efforts of the body to maintain it . Most foods are composed of diverse constituents. Consequently. These disorders are complex and multifactorial in their origin.G Model PBA-7217. 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 . 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. of Pages 15 ARTICLE IN PRESS V. 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 therefore. Also. 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. Advances in Transcriptomics In the expression process of genomic information. representing thousands of genes. protein expression and metabolite production will be more useful than single markers.) through diet. 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.. obesity (Nutrigenetics).13]. Transcriptomics. Nutrigenomics. cardiovascular diseases. To attain this. the production of metabolites [8.9]. the expression of individual genes has been determined by quantiﬁcation of mRNA with Northern blotting. This is supported by the increasingly growing number of studies in humans. Pharm. (2009). The development of Genomics. Proteomics and Metabolomics. Proteomics and Metabolomics  has created extraordinary opportunities for increasing our understanding about (i) the biochemical. 2. the importance of the biological homeostasis maintenance for disease prevention has been underlined. DNA microarrays are collections of oligonucleotides or probes. and the other group of techniques is based on DNA sequencing . (ii) the identity of genes that are involved in the previous stage to the onset of the disease. J.5]. García-Ca˜as et al. Proteomics and Metabolomics [12.18]. In the following sections. associated with a healthy status and how diet affects this homeostatic control . each combination making a relatively small contribution to overall homeostasis. attached to a substrate. In addition to this. bioactive food compounds also interact with each other. the discovery of such biomarkers is not easy since diet–gene interactions are complex. As a consequence. 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. however. For years. protein expression and metabolite production. 2 No. the availability of advanced analytical techniques will be essential for the investigation of complete biomarker proﬁles of gene expression. molecular and cellular mechanisms that underlies the beneﬁcial or adverse effects of certain bioactive food components. To do this. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n reducing the process of sustained inﬂammation that accompanies chronic disease [4. etc. to determine health status and reﬂecting the functional response to a bioactive food component . function and health. the pre-disease state . 2. 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..g. there is a need for molecular biomarkers that allow early detection of the onset of disease or. at predeﬁned locations within a grid pattern. Anal. One approach is based on microarray technology.1. can only analyze gene expression for a limited number of candidate genes at a time.19.1016/j.019 . 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. The topic of this review. little is known about their molecular functions and the biological processes involved. several steps may be regulated by nutrients and other bioactive compounds in food. 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 . ideally. Nowadays.2009. In the past decade. cancer. García-Canas. One of the main interests in Nutrigenomics research relates to health and prevention of chronic diseases (such as e.21]. Unlike the comparative simplicity of the single-gene disorders.04. On the opposite. ˜ Please cite this article in press as: V. metabolic syndromes. 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 loss of homeostasis and altered biochemical composition of cells and tissues can be a primary cause in disease . However.11]. This integrated analytical approach puts together Transcriptomics. making even more difﬁcult any effort to identify their biological activity . and providing new means for discovering biomarkers for efﬁcacy testing of bioactive functional food ingredients . Owing to the lack of effective analytical approaches that take into account multiple aspects of the biological effects of food compounds. there is a necessity for understanding the molecular mechanisms that describe homeostasis at biochemical. many of which have speciﬁc biological activity. As a result. Biomed. possible molecular biomarkers. animals and cell cultures demonstrating that nutrients and other bioactive compounds in food can regulate gene expression in diverse ways . the main characteristics of these -omics techniques and their use in Nutrigenomics are reviewed. More speciﬁcally. et al. 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.
aimed to improve the data sharing between platforms. ArrayExpress Atlas  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 . and determination of the biological meaning of both individual genes and group genes. Anal. The most common approaches adopted for normalization are total intensity normalization. this database accepts processed data ﬁles generated with the very recent RNA-Seq technologies that will be discussed later. Data acquisition and pre-processing are important posttechnical steps in microarray experiments. principal component analysis and self-organizing maps are the most widely used [32. Regardless of the platform used for the analysis. García-Ca˜as et al. The molecular mechanisms of cer- ˜ Please cite this article in press as: V. including fold change and statistical signiﬁcance determined by comparison statistics is necessary in order to identify candidate genes that are differentially expressed. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n 3 Fig. identiﬁcation of global patterns of gene expression. the ﬂuorescent signal of derivatized-nucleic acids bound to any probe is a function of their concentration. the ArrayExpress microarray database  has been created as a repository for Transcriptomics experimental data. Data analysis process can be divided into three main parts: identiﬁcation of signiﬁcantly regulated genes. or speciﬁcity between different microarray platforms. After ﬁltering data. In order to alleviate in part cross-comparison problems among distinct microarray platforms. mRNA is converted into cDNA and subsequently ampliﬁed in a PCR by speciﬁc primers in the presence of a ﬂuorescent dye or internal probes. 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. of Pages 15 ARTICLE IN PRESS V.51]. The fundamental goal of microarray expression proﬁling is to identify genes that are differentially expressed in the condition of interest . a careful study of the experimental design is required . quantitative reverse transcriptase polymerase chain reaction (RT-PCR) [34–38]. the differentially expressed genes are then classiﬁed into discrete groups or clusters.or low-carbohydrate intake [50. or other materials). through routine Western blotting . and possible differences in isoforms detected by the two technologies . In general. although other methods such as Northern blotting . In order to fully exploit the possibilities of DNA microarrays. often housekeeping genes. immunohistochemical or immunocytochemical assays has also been used [31.jpba. indicating the ability of the technology to produce reliable results . doi:10.41]. 1. which consists of adjusting the individual hybridization intensities in order to remove variation derived from unequal quantities of starting RNA. cells. The relative ﬂuorescence intensity for each gene is extracted and transformed to a numeric value . Early applications of microarray to Nutrigenomics were related to the effects of caloric restriction on aging [42–45].019 . This step allows the identiﬁcation of groups of co-regulated genes. strict quality criteria for array-PCR data analysis. et al. Moreover. Most of the microarray studies published to date corroborate the gene expression data by an alternative sensitive technique.25]. 1). the Microarray Gene Expression Data (MGED) organization  has established Minimum Information About a Microarray Experiment (MIAME) guidelines for microarray data annotation and reporting.04. Furthermore. In a recent study. J. but variability arising throughout the measurement process can obscure the biological signals of interest. The validation is generally performed by analyzing a selection of differentially expressed genes of interest in the samples using real-time. Redrawn from . 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. Differences in precision. First. the mechanisms of dietary long chain polyunsaturated fatty acids in molecular function cancer and normal cells [48.. there is a good correlation between microarray results and other more traditional methods. have been adopted by a number of scientiﬁc journals. Once hybridization is complete. Selecting proper housekeeping genes is one of the most critical aspects of the analysis. (2009). ﬁltering criteria. the typical experimental procedure is based on the same analytical steps: RNA is extracted from a source of interest (tissue. quantitative radioactive in situ hybridization and veriﬁcation at the level of the protein. No. These guidelines. Theoretically. sensitivity. and systematic biases in the measured expression levels.49] and the effects on transcriptome of a high. Also. To achieve this. In addition. García-Canas. ﬂuorescent dye) and allowed to hybridize to the microarrays with individual DNA sequences hybridizing to their complementary gene-speciﬁc probes on the microarray.G Model PBA-7217. labelled with a detectable marker (typically.1016/j. Soon. Gene expression microarrays are powerful.2009. the technology was extended to study other interesting aspects in Nutrigenomics including the effects of dietary protein in the gene expression of cells [46.33]. The latter involves data normalization. the annotations across different platforms are not represented by exactly the same gene sequence regions. Locally Weighted Scatterplot Smoothing (LOWESS) and the use of housekeeping genes [24. In RT-PCR analysis. in an effort to make MIAME-compliant data publicly available. Pharm. Biomed. based on expression pattern. samples are washed and imaged using a confocal laser scanner (Fig. The expression level of the target gene is computed relative to the expression level of one or more reference genes. The analysis of the vast amount of microarray data for extracting biologically meaningful information is perhaps the most challenging and daunting tasks .47]. sophisticated bioinformatics tools such as unsupervised clustering. differences in labelling or detection efﬁciencies between the ﬂuorescent dyes used. make comparisons between experimental platforms unreliable. since they need to be constantly expressed between all samples and conditions in the experiment.
jpba. vegetables. Anal. García-Canas. García-Ca˜as et al.4 PBA-7217. Modulation of glucose sensitivity in humans Prevention of the oxidative DNA damage.   ARTICLE IN PRESS Chlorella algae intake Diet Healthy men (blood cells) Custom diabetes-related microarray (Hitachi)  Epicatechin Cocoa Human colon adenocarcinoma Caco-2 cells Clontech Human Haematology microarray  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        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     . (2009). red wine Fish. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n Table 1 Applications of expression DNA microarrays in Nutrigenomics research. doi:10.. 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. et al. G Model ˜ Please cite this article in press as: V. Pharm.1016/j. J. Biomed.2009. 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. Bioactive compound/food ingredient Anthocyanins C3G and cyanidin Cy Astaxanthin Food/beverage Fruits. of Pages 15 V.04.019 No.
These techniques include Serial Analysis of Gene Expression (SAGE)  and some of its variants. allows to systematically analyze large gene lists in an attempt to assemble a summary of the most enriched and signiﬁcant biological aspects . high-throughput technologies in Transcriptomics usually generate large lists of differentially expressed genes as ﬁnal output. etc. Onto-Express. mapping RNA splice isoforms and discovery of small RNAs [78–80]. quantify the gene expression . doi:10. Gene Identiﬁcation Signature (GIS) . Cap Analysis of Gene Expression (CAGE) . many SAGE tags are shared by the transcripts from different genes. which with Sanger sequencing would not have been economically or logistically practical before [70. In addition to the aforementioned Gene Ontology database. and it is likely to increase further in the future. to comprehensively map and quantify transcriptomes. The read lengths typically achievable by these technologies range from 30 to 300 bp. Gene Ontology database and information from Dragon Estrogen Responsive Genes Database . the principle behind these novel techniques for Transcriptomics. it uses a different strategy that does not involve propagation in bacteria neither Sanger sequencing . Although they have not been applied in Nutrigenomics yet. in three key biological domains that are shared by all organisms: molecular function. is based on a global sequence census approach. et al. cDNA molecules. DAVID. shows the potential of exploiting bioinformatics tools for extracting valuable biological information from microarray experiments. Following sequencing. A summary of some representative applications of microarray in Nutrigenomics ﬁeld is given in Table 1.65]. 2. Anal. These short reads will represent a challenge for either mapping them to reference genome or de novo assembling without genomic reference . based on arginine-sensitive regulation that coordinates aspects related to nutrient availability in hepatic cells .019 . Nevertheless. referred to as terms.G Model PBA-7217. the development of “next-generation” sequencing methods. Moreover. An additional limitation of these techniques is that owing to the short length. FatiGO.2. and for the comparative analysis between different disease states or conditions . Generally. biological process and cellular component. recent enrichment tools also integrate information extracted from other databases as for instance. cloning and bacterial cell propagations that these techniques involve may result in a quantitative bias for different tags . the biological interpretation of such results is very challenging. which are then concatenated. In their work. These novel technologies have had an enormous impact on research in a short time period.3. a complex RNA sample is converted to a library of cDNA with adaptors attached to one or both ends.04. Biomed. Unfortunately. the use of biological knowledge accumulated in public databases by means of bioinformatics. Sequencing-based technologies In contrast to microarray technology. the sequence hits are counted to determine their density and distribution and then. for the consistent description of attributes of genes and gene products. Bioinformatics and Gene Ontology database As mentioned. the Kyoto Encyclopedia of Genes and ˜ Please cite this article in press as: V. which are applied to genes and proteins are then. Despite the more statistical robustness and less stringent standardization and replication requirements than those used for microarrays. 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]. however. No. The new features of next-generation sequencers have stimulated the development of new techniques that have expanded their applications. standard biological phrases. More speciﬁcally. Thus. Enrichment analysis would not be possible without appropriately structured databases such as Gene Ontology . Massively Parallel Signature Sequencing (MPSS) is a more sophisticated “clone-andcount” technique that also generates small tags of each mRNA species. García-Canas. with reference genomes available. to a reference genome or reference transcripts. is changing the way in which gene expression is studied [20. linked or associated with other Gene Ontology terms by trained curators at genome databases . which have been termed RNA-Seq methods. depending on the sequencing platform used. are subjected to massively parallel sequencing in short reads without bacterial cloning as a pre-requisite. the MPSS technology has been restricted to only a few specialized laboratories . which can complicate gene identiﬁcation . the many PCR ampliﬁcations. First.2009. including LongSAGE .g. or ontology. The principle behind enrichment analysis is that if a certain biological process is occurring in a given study. for example. That is. usually derived from one end of an mRNA. the use of Affymetrix microarray platform and stringent data analysis provided interesting insights into a homeostatic mechanism. However. J. Similarly to the concept adopted in SAGE and MPSS.. Last advances in emulsion PCR and non-cloning based methods for DNA ampliﬁcation. cloned and sequenced to determine the expression proﬁles of their corresponding mRNAs. a variety of high-throughput enrichment tools (e. 2. based on gene expression microarray data. This approach increases the probability for researchers to identify the correct biological processes most pertinent to the biological mechanism under study . 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).jpba. EASE. of Pages 15 ARTICLE IN PRESS V. ProfCom. the co-functioning genes involved should have a higher (enriched) potential to be selected as a relevant group by high-throughput screening technologies. Thus. 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. In the past few years. with or without ampliﬁcation (depending on the sequencing platform). most of these methods are based on expensive Sanger sequencing method and. the size of the sample tags should be increased in order to improve the precision and accuracy for detecting rare mRNAs . short reads are sufﬁcient to map their locations rather than sequencing the entire large genome. the abundance of a particular mRNA species is estimated from the count of tags derived from one of its ends. A recent work by Leong et al. Pharm. depending on the sequencing platform and method used.71]. also referred to as either “massive parallel” or “ultra-deep” sequencing. Over the last years. A recent study of the effects of soy isoﬂavones. their potential is underlined by their adoption in studies of transcriptomes during physiological changes. has revealed potential mechanisms of action for genistein by the combined use of GoMiner as the enrichment tool. (2009). GOminer..) have been developed since 2002 in order to assist microarray end user to understand the biological mechanisms behind the large set of regulated genes. therefore. García-Ca˜as et al. sequenced SAGE library rarely exhibits saturating tag counts that would indicate complete representation of the cellular transcriptome . sequencing-based techniques consist of counting tags of DNA fragments to provide “digital” representation of gene expression levels using sequencing.1016/j. Once mapped. Next-generation sequencing technologies apply distinct concepts and procedures aimed to increase sequencing throughput in a cost-effective and rapid manner. restriction enzymes are used to obtain short tags of 14–21 bp. powerful bioinformatics tools are needed in order to align the resulting short reads (30–300 bp). In SAGE-based methods. Gene Ontology provide a systematic and controlled language. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n 5 tain bioactive food constituents have also been investigated by microarray technology.
They differ among individuals. In addition. In a ﬁrst instance. 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 . Another important drawback in the proteome study is the huge dynamic concentration range of proteins in biological ﬂuids or tissues. peptides are in general better separated and analyzed by MS than proteins. Bottom-up approach The bottom-up approach is the most widely used in Proteomics since it can apply conventional or modern methodologies. fractionation of the proteome can be done by differential centrifugation into different populations of organelles  and also attending solubility properties into different cellular compartments. the proteome provides a “picture” of the impact of speciﬁc bioactive nutrients and diets in a certain organism. Chromatographic  and electrophoretic  strategies have also been applied to the pre-fractionation of the proteome.2009. being still in not enough concentration for subsequent analysis.96]. combined with the everyday more powerful. a 1010 dynamic range has been estimated in serum for protein concentration . nutrients or diets on the proteome. It is already known that dietary components can also modify the translation of RNA to proteins and the post-translational procedures . These studies are summarized in Table 2. This separation step is followed by analysis of the isolated proteins (or peptides) by mass spectrometry (MS) via the so-called “soft ionization” techniques. For this reason.1. Currently. ubiquitination. a compound found in processed garlic. such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). 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. rapid and automatic identiﬁcation of complex protein mixtures. The chemical tagging strategies. ˜ Please cite this article in press as: V. the introduction of difference gel electrophoresis (DIGE). 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. In a conventional approach. 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 . phosphorylation. in Proteomics fractionation and subsequent concentration of the proteome is often needed [95. Physical and chemical diversity of proteins are also higher than nucleic acids. involving the modiﬁcation of functional groups of amino acid residues. After image analysis of the 2DE gels. Thus. the importance of Proteomics as a tool to understand the effect of the diet on health has already been recognized by several authors [18. In this sense. tissue or cell in a particular moment. which evidently will inﬂuence chemical properties and functions of proteins. more precisely in classical 2DEMS. Moreover. although also LC-MS/MS has been applied with this purpose.. easy to use and affordable mass spectrometers. has brought about an important improvement. there are hundreds of different types of post-translational modiﬁcations (PTMs). Namely. In general. of Pages 15 ARTICLE IN PRESS V. DIGE methodology is based on the use of novel ultra high sensitive ﬂuorescent dyes (typically. the remaining proteins (in case of depletion) or the proteins in the fractions of interest remain dilute. in which gel variation is eliminated by loading different samples in the same gel . glycosylation. García-Ca˜as et al. In other cases post-translational regulation of proteins by dietary components can involve the modiﬁcation of their thiol groups . 3. however. There are limited studies on the effect of speciﬁc natural compounds. (2009).85–90]. sumoylation. including PTM in proteins (and peptides) is the methodology of choice when a speciﬁc population of the proteome is under study . it has been observed that dietary components such as diallyl disulﬁde. 3. It has become the alternative in certain application. time-consuming and presents low sensitivity. This situation causes many detection difﬁculties since a large number of proteins are below the level of sensitivity of the most advanced instruments. Both methodologies differ on the separation requirements and the type of MS instrumentation. it is dynamic and varies according to the cell type and functional state. 6 No.104].G Model PBA-7217. being the number of review papers higher than research papers . however. In the case of a Nutrigenomic study. up to now the application of Proteomics in Nutrigenomics is still rather limited. more than a single electrophoretic or chromatographic step is used to separate the thousands of proteins found in a biological sample. et al. J. it has been described the use of a library of combinatorial ligands. The selected analytical strategy for the detection of PTMs will depend on the type of modiﬁcation: acetylation. in order to reduce the complexity of the sample. PTMs are key to the control and modulation of many processes inside the cell. large-scale analyses of proteomes are accomplished by the combination of two-dimensional gel electrophoresis (2DE) followed by MS analysis . Most recently. and within the same cell depending on cell activity and state. Cy5 and Cy2) to label up to three different protein samples that will be separated in the same 2DE run. 2DE is the methodology that currently provides the highest protein species resolution capacity with low-instrumentation cost. depending strongly on staining and visualization techniques.jpba. loosing information about PTM. One of the main differences when working with proteins is that there is not an ampliﬁcation methodology for proteins comparable to PCR. Anal. the protein spots of interest are subsequently submitted to an ingel digestion step with a protease enzyme. Shotgun Proteomics is an advanced bottom-up approach in which a complex protein mixture is digested with endoproteinases of known speciﬁcity .019 . providing a comprehensive. cell types. two fundamental analytical strategies can be employed: the bottom-up and the top-down approach. 2DE is laborious.04. Biomed. MS and databases of protein sequences are then use in different ways for protein identiﬁcation. Although Proteomics has been scarcely applied to study the effect of nutrients on health. the main problem of the mentioned strategies is that after fractionation or depletion. / 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 . However. in which the molecular masses of the peptides from the protein digest are compared with those simulated of already sequenced proteins. Pharm. In spite of the higher sample complexity due to the huge number of peptidic species obtained from the protein mixture hydrolysate. doi:10. 2DE has also some limitations to separate highly hydrophobic biomolecules or proteins with extreme isoelectric point or molecular weight values. following: (i) the peptide mass ﬁngerprint (PMF) approach using MS data. most of them being based in the bottom-up approach. Advances in Proteomics The proteome is the set of expressed proteins at a given time under deﬁned conditions.1016/j. Cy3. The main limitation of the bottom-up approach is that information obtained is related to a fraction of the protein. García-Canas. the main drawback regarding this approach is that co-depletion of a certain fraction of the proteome with the abundant proteins can occur. Thus. has been shown to post-translationally modify proteins . An essential part of shotgun Proteomics is the use of powerful and highly resolving separation methods prior to MS analysis. etc.
TOF-TOF and the recently introduced hybrid linear ion trap (LTQ)-Orbitrap (OrbitrapTM ) . it permits the study of 2DE. coupled with MS or tandem MS [125. 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 .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. Some proteomic studies have also focused on the use of MALDI interface for chromatographic separations and MS. MALDI-TOF/TOF-MS 2DE. The vast majority is based on the combination of ion exchange chromatography and reversed-phase (RP) chromatography. of Pages 15 ARTICLE IN PRESS V. No. Pharm. nLC-ESI-Q-TOF-MS/MS 2DE. Special attention is currently being paid to the use of CE in microchips since it provides much shorter separation times . Liquid chromatography (LC)  and capillary electrophoresis (CE) [121. Biomed. García-Ca˜as et al. doi:10. a relatively new approach compared to bottomup. García-Canas. et al. lower sample and reagent consumption than nLC and CE. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n   [109–113]      7 Table 2 Applications of Proteomics in Nutrigenomics research. (2009). MALDI-TOF-MS 2DE. 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 .jpba.1016/j. as in the case of ultra performance LC systems (an LC system using sub-2 m packing columns combined with high operating pressures).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.126] (Fig. 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. On the other hand. Top-down approach A growing number of researchers are focusing on the use of topdown Proteomics. LC-MS/MS SELDI-TOF   Ref. 3. The main advantages over the bottom-up approach are that higher sequence coverage is obtained. MALDI-TOF-MS 2DE. Other novel couplings have been described. in which structure of proteins is studied through measurement of their intact mass followed by direct ion dissociation in the gas phase . as well as in multidimensional protein identiﬁcation technology (MudPIT). reversed phase (RP) and aqueous mobile phases are normally used for the chromatography of peptides. as for instance those based on hydrophilic interaction liquid chromatography (HILIC) . 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. MALDI-Q-TOF-MS/MS 2DE. since CE is a robust system that provides fast and high-resolution separations employing inexpensive capillaries . Anal. MALDI-TOF-MS 2DE. Special attention has been put in miniaturization of columns through the reduction of column diameters and sorbent particles.. Due to the analytical characteristics of these micro-devices further high-throughput in shotgun Proteomics is expected in the near future. 2).2009. to the more advanced hybrid instrumentation such us quadrupoletime of ﬂight (Q-TOF). 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  creating new and encouraging perspectives in the discovery of biomarkers in Nutrigenomics studies. Although physico-chemical diversity of peptides makes them well suited to be separated by different liquid-based separation modes.04. J. 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). MALDI-TOF-MS. MALDI-TOF-MS 2DE. CE combined with MS has also come out as an effective strategy for proteomic studies.G Model PBA-7217. 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. 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. .2.
CE coupling with high-resolution MS is one of the most promising methodology in top-down studies .2009. J. can help to save time and research effort in studies about the effect of bioactive food components on the proteome. of Pages 15 ARTICLE IN PRESS V. There are two major classes of protein micro. A key to the top-down approach is the capacity to fragment intact proteins inside the mass spectrometer. rapid and high-throughput Proteomic approach to identify differentially expressed peptides and proteins and. precludes its general use. Nevertheless. they can be subjected to a limited proteolysis to produce large polypeptides that are then analyzed employing topdown Proteomic approaches. 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. 8 No. After import data ﬁles from the microarray scanning sources and normalization and quantiﬁcation of protein microarrays. resolving power.(or nano) arrays: analytical and functional protein microarrays. mostly derived from the expensive superconducting magnet and liquid helium supply required. This technology enables to speed up the process signiﬁcantly with the capacity to run and analyze several hundreds samples in parallel per day. resulting in easier identiﬁcation of the modiﬁcation sites. García-Ca˜as et al. 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. Top-down Proteomics approaches are usually limited to simple protein mixtures since very complex spectra are usually generated by multiple charged proteins.G Model PBA-7217. This has been called the middle-down approach. PTM and it makes possible to discern between biomolecules with a high degree of sequence identity. improved ECD and ETC speed will be needed for future high-throughput CE-MS (and also LC-MS) applications in top-down Proteomics. Different SELDI chip types have different surfaces. Surface enhanced laser desorption ionization time-of-ﬂight mass spectrometry (SELDI-TOF-MS). and maintenance costs. although ultraviolet photodissociation (UVPD) . In this regard. Biomed. (2009). which also uses charge reduction by electron transfer . or detailed molecular ﬁngerprints. In general.jpba. depending on the type of proteins to be analyzed. such us MALDI-TOF/TOF. a variety of instruments have been used for top-down Proteomics. 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. Protein microarrays can be composed by recombinant protein molecules or antibodies immobilized in a high-density format on the surface of a substrate material. due to the capabilities of CE for complex protein separation. therefore. Workﬂow of multidimensional separation-MS/MS-based shotgun Proteomics strategy. ESI-IT and the novel OrbitrapTM . while consuming only a few microliters of sample. with a growing increase of works focused on the detection of protein biomarkers. a protein isolate from a previous fractionation or puriﬁcation step is directly infused to the high-resolution MS. Other Proteomic approaches New Proteomic approaches are under development.1016/j. This alternative technique is based on the fragmentation of multiple charged protein cations due to interaction with low-energy electrons. García-Canas. et al. maintaining the advantages of high sequence coverage and PTM information. antibodies. Pharm. and blackbody infrared radiative dissociation (BIRD)  can be found in literature. a statistical analysis has to be carried out. revealing the composition of the proteome. low-energy multiple collision-induced dissociation (CID) is used. Thus. Usually. doi:10. 2. One of the approaches that are gaining popularity is based on the array technology. receptors. to surfaces with a speciﬁc biomolecular afﬁnity (e. Anal. Today. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n Fig. infrared multiphoton dissociation (IRMPD) . being the antibody-based microarray the most common platform in proteomic studies . accuracy and sensitivity among present MS technologies .3. 3. A newer dissociation method similar to ECD is electron transfer dissociation (ETD). Fourier transform ion cyclotron resonance (FTICR) MS offers the highest mass resolution. an emerging tool for protein proﬁling and biomarker discovery applicable to minute amounts of starting material  can also be used as a complementary. Both charge reduction dissociation techniques. ESI-Q/TOF. N is the sample fraction. In those cases in which the molecular mass of proteins exceeds the analytical power of the mass spectrometer. its high purchase.g. Redrawn from . Alternative dissociation techniques such us electron capture dissociation (ECD) represent one of the most recent and signiﬁcant advances in tandem MS . However. range from chromatographic chemistries.. The microarray patterns generated can then be transformed into proteomic maps.04. enzymes and ligands) that bind one speciﬁc protein or group of proteins.. suitable for routine topdown Proteomics as recently reported .019 .
vitamins. and soybased estrogen analogues in cancer . vitamin A-deﬁcient Mr proﬁle. tissue. There are three basic approaches used in Metabolomic research. Metabolic proﬁling is a non-targeted strategy that focuses on the study of a group of related metabolites or a speciﬁc metabolic pathway. the metabolome can be described as the full set of endogenous or exogenous low molecular weight metabolic entities of approximately <1000 Da (metabolites). To date.1. In general. even more complex than in other areas of research. García-Canas.2009. such as a speciﬁc biomarker or reaction product. metabolism is dynamic and depends on the different cellular environments and physiological situation.. Metabolomics is an emerging technology. Some examples are the potential beneﬁts of ﬂavones in heart diseases.04. Application of Metabolomics in Nutrigenomics (or Nutritional Metabolomics) is. sion changed in response to vitamin A status from retinol-sufﬁcient and retinol-deﬁcient rats  (Fig. No. 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.1016/j. diet and other lifestyle parameters give rise to high metabolic variability [153–156]. Therefore. In this regard. metabolic ﬁngerprinting does not aim to identify all metabolites. 3. 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. dotted line. so new analytical techniques and methods are continuously being developed and will continue in the near future in order to achieve its goals. making it easy to exceed the linear range of the analytical technique employed. small peptides or carbohydrates. Pharm. so it is hard to understand the effects separately and to link directly metabolites to genes and proteins . 15. Redrawn from . As no single technique can be expected to meet all these requirements. Meanwhile.693. as a general result it can be stated that the inﬂuence of ˜ Please cite this article in press as: V. Humans feed on other organisms. Analytical techniques in Metabolomics Unlike Transcriptomics or Proteomics. . most Metabolomics studies in mammals have used urine as the vehicle for investigation and. Metabolomics has to deal with very different compounds of very diverse chemical and physical properties. many Metabolomics approaches can employ several analytical techniques [12. any change in metabolite concentration may describe better the biochemical state of a biological system than proteomic or transcriptomic variations. the biological activity of these compounds is an important topic of investigation in Nutrigenomics .G Model PBA-7217. (2009). 4. 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. according to some authors . but to compare patterns of metabolites that change in response to the cellular environment . each with its own metabolome. and its association with health beneﬁts.jpba. 4. Metabolites are the real endpoints of gene expression and of any physiological regulatory processes. The most common metabolites are amino acids. of stannols in cholesterol metabolism. metabolic proﬁling and metabolic ﬁngerprinting.151]. the relative concentration of metabolites in the bioﬂuids varies from millimolar level (or higher) to picomolar. Advances in Metabolomics As deﬁned by Trujillo et al. in the ﬁeld of Nutrigenomics blood and urine are the most likely candidates for sample choice . 4). which intend to determine a single chemical class of compounds (mRNA or proteins). 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 . doi:10. So far. J. so the number of different metabolomes that conform our diet is signiﬁcant enough to make Nutrigenomics a very complex discipline . / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n 9 Fig. organ. 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. vitamin A-sufﬁcient Mr proﬁle. Anal. but also on the identiﬁcation of certain metabolites such as cholesterol or glucose as biomarkers for health or disease status . Biomed. Because of the extensive consumption of polyphenols in the diet. 3). and the small pathway motifs that are present in a biological system (cell. lipids. The two most common analytical techniques used so far in Metabolomics are nuclear magnetic resonance (NMR) and MS. Moreover. One of the advantages of Nutritional Metabolomics is that there are several accessible body ﬂuids that contain possible biomarkers in human body. Thus. Metabolomics has diverse applications such as biomarker discovery. 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  (Fig. there are several challenges that Metabolomics has to face in human nutrition. Bold line. of Pages 15 ARTICLE IN PRESS V. García-Ca˜as et al.720. et al. however. The most relevant application of Metabolomics in Nutrigenomics is the possible health beneﬁts provided by the ingest of functional compounds.203 and 18. 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. determination of the metabolic effects of environmental changes in the body or early disease detection . target analysis. organism or species). In order to gain a detailed knowledge on human metabolism by means of Metabolomic tools.019 . In Nutritional Metabolomics the complex interactions between gut microﬂora and host metabolism as well as other extrinsic factors such as food habits. the effects of phytochemicals in human health are the most studied. Target analysis aims the quantitative measurement of selected analytes. Therefore.
As for Proteomics. García-Ca˜as et al. 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 . 3-(3-hydroxyphenyl) propionic acid. of Pages 15 ARTICLE IN PRESS V. like 1 H-detection triple resonance cold probes. J.  in the metabolomic identiﬁcation of biomarkers of individuals at risk of diabetes. other separation techniques have also been successfully coupled to MS instruments to carry out Nutritional Metabolomics. derivatization and analysis by GCTOF-MS have been optimized.. In another study. (2009). A summary of main Metabolomic applications in Nutrigenomics ﬁeld is listed in Table 3. 5).1016/j.2009. and 3hydroxycinnamic acid in rat urine using reverse phase gradient HPLC coupled with 1 H NMR . 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. have provided an improvement in detection sensitivity that will increase even further the number of applications of NMR in metabolomic studies . the most usual ionization techniques are the ones that use atmospheric pressure ionization. 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. This could be explained through the modulation of microbial gut metabolism produced by the polyphenols present in wine. providing limits of detection below 0. 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  (see Fig. Hybrid analyzers. et al. doi:10. Pharm. the effect of diet on urea cycle and purine metabolic pathways was demonstrated using both 1 H NMR and ion trap MS . Mass spectrometry (MS) approaches MS can be used as a standalone technique or most commonly combined with a preliminary chromatographic separation technique. Yin et al. The lack of sensitivity of NMR. Thus.  which stated that different strains of Salinibacter ruber could be differentiated by some characteristic metabolites. Anal. Apart of GC. an improvement in renal function and an increase in the urinary excretion of the osmolyte trimethylamine-N oxide has been proven . Biomed. especially ESI interfaces. or different approaches such as dynamic nuclear polarization. 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 . thus suggesting a metabolic alteration due to soy consumption . In this method. amino acids and carbohydrate levels in plasma from ﬁve healthy pre-menopauseal women.2. 4. Metabolic proﬁling after dietary intervention with soy isoﬂavones has also been determined by 1 H NMR differences in lipoprotein. However. 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. A complete methodology for metabolite elucidation is provided by Chen et al. has been one of the main problems of this analytical technique.1. high performance chromatography (HPLC) with MS (HPLC-MS) or CE-MS . 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. In another 1 H NMR study on the effect of soy isoﬂavones. in the range of g.1 g/ml for most phenolic acids. García-Canas.G Model PBA-7217. For example. 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. provide information about the structure of the detected metabolites . Using this analytical technique.1. A GC-MS method for the analysis of ﬂavonoids. the recent introduction of new instrumentation. UPLCTM -MS has been used in the determination of metabolic proﬁles in human urine . but can also give interesting results in Metabolomics. as for example Q-TOF. These characteristics make them ideal tools for metabolomic studies as was shown by Rosello-Mora et al. 4. in urine and plasma has been developed . GC-MS is so far the most common coupling for metabolomic studies. 10 No. ˜ Please cite this article in press as: V. Thus. These kinds of analyzers should lead the metabolomic studies in the near future. either gas chromatography (GC). have studied the relationship between the consumption of chamomile tea and some human biological responses. Coupling of NMR with separation techniques is less extended than in MS. Ofﬂine coupling with solid-phase extraction (SPE) or on-line coupling with HPLC are the most extended in metabolomic research. Wang et al. Metabolomics workﬂow in Nutrigenomics research. 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. liquid–liquid extraction. This method identiﬁed 11 phenolic acids in urine samples whose concentration increased after tea consumption.  have recently developed a reliable metabolic proﬁling method for human serum analysis using a C-18 column coupled to a Q-TOF-MS analyzer. diet in metabolism has been clearly established using these techniques. Nuclear magnetic resonance (NMR) approaches NMR is a high reproducibility technique that has demonstrated its great potential in Metabolomics studies . Fig.04. as it can work with a wide range of polarities . To overcome these limitations MS analyzers are usually coupled to separation techniques for metabolomic studies.jpba. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n polyphenols. a sub class of 4.1. have the advantages of mass accuracy given by a TOF analyzer combined with the possibility to fragment the ions and thus.019 . For example HPLC NMR has been used for the identiﬁcation of hippuric acid.
. organ) giving rise to the growing of a new area of biology called Systems Biology. there are different large-scale databases available on the web. which can be caused by either a biological difference or a methodological bias..1016/j. 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. J. 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 . Thus. (2009). protein. their very different concentrations and the numerous targets with different afﬁnities and speciﬁcities that they may have.jpba. Biomed. organs. tissue. NMR proﬁles of faeces from different subjects using two different extraction methods (water and methanol). 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. cell. García-Canas. García-Ca˜as et al. In particular for Nutritional Metabolomics the most interesting biochemical databases are the ones operated by the European Nutrigenomics Organization  that allows to upload and edit scientiﬁc information. from food complexity. the following challenge is to identify it and determine its biological signiﬁcance . ‘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.177]. Proteomics and Metabolomics represents powerful analytical platforms developed for the analysis of genes. proteins and metabolites. Metabolomic data reduction is normally accomplished using principal component analysis (PCA) or other related techniques.2. among others. doi:10. it has been necessary to develop strategies to convert the complex raw data obtained into useful information. The aim of PLSDA is to discriminate the complete peak list and reduce it with the most relevant ones .g.019 . Transcriptomics. The most common is partial least square discriminant analysis (PLS-DA). Once an unknown compound is determined. As described above.2009. Modern Systems Biology is the analysis of the relationships among the elements in a system in response to genetic or environmental perturbations. Redrawn from . 4. of Pages 15 ARTICLE IN PRESS V. the large number of different nutrients and bioactive food compounds. et al. For this matter. 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. However. Systems Biology exploits global data sets to derive useful information . which provides information on more then 2180 metabolites from human. Unlike any reductionist approach that would take these techniques individually. No.G Model PBA-7217. cells. individuals. with the goal of understanding the system or the emergent properties on the system [178–180]. / Journal of Pharmaceutical and Biomedical Analysis xxx (2009) xxx–xxx n 11 Fig. One of the most accessed databases available online is KEGG . or even ecosystems and it is regarded as an integrative approach of all information at the different levels of genomic expression (mRNA. metabolite). For all these reasons. Systems Biology approaches may encompass molecules. 5. in Nutrigenomic studies biologic responses to a bioactive food component may be subtle and.04. However. It is usually used as a ﬁrst step to have information about the quality of the data. therefore. PCA is a tool for exploratory data analysis that determines correlation differences among sample sets. Anal. 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. After data reduction a multivariate analysis is usually performed. Systems Biology Nutrigenomics has to face important difﬁculties derived. Pharm.   H NMR and HPLC-ESI-Q-TOF GC-TOF-MS Polyphenols Polyphenols Isoﬂavones Isoﬂavones 1 H NMR H NMR H NMR H NMR     1 1 1 ˜ Please cite this article in press as: V. 5. and the Human Metabolome Database [176.
bioinformatics and complete coverage at reasonable costs. it affects the ability of the technique to accurately detect differences in gene expression levels . 12 No.G Model PBA-7217. CE and MS have become the most used methodologies. providing unique opportunities to establish interrelationship between dietary components and the development and/or progression of diseases. 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. Also. As can be seen in the low number of proteomic applications in Nutrigenomic studies. This will aid in rapidly identifying new biomarkers for nutritional status and disease progression. J. Rapid advances in MS instrumentation. problems derived from element-to-element differences within a microarray and microarray-to-microarray differences need also to be solved [23. a Systems Biology approach has been applied to improve our knowledge about carbohydrate metabolism in yeast . although much less employed than the methodologies used today in Proteomics. doi:10. of Pages 15 ARTICLE IN PRESS V. García-Canas.jpba. RNA-Seq has very low background signal because DNA sequences can be unambiguously mapped to unique regions of the genome. On the other hand. 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. Although microarrays is currently the technique of choice for proﬁling RNA populations under different conditions. MS is essential for the systematic investigation in Proteomics. Despite these advantages. and to optimize and standardize microarray procedures .2009. 6. In this sense it has been proved the robustness of MudPIT for the analysis of complex mixtures of peptides. 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. 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. to identify critical factors affecting data quality. the MicroArray Quality Control (MAQC) Consortium establishes quality control criteria to ensure data quality. Apart from the everyday more sophisticated sample treatments and separation techniques. allowing detection of very low expressed mRNA. For instance. as well as with label-free hybridization strategies are being investigated as cheaper and sensitive alternatives to current optical detection systems [184. Sequencing depth is another important aspect that affects sequence coverage (percentage of transcripts surveyed). In addi- tion. RNA-Seq does not require cloning. In this sense. redox-active indicators. Fortunately.04. 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. Pharm. it is expected that new innovations in proteomic technology will help proteomic proﬁling to become standard practice in the Nutrigenomic ﬁeld. 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. For that. without prior knowledge. spliced transcript isoforms  and sequence variations such as single nucleotide polymorphisms (SNPs) in the transcribed regions .e. and designing a novel concept for dietary prevention and intervention of disease . low signal-to-noise ratios). however.. / 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. Also. (2009). the library construction involves some manipulation stages and procedures that could originate some bias .194]. conventional mass spectrometers are giving way to the more sophisticated and compact mass spectrometers. There is an evident need of development of improved or alternative technologies to become the routine analysis for proteome research a reality. some advantages of RNA-Seq over existing technologies make this technique valuable for comprehensive transcriptome studies. unlike hybridization-based approaches. Systems Biology. Future needs and developments in Nutrigenomics Despite the use of DNA microarrays is a powerful analytical approach. or nanoparticle labels.182]. Another important advantage of RNA-Seq relative to gene expression microarray is their ability to identify. In this regard. LC. especially if an important fraction of sequence reads match multiple locations in the genome. For large and complex transcriptomes. Although Systems Biology has been scarcely applied in Nutrigenomic studies.188]. 2DE.185]. In Proteomics.019 . based on sequencing both ends of each interrogated DNA fragment. determines the number of expressed genes and rare spliced isoforms detected and is directly proportional to the sequencing costs . 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 [72.1016/j. These probes offer high speciﬁcity and appear as good candidates for mismatch discrimination. longer reads and paired-end reads sequencing. Novel approaches focused on the use of electrochemical transducers in combination with either enzymatic. both in terms of hardware and software. Some of these problems are associated with the high background noise that specially hinders the detection of low signals (i. In a recent work. 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 . however. To achieve this. 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. First. there are technical limitations that have to be addressed for optimal implementation. mapping the short reads to the reference transcriptome can be complicated. Contrary to microarray. some interesting alternatives to typical linear probes have been proposed such as molecular beacon probes and peptide nucleic acids . 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) . Still. their potential is underlined by their adoption by other disciplines. the cost is likely to keep falling.. In such case. most of them hybrid instruments in a combination of two or more analyzers. et al. Next-generation methods in Transcriptomics will undoubtedly continue to technically improve in several ways within the next years. Anal. ˜ Please cite this article in press as: V. García-Ca˜as et al. however.186]. appropriate statistical models have to be used in order to ﬁlter through the large data sets and highlight only those important changes. have been proposed to help alleviate the problem . improvements in the resolution of peptides are desirable to provide increased protein coverage. one of the main limitations of RNA-Seq stems from the short length of the sequence reads provided. provided that sequencing depth is sufﬁcient . Biomed. which rely on continuous signals. A second problem is related to the efﬁciency and speciﬁcity of the hybridization reaction that is subject to variability and cross-hybridization and thus. Kohanski et al. are improving current technologies and have catalyzed the development of new Proteomic approaches. RNA-Seq currently faces several challenges including those concerning library construction issues.
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