- Email: [email protected]
Special Issue “Genome Regulation”
J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 33 8 1 –3 38 5
Available online at www.sciencedirect.com
www.elsevier.com/locate/jprot
Special Issue “Genome Regulation” ☆
Lausanne, March 2012 Dear Readers, It is our pleasure to introduce the present special issue on “Understanding genome regulation and diversity by mass spectrometry”. We cover mass spectrometry developed for and applied to genetics and epigenetics; RNA transcript analysis (rather than genome-wide transcriptomics); characterisation of protein complexes; validation of biomarkers; and to studies of nutritional bioactives. In other words we review many facets of biological mass spectrometry except: classical proteomics fields like protein expression profiling and biomarker/target discovery; metabolomics; and technology development for these two latter applications. So you may ask: why do I find such a collection of articles in the “Journal of Proteomics”? Well, two short answers are the following: 1. We intend to (re-)position proteomics as the key systems biology platform reaching beyond discovery of protein/ peptide biomarkers, targets or candidates thereof; and 2. We aim at (re-)positioning mass spectrometry as the key analytical technology for any kind of comprehensive biomolecular analysis, hereby focusing on DNA, RNA and proteins/ peptides. The human genome sequence has given us “the words” (only ~20,000 protein-coding genes though) but we are far from understanding the grammar and from being able to speak the language of human genomics. The complexity of life clearly scales with non-coding RNA and proteins and not with numbers of protein-expressing genes. The genomic individuality of humans can be partly explained by genetic variation in the form of single-nucleotide polymorphisms (SNPs) and copy number variants (CNVs) but these variants have so far only accounted for a relatively small percentage of interindividual variability in complex phenotypes and diseases. Epigenetics appears to represent a format and mechanism of how ☆
This article is part of a Special Issue entitled: Genome regulation and genetic diversity. Item group: IG000019. 1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2012.03.036
the environment, including nutrition, shapes the packaging and accessibility of the genome and influences long-term gene expression and thereby possibly even phenotype. The intuitive observation in life that environment shapes phenotype and experience impacts behavior may now be tracked down to the molecular mechanisms of e.g. DNA methylation and chromatin packaging by histone proteins, which have been known to be implicated in development. In view of all these facets of genomic sciences, the present issue attempts to address main regulation and diversity levels of the genome, i.e. where most of the complexity of higher organisms and their inter-individual variability emerge. We believe in and pursue the integration of genetics and epigenetics with the -omics to help understand this complexity. Mass spectrometry, proteomics and related disciplines are key players in this undertaking. The scope of the invited papers ranges therefore from assessing genetic predisposition (in our case: SNPs), via epigenetic regulation (histone codes and DNA methylation) and mRNA characterisation, to protein interactions and bioactive proteins and peptides. Consequently, our issue is markedly distinct from others typically found in proteomics journals, as it does neither contain any technology review, nor does it address biomarker discovery, nor does it focus on a particular class of molecules, a certain pathway or a given disease/condition. We herewith line up the distinguished contributions along the themes of predisposition (genetics), programming (epigenetics), transcription (RNA analysis), networks (protein interactions), diagnostics (biomarker validation) and bioactives (nutritional peptidomics).
1.
Predisposition — Genetics
Dominique Richter, Simone Harsch and colleagues from the Eduard F. Stange group at the Robert-Bosch Hospital, in Stuttgart, Germany, begin this issue by describing MALDI-TOF mass spectrometric screening of cholelithiasis risk markers in the HNF1α gene. The presented study complements their preceding work on gallstone disease (Bergheim et al., 2006; Renner et al., 2008; 2009; 2010), now focusing on the mutational analysis of hepatocyte nuclear factor 1α (HNF1α) using MALDI-TOF mass
3382
J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 33 8 1 –33 8 5
spectrometry for genotyping. Richter et al. identify HNF1α as a novel intestinal risk factor for cholesterol gallstones, thereby placing mass spectrometry as a genetic diagnostic tool in the highly health-care relevant field of clinical gastroenterology.
2.
Programming — Epigenetics
2.1.
DNA methylation
Allan Sheppard, Cameron McLean and Peter Gluckman from the Liggins Institute in Auckland, New Zealand, highlight the utility of mass spectrometry for DNA analysis which generates insights into phenotypic diversity and epigenomic variation. Epigenomic variation may underlie the phenotypic diversity that is not attributable to genomic sequence differences. Such processes provide the organism with the flexibility to respond to a changing environment within its lifetime, and perhaps even its offspring's lifetime, and may therefore confer a selective evolutionary advantage. A key molecular indicator of epigenomic variation in organisms is the chemical modification of DNA by methylation at specific nucleotide residues. Sheppard et al. discuss how mass spectrometry can be adapted and deployed to provide quantitative analysis of DNA methylation patterns across human and animal populations. Following this introductory review, Babu, Zhang, Sheppard et al. present their study on epigenetic regulation of the ABCG2 gene and its association with susceptibility to xenobiotic exposure, and this in a farm animal health context. Cells are protected from oxidative stress and xenobiotic exposure by a network of xenobiotic metabolizing enzymes (XME) that convert free oxidative radicals to less damaging metabolites, whereas efflux pumps remove toxins and XME derivatives from the cell. These mechanisms are well documented for hypoxia and multidrug resistance. Another relevant context is the exposure of ruminants to fungal toxins, which leads to hepatotoxicosis and skin eczema. Applying toxin challenge in sheep, Babu et al. investigated possible epigenetic regulations in cellular responses to such xenobiotic exposure, concentrating on the phase III defense efflux protein ABCG2. The Sheppard group shows that resistance to skin eczema positively correlates with ABCG2 gene expression, and inversely associates with DNA methylation at CpG sites in the regulatory region of ABCG2. Mass spectrometric DNA methylation analysis resolved individual CpG sites varying with disease progression, enabling fine mapping of underpinning transcription factor bindings, thereby revealing epigenetic mechanisms as being important to xenobiotic responses.
2.2.
Histone codes
Ole N. Jensen and his group members Simone Sidoli and Lei Cheng, from the University of Southern Denmark in Odense review proteomics in chromatin biology and epigenetics and the role of mass spectrometry in the elucidation of post-translational modifications of histone proteins. Histone proteins contribute to the maintenance and regulation of the dynamic chromatin structure, to gene activation, DNA repair and many other processes in the cell nucleus. Sitespecific reversible and irreversible post-translational modifications of histone proteins mediate biological functions,
including recruitment of transcription factors to specific DNA regions, assembly of epigenetic reader/writer/eraser complexes onto DNA, and modulation of DNA–protein interactions. Histones thereby regulate chromatin structure and function, propagate inheritance and provide memory functions in the cell. Sidoli et al. discuss a range of analytical methods and various mass spectrometry-based approaches for histone analysis, from sample preparation to data interpretation.
3.
Transcription — RNA analysis
Transcriptomics has typically been performed with wholegenome DNA chips or targeted oligonucleiotide arrays and is now advancing from hybridization-based to sequencing-bysynthesis platforms such as RNA Seq. This development results in a quantum leap in data quantity output requiring substantial bioinformatic resources and putting personal genomics (also at DNA level) within reach of laboratories, clinics and eventually even individual patients or consumers. Mass spectrometry has and will not compete with this kind of impressive genome-wide throughput. However, with its unique information richness in terms of molecular structures and quantities, it significantly contributes to RNA transcript research. Anders Giessing and Finn Kirpekar from the University of Southern Denmark, Odense, discuss the role of mass spectrometry in studying the biology of RNA and its modifications. They focus on questions in RNA biology, which have been answered by mass spectrometry. The biological applications are divided into analyses performed at the “building block” level and investigations involving mass spectrometry at the “oligomer” level. The same biological issues appear in the “building block” and the “oligomer” sections, a structure that reflects the intention of their review: it is the biology that drives and determines the applied technology and not vice versa. Transfer ribonucleic acids (tRNAs) are the non-coding RNAs that link gene transcription with protein translation. Complementing our view of RNA, Collin Wetzel and Patrick A. Limbach from the University of Cincinnati, Ohio, USA have contributed an article on global identification of tRNAs by liquid chromatography coupled to mass spectrometry (LC–MS). Few techniques exist for the global identification of tRNAs at RNA and posttranscriptional RNA levels. Patrick Limbach's laboratory introduced the concept of signature enzymatic digestion products (SDPs) for tRNA identification using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Here, they examine the applicability of LC–MS and LC–MS/MS for global identification of bacterial tRNAs via their SDPs using Escherichia coli as the model system. The deployment of LC– MS/MS was found to improve the accuracy of SDP assignments through confirming sequence information.
4.
Networks — Protein interaction
Proteins do not act alone. Rather, they form complexes with each other, with DNA and RNA, with nutrients, metabolites, substrates, ligands, drugs and so forth. Protein interaction studies at large scale, high throughput and with a global perspective
J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 33 8 1 –3 38 5
have therefore developed into a key discipline in proteomics leveraged into systems biology, thereby providing insights into another level of genome complexity and regulation.
4.1.
Protein–protein interactions
As an example of such investigations, Simone Lemeer and coauthors from Bernhard Küster's team at the Technical University Munich, Germany, present a study on phosphotyrosinemediated protein interactions of the discoidin domain receptor 1. The receptor tyrosine kinase DDR1 has been implicated in multiple human cancers and fibrosis and is targeted by the leukemia drug Gleevec. This suggests that DDR1 might be a new therapeutic target. Lemeer et al. investigated DDR1 proximal signaling by proteomic analysis of protein–protein interactions. They identified all known interactors of DDR1 and localized them to specific phosphotyrosine residues on the receptor. In addition, they identified numerous signaling proteins as new putative phosphotyrosine mediated interactors. These identified proteins have roles in early steps of the signaling cascade, propagating the signal from the DDR1 receptor into the cell.
4.2.
Protein–RNA interactions
Protein–RNA complexes play important roles in many cellular functions such as growth and differentiation along the various cell cycle stages. With their function and activity being directly coupled to the structural arrangement of their components – namely the proteins and ribonucleic acids – the investigation of protein–RNA interactions is of high functional and structural importance. In this perspective, Carla Schmidt et al. from Henning Urlaub's team from the Max-PIanck Institute in Göttingen, Germany, review the current investigation of protein–RNA interactions by mass spectrometry and discuss techniques and applications: they address prominent examples of protein–RNA complexes, describe purification strategies, plus various mass spectrometric techniques and applications to study protein–RNA complexes. The authors cover both the analysis of intact complexes and proteome- and crosslinking-based approaches.
5. Targets and biomarkers — Deeper proteome insights and candidate validation Classical discovery-mode proteomic technologies have greatly expanded our views on complex proteomes and their variation as a function of condition or stimulus. However, due to their stochastic nature of data acquisition, both reproducibility and sensitivity of shotgun techniques have reached certain limits when studying proteomes of higher organisms. Therefore, such workflows have been complemented by targeted proteomic approaches which can be understood as MS-based ELISAs: in a highly multiplexed fashion, several target proteins can be analyzed and quantified against a complex proteome background. Alessio Maiolica and co-workers from Ruedi Aebersold's group at the Swiss Federal Institute of Technology (ETH), Zurich, Switzerland, deliver an overview on targeted proteome investigation via selected-reaction monitoring (SRM) mass spectrometry
3383
and compare these approaches to antibody-based platforms. The emphasis of their review is on SRM-MS, emerging tools to control error rates in targeted proteomic experiments, and representative application examples. Another application of the above mentioned “MS-ELISAs” is validation: once candidate targets and markers have been discovered (e.g. by shotgun workflows), they need to be validated at several levels. A first important such validation step is the confirmation of condition-specific variation of protein levels by the above mentioned targeted MS techniques. Dominik Domanski, and co-authors from Christoph H. Borchers' group at the University of Victoria, British Columbia, Canada, report in this context on multiplexed MRM for biomarkers that distinguish iron-deficiency anemia from anemia of inflammation among normal and iron-metabolism disorder mouse models. Their initial panel of potential biomarkers was based on the analysis of 14 proteins expressed by candidate genes involved in iron transport and metabolism. Based on this study, Domanski et al. consolidated a panel of eight protein markers that clearly distinguishes the normal from the disease models. The group now suggests further validation of this candidate biomarker panel in human samples in an effort to differentiate anemia, inflammation, and combinations thereof. A second clinical application addresses three common urological diseases, namely bladder cancer, urinary tract infection, and hematuria. Yi-Ting Chen and collaborators, including as partner the Borchers group, present a multiplexed MRM-based quantification of 63 proteins in human urine for discovery of potential bladder cancer biomarkers. Seventeen bladder cancer biomarkers were previously discovered by shotgun discovery proteomics including multiplexed stable-isotope labeling. These findings were validated by MRM-MS data that were translated into a six-peptide marker panel.
6.
Bioactives — Nutritional peptidomics
Understanding nature and bioactivity of nutritional peptides means comprehending an important level of environmental regulation of the human genome, because diet is the environmental factor with the most profound life-long influence on health. Other dietary ingredients such as fatty acids bind to transcription factors by which they modify acute gene expression and dietary methyl donors can change promoter methylation status thereby altering long-term expression. Hence, nutrition has a strong impact on short- and long-term gene regulation. Alexandre Panchaud, Michael Affolter from the the Nestlé Research Center and Martin Kussmann from the Nestlé Institute of Health Sciences, both located in Lausanne, Switzerland, have therefore contributed an article on bioactive nutritional peptides and proteins and describe how to analyze food bioactives and their health effects by “nutripeptidomics”. They approach the theme from three angles, namely the analysis, the discovery and the biology perspective. Food peptides derive from parent food proteins via in vitro hydrolysis (processing) or in vivo digestion by various unspecific and specific proteases, as opposed to the tryptic peptides typically generated in biomarker proteomics. Moreover, many food genomes are less well annotated than e.g. the human genome. Bioactive peptides can be discovered either empirically or by prediction. Panchaud
3384
J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 33 8 1 –33 8 5
et al. explain both the classical hydrolysis strategy and the bioinformatics-driven reversed genome engineering. In order to exert bioactivity, food peptides must be either ingested and then reach the intestine in their intact form or be liberated in situ from their parent proteins to act locally, that is in the gut, or even systemically, i.e. through the blood stream. With this collection of articles about predisposition, programming, transcription, interactions, biomarkers and bioactivity, all laid down in and emerging from complex genomes, we hope to have given you a refreshing perspective on how mass spectrometry can help understand regulation and variability of such complex genomes, beyond classical discovery proteomics. We thereby intended to reinforce the position of mass spectrometry and proteomics at the center of systems biology-oriented, technology-rooted, but not technology-driven and thereby integrated approaches to understand health and disease. We hope you will appreciate this different view and enjoy browsing and reading this special issue. Thank you for your kind interest, with our best wishes, Alexandre, Michael and Martin.
Prof. Martin Kussmann, PhD. As of February 2011, Martin has joined the Nestlé Institute of Health Sciences at the EPFL (Ecole Polytechnique Fédérale Lausanne) as Head of the Proteomics and Metabonomics Core, which he is building from scratch and managing. Since June 2009, Martin is Honorary Professor for Nutritional Science at the Faculty of Science, Aarhus University, Denmark. From March 2003 to January 2011, Martin had been leading the Functional Genomics Group at the Nestlé Research Centre and was responsible for NutriGenomics and NutriEpiGenetics. Being educated as an analytical biochemist, Martin has acquired research experience in the pharmaceutical (PharmaciaUpjohn/Sweden, Cerbios-Pharma/Switzerland), biotechnological start-up (Prof. Hochstrasser, GeneProt/ Switzerland) and nutritional (Nestlé) industry. Martin holds a B.Sc. from the University of Aachen, Germany, and a M.Sc. from the University of Konstanz, Germany. He performed his doctoral research in Konstanz and at the University of California, San Francisco, USA (Prof. Burlingame). During his doctorate and post-doctorate (Prof. Roepstorff, University of Southern Denmark, Odense), he has specialized in analytical biochemistry, proteomics and genomics. Martin has (co-) authored 70 peer-reviewed publications, has edited books and journal issues, and is an internationally requested author and speaker. He is a member of the ASBMB (Am. Soc. Biochem. Mol. Biol.); DGMS (German Soc. Mass Spectrom.); EuPA (Eur. Proteome Assoc.); NuGO (European Nutrigenomics Organization); the Nutrition Society; SGMS (Swiss Soc. Mass Spectrom.); and SPS (Swiss Proteomics Soc.). He is an Editorial Board Member of Frontiers in Nutrigenomics; the Journal of Proteomics; The Open Journal of Proteomics; Proteomics Insights; and Endocrine, Metabolic and Immune DisordersDrug Targets (EMID-DT).
Alexandre PANCHAUD, Ph.D. Alexandre has a broad interdisciplinary and international background with experience from food industry and academia. Alexandre obtained his PhD in a joint collaboration between University of Lausanne and Nestlé Research Center with the focus of developing quantitative proteomics methods to be applied to the field of nutrigenomics or microbial pathogenicity. In 2008, Alexandre obtained a beginner fellowship from the Swiss National Science Foundation and moved to Seattle to join the group of Prof. David Goodlett. In 2009, he was granted an advanced fellowship from the same foundation to pursue his work. During this time Alexandre was involved in the development of proteomics techniques using data-independent acqisition technique referred to as PAcIFIC. In parallel he was aslo involved in protein structure analysis by cross-linking and mass spectrometry where he for example developed a tool called xComb to interrogate cross-linked peptides. In 2010, Alexandre moved back to Switzerland where he joined the Nestlé Research Center as an R&D specialist to lead projects in the field of biomarker discovery in nutritional interventions as well as food peptidomics for the characterization of food ingredients. In 2011, he was also appointed Protein and Peptide pipeline manager in which he deals with the management of new ingredients for functional foods.
Michael Affolter, PhD. Michael is a senior research scientist in the Functional Genomics Group at the Nestlé Research Centre (Lausanne, Switzerland) and is heading the proteomics platform with a special focus to implement and apply new technologies for protein and peptide analyses in the context of Nutrigenomics. His team develops and integrates qualitative and quantitative mass spectrometry based protein and peptide profiling with bioinformatics and computational molecular science. Major application fields are immunology, diabetes, obesity, and digestive health. Being educated as an analytical biochemist, Michael has acquired extensive research experience over the last eighteen years in the biotechnological and nutritional industry. Michael holds a M.Sc. and a Ph.D. in Biochemistry, obtained at the University of Berne, Switzerland, with experience in the field of protein sequence analysis (Edman sequencing). During his post-doctorate work with Prof. R. Aebersold (Biomedical Research Centre, University of British Columbia, Vancouver, Canada), he has specialized in analytical protein biochemistry and mass spectrometry based proteomics. Michael is an active board member of the SPS (Swiss Proteomics Society) and the SGMS (Swiss Group for Mass Spectrometry). He has (co-) authored more than 40 peerreviewed publications and book chapters, and filed over 10 patent applications.
J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 33 8 1 –3 38 5
Martin Kussmann Guest Editor Proteomics & Metabonomics Core, Nestlé Institute of Health Sciences, Lausanne, Switzerland and Faculty of Life Sciences, Aarhus University, Aarhus, Denmark. Corresponding author. Postal address: EPFL Campus, Quartier de l'innovation, Bâtiment G, 1015 Lausanne, Switzerland E-mail address: [email protected]. Michael Affolter Guest Editor Functional Genomics Group, Nestlé Research Center, Lausanne, Switzerland
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Alexandre Panchaud Guest Editor Functional Genomics Group, Nestlé Research Center, Lausanne, Switzerland
Available online at www.sciencedirect.com
www.elsevier.com/locate/jprot
Special Issue “Genome Regulation” ☆
Lausanne, March 2012 Dear Readers, It is our pleasure to introduce the present special issue on “Understanding genome regulation and diversity by mass spectrometry”. We cover mass spectrometry developed for and applied to genetics and epigenetics; RNA transcript analysis (rather than genome-wide transcriptomics); characterisation of protein complexes; validation of biomarkers; and to studies of nutritional bioactives. In other words we review many facets of biological mass spectrometry except: classical proteomics fields like protein expression profiling and biomarker/target discovery; metabolomics; and technology development for these two latter applications. So you may ask: why do I find such a collection of articles in the “Journal of Proteomics”? Well, two short answers are the following: 1. We intend to (re-)position proteomics as the key systems biology platform reaching beyond discovery of protein/ peptide biomarkers, targets or candidates thereof; and 2. We aim at (re-)positioning mass spectrometry as the key analytical technology for any kind of comprehensive biomolecular analysis, hereby focusing on DNA, RNA and proteins/ peptides. The human genome sequence has given us “the words” (only ~20,000 protein-coding genes though) but we are far from understanding the grammar and from being able to speak the language of human genomics. The complexity of life clearly scales with non-coding RNA and proteins and not with numbers of protein-expressing genes. The genomic individuality of humans can be partly explained by genetic variation in the form of single-nucleotide polymorphisms (SNPs) and copy number variants (CNVs) but these variants have so far only accounted for a relatively small percentage of interindividual variability in complex phenotypes and diseases. Epigenetics appears to represent a format and mechanism of how ☆
This article is part of a Special Issue entitled: Genome regulation and genetic diversity. Item group: IG000019. 1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2012.03.036
the environment, including nutrition, shapes the packaging and accessibility of the genome and influences long-term gene expression and thereby possibly even phenotype. The intuitive observation in life that environment shapes phenotype and experience impacts behavior may now be tracked down to the molecular mechanisms of e.g. DNA methylation and chromatin packaging by histone proteins, which have been known to be implicated in development. In view of all these facets of genomic sciences, the present issue attempts to address main regulation and diversity levels of the genome, i.e. where most of the complexity of higher organisms and their inter-individual variability emerge. We believe in and pursue the integration of genetics and epigenetics with the -omics to help understand this complexity. Mass spectrometry, proteomics and related disciplines are key players in this undertaking. The scope of the invited papers ranges therefore from assessing genetic predisposition (in our case: SNPs), via epigenetic regulation (histone codes and DNA methylation) and mRNA characterisation, to protein interactions and bioactive proteins and peptides. Consequently, our issue is markedly distinct from others typically found in proteomics journals, as it does neither contain any technology review, nor does it address biomarker discovery, nor does it focus on a particular class of molecules, a certain pathway or a given disease/condition. We herewith line up the distinguished contributions along the themes of predisposition (genetics), programming (epigenetics), transcription (RNA analysis), networks (protein interactions), diagnostics (biomarker validation) and bioactives (nutritional peptidomics).
1.
Predisposition — Genetics
Dominique Richter, Simone Harsch and colleagues from the Eduard F. Stange group at the Robert-Bosch Hospital, in Stuttgart, Germany, begin this issue by describing MALDI-TOF mass spectrometric screening of cholelithiasis risk markers in the HNF1α gene. The presented study complements their preceding work on gallstone disease (Bergheim et al., 2006; Renner et al., 2008; 2009; 2010), now focusing on the mutational analysis of hepatocyte nuclear factor 1α (HNF1α) using MALDI-TOF mass
3382
J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 33 8 1 –33 8 5
spectrometry for genotyping. Richter et al. identify HNF1α as a novel intestinal risk factor for cholesterol gallstones, thereby placing mass spectrometry as a genetic diagnostic tool in the highly health-care relevant field of clinical gastroenterology.
2.
Programming — Epigenetics
2.1.
DNA methylation
Allan Sheppard, Cameron McLean and Peter Gluckman from the Liggins Institute in Auckland, New Zealand, highlight the utility of mass spectrometry for DNA analysis which generates insights into phenotypic diversity and epigenomic variation. Epigenomic variation may underlie the phenotypic diversity that is not attributable to genomic sequence differences. Such processes provide the organism with the flexibility to respond to a changing environment within its lifetime, and perhaps even its offspring's lifetime, and may therefore confer a selective evolutionary advantage. A key molecular indicator of epigenomic variation in organisms is the chemical modification of DNA by methylation at specific nucleotide residues. Sheppard et al. discuss how mass spectrometry can be adapted and deployed to provide quantitative analysis of DNA methylation patterns across human and animal populations. Following this introductory review, Babu, Zhang, Sheppard et al. present their study on epigenetic regulation of the ABCG2 gene and its association with susceptibility to xenobiotic exposure, and this in a farm animal health context. Cells are protected from oxidative stress and xenobiotic exposure by a network of xenobiotic metabolizing enzymes (XME) that convert free oxidative radicals to less damaging metabolites, whereas efflux pumps remove toxins and XME derivatives from the cell. These mechanisms are well documented for hypoxia and multidrug resistance. Another relevant context is the exposure of ruminants to fungal toxins, which leads to hepatotoxicosis and skin eczema. Applying toxin challenge in sheep, Babu et al. investigated possible epigenetic regulations in cellular responses to such xenobiotic exposure, concentrating on the phase III defense efflux protein ABCG2. The Sheppard group shows that resistance to skin eczema positively correlates with ABCG2 gene expression, and inversely associates with DNA methylation at CpG sites in the regulatory region of ABCG2. Mass spectrometric DNA methylation analysis resolved individual CpG sites varying with disease progression, enabling fine mapping of underpinning transcription factor bindings, thereby revealing epigenetic mechanisms as being important to xenobiotic responses.
2.2.
Histone codes
Ole N. Jensen and his group members Simone Sidoli and Lei Cheng, from the University of Southern Denmark in Odense review proteomics in chromatin biology and epigenetics and the role of mass spectrometry in the elucidation of post-translational modifications of histone proteins. Histone proteins contribute to the maintenance and regulation of the dynamic chromatin structure, to gene activation, DNA repair and many other processes in the cell nucleus. Sitespecific reversible and irreversible post-translational modifications of histone proteins mediate biological functions,
including recruitment of transcription factors to specific DNA regions, assembly of epigenetic reader/writer/eraser complexes onto DNA, and modulation of DNA–protein interactions. Histones thereby regulate chromatin structure and function, propagate inheritance and provide memory functions in the cell. Sidoli et al. discuss a range of analytical methods and various mass spectrometry-based approaches for histone analysis, from sample preparation to data interpretation.
3.
Transcription — RNA analysis
Transcriptomics has typically been performed with wholegenome DNA chips or targeted oligonucleiotide arrays and is now advancing from hybridization-based to sequencing-bysynthesis platforms such as RNA Seq. This development results in a quantum leap in data quantity output requiring substantial bioinformatic resources and putting personal genomics (also at DNA level) within reach of laboratories, clinics and eventually even individual patients or consumers. Mass spectrometry has and will not compete with this kind of impressive genome-wide throughput. However, with its unique information richness in terms of molecular structures and quantities, it significantly contributes to RNA transcript research. Anders Giessing and Finn Kirpekar from the University of Southern Denmark, Odense, discuss the role of mass spectrometry in studying the biology of RNA and its modifications. They focus on questions in RNA biology, which have been answered by mass spectrometry. The biological applications are divided into analyses performed at the “building block” level and investigations involving mass spectrometry at the “oligomer” level. The same biological issues appear in the “building block” and the “oligomer” sections, a structure that reflects the intention of their review: it is the biology that drives and determines the applied technology and not vice versa. Transfer ribonucleic acids (tRNAs) are the non-coding RNAs that link gene transcription with protein translation. Complementing our view of RNA, Collin Wetzel and Patrick A. Limbach from the University of Cincinnati, Ohio, USA have contributed an article on global identification of tRNAs by liquid chromatography coupled to mass spectrometry (LC–MS). Few techniques exist for the global identification of tRNAs at RNA and posttranscriptional RNA levels. Patrick Limbach's laboratory introduced the concept of signature enzymatic digestion products (SDPs) for tRNA identification using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Here, they examine the applicability of LC–MS and LC–MS/MS for global identification of bacterial tRNAs via their SDPs using Escherichia coli as the model system. The deployment of LC– MS/MS was found to improve the accuracy of SDP assignments through confirming sequence information.
4.
Networks — Protein interaction
Proteins do not act alone. Rather, they form complexes with each other, with DNA and RNA, with nutrients, metabolites, substrates, ligands, drugs and so forth. Protein interaction studies at large scale, high throughput and with a global perspective
J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 33 8 1 –3 38 5
have therefore developed into a key discipline in proteomics leveraged into systems biology, thereby providing insights into another level of genome complexity and regulation.
4.1.
Protein–protein interactions
As an example of such investigations, Simone Lemeer and coauthors from Bernhard Küster's team at the Technical University Munich, Germany, present a study on phosphotyrosinemediated protein interactions of the discoidin domain receptor 1. The receptor tyrosine kinase DDR1 has been implicated in multiple human cancers and fibrosis and is targeted by the leukemia drug Gleevec. This suggests that DDR1 might be a new therapeutic target. Lemeer et al. investigated DDR1 proximal signaling by proteomic analysis of protein–protein interactions. They identified all known interactors of DDR1 and localized them to specific phosphotyrosine residues on the receptor. In addition, they identified numerous signaling proteins as new putative phosphotyrosine mediated interactors. These identified proteins have roles in early steps of the signaling cascade, propagating the signal from the DDR1 receptor into the cell.
4.2.
Protein–RNA interactions
Protein–RNA complexes play important roles in many cellular functions such as growth and differentiation along the various cell cycle stages. With their function and activity being directly coupled to the structural arrangement of their components – namely the proteins and ribonucleic acids – the investigation of protein–RNA interactions is of high functional and structural importance. In this perspective, Carla Schmidt et al. from Henning Urlaub's team from the Max-PIanck Institute in Göttingen, Germany, review the current investigation of protein–RNA interactions by mass spectrometry and discuss techniques and applications: they address prominent examples of protein–RNA complexes, describe purification strategies, plus various mass spectrometric techniques and applications to study protein–RNA complexes. The authors cover both the analysis of intact complexes and proteome- and crosslinking-based approaches.
5. Targets and biomarkers — Deeper proteome insights and candidate validation Classical discovery-mode proteomic technologies have greatly expanded our views on complex proteomes and their variation as a function of condition or stimulus. However, due to their stochastic nature of data acquisition, both reproducibility and sensitivity of shotgun techniques have reached certain limits when studying proteomes of higher organisms. Therefore, such workflows have been complemented by targeted proteomic approaches which can be understood as MS-based ELISAs: in a highly multiplexed fashion, several target proteins can be analyzed and quantified against a complex proteome background. Alessio Maiolica and co-workers from Ruedi Aebersold's group at the Swiss Federal Institute of Technology (ETH), Zurich, Switzerland, deliver an overview on targeted proteome investigation via selected-reaction monitoring (SRM) mass spectrometry
3383
and compare these approaches to antibody-based platforms. The emphasis of their review is on SRM-MS, emerging tools to control error rates in targeted proteomic experiments, and representative application examples. Another application of the above mentioned “MS-ELISAs” is validation: once candidate targets and markers have been discovered (e.g. by shotgun workflows), they need to be validated at several levels. A first important such validation step is the confirmation of condition-specific variation of protein levels by the above mentioned targeted MS techniques. Dominik Domanski, and co-authors from Christoph H. Borchers' group at the University of Victoria, British Columbia, Canada, report in this context on multiplexed MRM for biomarkers that distinguish iron-deficiency anemia from anemia of inflammation among normal and iron-metabolism disorder mouse models. Their initial panel of potential biomarkers was based on the analysis of 14 proteins expressed by candidate genes involved in iron transport and metabolism. Based on this study, Domanski et al. consolidated a panel of eight protein markers that clearly distinguishes the normal from the disease models. The group now suggests further validation of this candidate biomarker panel in human samples in an effort to differentiate anemia, inflammation, and combinations thereof. A second clinical application addresses three common urological diseases, namely bladder cancer, urinary tract infection, and hematuria. Yi-Ting Chen and collaborators, including as partner the Borchers group, present a multiplexed MRM-based quantification of 63 proteins in human urine for discovery of potential bladder cancer biomarkers. Seventeen bladder cancer biomarkers were previously discovered by shotgun discovery proteomics including multiplexed stable-isotope labeling. These findings were validated by MRM-MS data that were translated into a six-peptide marker panel.
6.
Bioactives — Nutritional peptidomics
Understanding nature and bioactivity of nutritional peptides means comprehending an important level of environmental regulation of the human genome, because diet is the environmental factor with the most profound life-long influence on health. Other dietary ingredients such as fatty acids bind to transcription factors by which they modify acute gene expression and dietary methyl donors can change promoter methylation status thereby altering long-term expression. Hence, nutrition has a strong impact on short- and long-term gene regulation. Alexandre Panchaud, Michael Affolter from the the Nestlé Research Center and Martin Kussmann from the Nestlé Institute of Health Sciences, both located in Lausanne, Switzerland, have therefore contributed an article on bioactive nutritional peptides and proteins and describe how to analyze food bioactives and their health effects by “nutripeptidomics”. They approach the theme from three angles, namely the analysis, the discovery and the biology perspective. Food peptides derive from parent food proteins via in vitro hydrolysis (processing) or in vivo digestion by various unspecific and specific proteases, as opposed to the tryptic peptides typically generated in biomarker proteomics. Moreover, many food genomes are less well annotated than e.g. the human genome. Bioactive peptides can be discovered either empirically or by prediction. Panchaud
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et al. explain both the classical hydrolysis strategy and the bioinformatics-driven reversed genome engineering. In order to exert bioactivity, food peptides must be either ingested and then reach the intestine in their intact form or be liberated in situ from their parent proteins to act locally, that is in the gut, or even systemically, i.e. through the blood stream. With this collection of articles about predisposition, programming, transcription, interactions, biomarkers and bioactivity, all laid down in and emerging from complex genomes, we hope to have given you a refreshing perspective on how mass spectrometry can help understand regulation and variability of such complex genomes, beyond classical discovery proteomics. We thereby intended to reinforce the position of mass spectrometry and proteomics at the center of systems biology-oriented, technology-rooted, but not technology-driven and thereby integrated approaches to understand health and disease. We hope you will appreciate this different view and enjoy browsing and reading this special issue. Thank you for your kind interest, with our best wishes, Alexandre, Michael and Martin.
Prof. Martin Kussmann, PhD. As of February 2011, Martin has joined the Nestlé Institute of Health Sciences at the EPFL (Ecole Polytechnique Fédérale Lausanne) as Head of the Proteomics and Metabonomics Core, which he is building from scratch and managing. Since June 2009, Martin is Honorary Professor for Nutritional Science at the Faculty of Science, Aarhus University, Denmark. From March 2003 to January 2011, Martin had been leading the Functional Genomics Group at the Nestlé Research Centre and was responsible for NutriGenomics and NutriEpiGenetics. Being educated as an analytical biochemist, Martin has acquired research experience in the pharmaceutical (PharmaciaUpjohn/Sweden, Cerbios-Pharma/Switzerland), biotechnological start-up (Prof. Hochstrasser, GeneProt/ Switzerland) and nutritional (Nestlé) industry. Martin holds a B.Sc. from the University of Aachen, Germany, and a M.Sc. from the University of Konstanz, Germany. He performed his doctoral research in Konstanz and at the University of California, San Francisco, USA (Prof. Burlingame). During his doctorate and post-doctorate (Prof. Roepstorff, University of Southern Denmark, Odense), he has specialized in analytical biochemistry, proteomics and genomics. Martin has (co-) authored 70 peer-reviewed publications, has edited books and journal issues, and is an internationally requested author and speaker. He is a member of the ASBMB (Am. Soc. Biochem. Mol. Biol.); DGMS (German Soc. Mass Spectrom.); EuPA (Eur. Proteome Assoc.); NuGO (European Nutrigenomics Organization); the Nutrition Society; SGMS (Swiss Soc. Mass Spectrom.); and SPS (Swiss Proteomics Soc.). He is an Editorial Board Member of Frontiers in Nutrigenomics; the Journal of Proteomics; The Open Journal of Proteomics; Proteomics Insights; and Endocrine, Metabolic and Immune DisordersDrug Targets (EMID-DT).
Alexandre PANCHAUD, Ph.D. Alexandre has a broad interdisciplinary and international background with experience from food industry and academia. Alexandre obtained his PhD in a joint collaboration between University of Lausanne and Nestlé Research Center with the focus of developing quantitative proteomics methods to be applied to the field of nutrigenomics or microbial pathogenicity. In 2008, Alexandre obtained a beginner fellowship from the Swiss National Science Foundation and moved to Seattle to join the group of Prof. David Goodlett. In 2009, he was granted an advanced fellowship from the same foundation to pursue his work. During this time Alexandre was involved in the development of proteomics techniques using data-independent acqisition technique referred to as PAcIFIC. In parallel he was aslo involved in protein structure analysis by cross-linking and mass spectrometry where he for example developed a tool called xComb to interrogate cross-linked peptides. In 2010, Alexandre moved back to Switzerland where he joined the Nestlé Research Center as an R&D specialist to lead projects in the field of biomarker discovery in nutritional interventions as well as food peptidomics for the characterization of food ingredients. In 2011, he was also appointed Protein and Peptide pipeline manager in which he deals with the management of new ingredients for functional foods.
Michael Affolter, PhD. Michael is a senior research scientist in the Functional Genomics Group at the Nestlé Research Centre (Lausanne, Switzerland) and is heading the proteomics platform with a special focus to implement and apply new technologies for protein and peptide analyses in the context of Nutrigenomics. His team develops and integrates qualitative and quantitative mass spectrometry based protein and peptide profiling with bioinformatics and computational molecular science. Major application fields are immunology, diabetes, obesity, and digestive health. Being educated as an analytical biochemist, Michael has acquired extensive research experience over the last eighteen years in the biotechnological and nutritional industry. Michael holds a M.Sc. and a Ph.D. in Biochemistry, obtained at the University of Berne, Switzerland, with experience in the field of protein sequence analysis (Edman sequencing). During his post-doctorate work with Prof. R. Aebersold (Biomedical Research Centre, University of British Columbia, Vancouver, Canada), he has specialized in analytical protein biochemistry and mass spectrometry based proteomics. Michael is an active board member of the SPS (Swiss Proteomics Society) and the SGMS (Swiss Group for Mass Spectrometry). He has (co-) authored more than 40 peerreviewed publications and book chapters, and filed over 10 patent applications.
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Martin Kussmann Guest Editor Proteomics & Metabonomics Core, Nestlé Institute of Health Sciences, Lausanne, Switzerland and Faculty of Life Sciences, Aarhus University, Aarhus, Denmark. Corresponding author. Postal address: EPFL Campus, Quartier de l'innovation, Bâtiment G, 1015 Lausanne, Switzerland E-mail address: [email protected]. Michael Affolter Guest Editor Functional Genomics Group, Nestlé Research Center, Lausanne, Switzerland
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Alexandre Panchaud Guest Editor Functional Genomics Group, Nestlé Research Center, Lausanne, Switzerland