Proteomics in Nutrition: Status Quo and Outlook for Biomarkers and Bioactives Martin Kussmann,*,†,‡ Alexandre Panchaud,† and Michael Affolter† Functional Genomics Group, Department of BioAnalytical Sciences, Nestle´ Research Center, Lausanne, Switzerland, and Faculty of Science, Aarhus University, Aarhus, Denmark Received May 12, 2010
Food and beverages are the only physical matter we take into our body, if we disregard the air we inhale and the drugs we may have to apply. While traditional nutrition research has aimed at providing nutrients to nourish populations and preventing specific nutrient deficiencies, it more recently explores health-related aspects of individual bioactive components as well as entire diets and this at group rather than population level. The new era of nutrition research translates empirical knowledge to evidencebased molecular science. Modern nutrition research focuses on promoting health, preventing or delaying the onset of disease, optimizing performance, and assessing risk. Personalized nutrition is a conceptual analogue to personalized medicine and means adapting food to individual needs. Nutrigenomics and nutrigenetics build the science foundation for understanding human variability in preferences, requirements, and responses to diet and may become the future tools for consumer assessment motivated by personalized nutritional counseling for health maintenance and disease prevention. The scope of this paper is to review the current and future aspects of nutritional proteomics, focusing on the two main outputs: identification of health biomarkers and analysis of food bioactives. Keywords: Proteomics • nutrition • health • biomarker • nutrigenomics • nutrigenetics
Introduction Food and beverages are the only physical matter we take into our body, if we disregard the air we inhale and the drugs we may have to apply. It is therefore logical and natural that nutrition exerts the strongest life-long environmental impact on human health, and this interplay between nutrition and health has been known for centuries. While traditional nutrition research has aimed at providing nutrients to nourish populations and preventing specific nutrient, e.g., vitamin, deficiencies, it more recently explores health-related aspects of ingredients and diets, and this at group rather than population level. The new era of nutrition research translates empirical knowledge to evidence-based molecular science, because food components interact with our body at system, organ, cellular and molecular level.1 Modern nutrition research focuses on promoting health, preventing or delaying the onset of disease, optimizing performance and assessing risk. These objectives require holistic approaches because nutritional improvement of one health aspect must not be compromised by deterioration of another.2 Personalized nutrition is a conceptual analogue to personalized medicine and means adapting food to individual needs. While there are already food products available that address requirements or preferences of specific groups, these products * To whom correspondence should be addressed. Nestle´ Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland. Email: martin.
[email protected]. † Nestle´ Research Center. ‡ Aarhus University.
4876 Journal of Proteome Research 2010, 9, 4876–4887 Published on Web 08/19/2010
are based on empirical research rather than on molecular nutrition. Nutrigenomics and nutrigenetics build the science foundation for understanding human variability in preferences, requirements, and responses to diet and may become the future tools for consumer assessment motivated by personalized nutritional counseling for health maintenance and disease prevention. Whereas it has become apparent that consumers respond differently to diet, depending on their genetic makeup, life style, and environment, the related knowledge and understanding remain fragmentary.1 However, there is an increasing awareness in the population of understanding and assessing one’s individual health status and nutritional needs. Responding to these changing perceptions and demands, the nutrition business is developing products according to desires and requirements of specific consumer groups, be they healthy, at risk, or diseased, such as sportive, elderly, diabetic, obese, or allergic individuals.3 The scope of this paper is to review the current and future aspects of nutritional proteomics, focusing on the two main outputs: health biomarkers and food bioactives. Table 1 summarizes these two categories of analytical deliverables of nutritional proteomics and gives examples from recent research. Expanding from reviewing the past and present and previewing the future of nutritional proteomics, we also present our views on how proteomics in nutrition and health differs from pharmaceutical and medical applications.
Mass Spectrometry and Nutrition In modern nutrition research, mass spectrometry (MS) has developed into a tool to assess health and sensory as well as 10.1021/pr1004339
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Proteomics in Nutrition
Table 1. Two Main Outputs of Nutritional Proteomics: Identification of Health Biomarkers and Analysis of Food Bioactivesa proteomics target
health biomarkers
health aspect
immunity
inflammation
allergy
diabesity adipose tissue
protein turnover
deployment of proteomics as a platform for biomarker development in nutrition research inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) celiac disease and Crohn’s disease: disorders of gastrointestinal mucosal immune function in vivo proteomic studies of intestinal inflammation important allergy markers accepted to date are IL-10, TGF-β, TLRs, PD-1 and CTLA-4 food allergens are glycoproteins in the range from 14 to 40 kDa proteomics on diabesity biomarkers and mechanisms identification of factors secreted during in vitro differentiation of preadipocytes to adipocytes comparison of omental and subcutaneous fat depots study of nutritional influence on protein synthesis and degradation
references
28, 29 31 36 37 39, 40, 41 43 46 56 58 60, 61
proteomics target
raw materials
examples
references
food bioactives
bovine milk
bioactive peptides and proteins in milk and dairy products milk and dairy proteomics for composition, falsification and quality assessment beneficial effects of lactotripeptides on hypertension proteomics of mammalian milk sources for potential optimization of bovine milk-based infant formulas comparison of wheat proteins from different cultivars lunasin is a bioactive soybean peptide with a capability to stop cell division in a skin cancer mouse model and to inhibit core histone acetylation in mammalian cells cardioprotective effects attributed to soy isoflavones and soybean proteins antioxidative capacity of enzymatically released peptides from soybean protein isolates β-conglutin suggested to be hypocholesterolemic based on its high homology with the R subunit of soy β-conglycinin pea seed proteome map
76 81, 82, 83
human milk
wheat soybeans
lupin
pea a
examples
86, 87 88 18 102
104 107 110
114
Examples given address specific, nutritionally actionable health aspects and selected protein/peptide sources.
quality and safety aspects of food and is implicated in three health-related application areas: the discovery and quantification of biomarkers for predisposition, exposure and efficacy, and the characterization and quantification of bioactives.4 Practically all modern mass spectrometric techniques are being deployed as analytical platforms for nutrition research. The instruments are, for example, used to investigate the pleasure aspects of food, i.e., its sensory qualities. Flavor is the sum of taste and olfaction; LC-MS/MS techniques typically serve to characterize taste molecules, whereas GC-MS instruments analyze the volatile compounds that contribute to olfaction of food and drinks. Safety and quality assessments employ a wide range of mass spectrometers from isotope ratio MS (IR-MS) for authenticity checks to hyphenated MS techniques for ensuring quality, safety, and compliance of food products.4
From Food Proteins to Nutrigenomics Nutrition provides to cells and organisms the food necessary to support life. Proteins form a major class of macronutrients, because they participate in every cellular process. Indeed, enzymes are the proteins that catalyze virtually all biochemical reactions involved in metabolism with, for instance, pepsin and trypsin being active during digestion. Proteins also ensure structural and mechanical functions (e.g., the cytoskeleton
maintains cell shape, and actin and myosin facilitate body motion). In addition, proteins are involved in cell signaling and immune response. In consequence, the body needs relatively large amounts of protein in order to function efficiently and also because proteins are continuously synthesized and degraded (metabolized). Some of the amino acids cannot be synthesized by the body (so-called essential amino acids) and have therefore to be provided by the diet. Food proteins differ in their composition on the basis of their origin, i.e., animal or plant source. Hence, a balanced diet comprises proteins from different but complementary sources (e.g., meat, vegetables, cereals, grains, legumes) in order to avoid amino acid deficiencies. The nutritional quality of proteins is usually characterized by the amino acid composition, digestibility, and absorptive ability. Nutrients and genomes interact. Dietary components appear in complex mixtures, and hence not only the concentrations of single compounds but also interactions between them have an impact on final ingredient bioavailability and bioefficacy.5 Proteomics has therefore logically developed into a central platform in nutrigenomics, which attempts to holistically understand how our genome is expressed as a response to diet.6 Nutrigenetics focuses on our genetic predisposition and susceptibility toward diet7 and can be deployed to stratify cohorts Journal of Proteome Research • Vol. 9, No. 10, 2010 4877
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Kussmann et al. ficacy; we source genetic techniques to reveal predisposition and susceptibility, and we apply epigenetics to understand metabolic programming and imprinting.
Nutritional Biomarkers
Figure 1. Nutritional proteomics has been extended toward a metaproteomics approach comprising information from three different proteome levels: host, food, and microbes. This concept is analogous to and rooting in metagenomics approaches, with the proteomics-typical additional challenge of a (meta)proteome being much more complex than any (meta)genome. The host proteome has been and will be intensely assessed to reveal nutritional health biomarkers. Food proteomes have been extensively characterized, spanning a variety of animal and plant protein sources. By contrast, the intestinal microbiome, i.e., the complement of gut-residing bacteria, has only recently been recognized as a fundamental factor for host health affecting a variety of conditions such as energy balance and immunity.
of subjects enrolled in nutritional intervention studies and to discern responders from nonresponders among those subjects.3 Epigenetics encompasses the investigation of DNA sequenceunrelated biochemical modifications of both DNA itself and DNA-binding proteins and appears to provide a format for metabolic imprinting.8,9 Mass spectrometry and proteomics play a key role here, too, as they can address post-translational modifications (e.g., acetylations) of DNA-packaging proteins and thereby help decipher the so-termed histone code10,11 or quantitatively measure DNA methylation changes, thereby advancing our understanding of gene regulation.12 Overall, proteomics in nutrition delivers bioactives and biomarkers and answers questions of nutritional bioavailability and bioefficacy.13,14 Most recently, as depicted in Figure 1, nutritional proteomics has been extended toward a metaproteomics approach comprising information from three different proteome levels: host, food, and microbes. This concept is analogous to and rooted in metagenomics approaches, with the proteomics-typical additional challenge of a (meta)proteome being much more complex than any (meta)genome. The host proteome has been and will be intensely assessed to reveal nutritional health biomarkers, as discussed above. Food proteomes have been extensively characterized, spanning a variety of animal and plant protein sources.15–17 By contrast, the intestinal microbiome, i.e., the complement of gut-residing bacteria, has only recently been recognized as a fundamental factor for host health affecting a variety of conditions such as energy balance and immunity.18,19 While techniques such as microarrays and high-throughput sequencing20,21 enable now an in-depth characterization of the intestinal bacterial population structure,22 most recently the question has moved from “who is there ?” to “what is going on ?”, i.e., from a population census23 to an activity profiling, the latter being facilitated by metaproteomic analyses.24 We deploy genomic platforms, i.e., transcriptomics, proteomics and metabonomics, to demonstrate nutritional ef4878
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A biomarker is a measurable change related to a phenotype. Molecular biomarkers are, for example, a variation in mRNA, protein, or metabolite concentration, and these should be responsive, specific, and applicable. A valid nutritional biomarker can also function as a key measure linking a specific exposure of a dietary compound to a health outcome and thus offers great potential to understand the relationship between diet and health. Biomarkers help understand nutrient absorption, transport, and metabolism within an organism to produce an effective dose at target tissue. Biomarkers of susceptibility consider host, environmental and lifestyle factors, and in particular genetic predisposition.5 The above-mentioned processes can be envisioned as a continuum that link exposure, dose, and effect. Biological steps along the pathway (exposure, internal dose, biologically effective dose, early biological effect, altered structure and/or function, and clinical outcome) can potentially be observed, monitored, and quantified using biomarkers. Markers of internal dose are direct measures of a dietary compound or its metabolites at a systemic level, e.g., in body fluids. More specific but often difficult to obtain are biomarkers that describe the dose of a dietary compound or metabolite at target tissues.5 Markers of biologically effective dose assess the interaction of food constituents with their molecular targets, and markers of early biological effect assess the molecular sequence of target compound-cell interactions.25 Biomarkers of altered structure and/or function are useful for assessing morphological and/or functional changes following compound-cell interactions, which are strongly indicative for disease outcomes, while markers of susceptibility may influence the magnitude of each step in the pathway. Biomarkers useful for disease prevention and nutritional/ therapeutic intervention may appear anywhere along the pathway. Earlier markers have the greatest potential to avert disease; later markers are most closely related to the disease. The challenges of applying biomarkers to nutrition research are less related to the sensitivity of the analytical techniques than to a lack of accuracy and validation in terms of (i) understanding the parameters related to bioavailability and mechanism of bioactive food components; and (ii) an application in large population studies.5 Furthermore, most micronutrients at normal dietary dose levels are only weakly biologically active in the short term and have multiple targets. Depending on their dose, they can be both beneficial and deleterious, posing a further challenge in determining the net effect of food and its constituents. In most cases, a battery of biomarkers needs be measured to comprehensively describe the entire continuum, from exposure via effect to end point. Key points to consider when assessing the use of biomarkers in studies of nutrition and health include (a) timing of measurement in relation to bioavailability and bioefficacy must be considered; (b) biomarkers may correlate with intake, but often the marker is a combined result of intake, absorption, metabolism, and excretion; (c) in the case of homeostatic regulation of nutrient levels in body fluids, biomarkers may poorly correlate with amounts of intake; and (d) environmental factors and genetic predisposition may modulate the correlation between dietary intake and biomarkers.26
Proteomics in Nutrition In summary, biomarkers are indicators of molecular and cellular events in biological systems and help epidemiologists and clinicians better understand relationships between food constituents, whole food or diets, and human health effects. However, given the complex interplay between nutrition, other environmental factors, and interindividual variability on the host side, biomarkers alone will not explain the relationship between diet and health. Rather, a combined approach including questionnaires and noninvasive and invasive measurements will prove to be more valuable and practicable. Immunity. Nutrition has a strong influence on immune status, development, and decline. Consequently, nutritional modulation of immunity is a major axis in nutrition and health research with the objectives to favorably “programme” neonate immunity, maintain immune homeostasis throughout life, and reinforce immunity in elderly. Modern immune-modulating nutrition accompanies the consumer through their life stages and styles. Although mass spectrometry is certainly a most powerful tool to assess immune status and nutritional immune modulation, it is to date largely under-deployed in this context. As the mature and diverse technology platforms deliver quantitative, information-rich data and are highly accurate and sensitive, mass spectrometry in immunology and nutrition means discovery of biomarkers for immune status and nutritional intervention and mass spectral monitoring of nutritional intervention and bioavailability/bioefficacy studies. Immune-relevant food sources such as milk have been extensively investigated by MS in terms of their protein and peptide complement. A few nutritional interventions have been monitored by regarding their immune effects, mainly assessing the peripheral blood mononuclear cell (PBMC) proteome, with the latter serving in general as an accessible and relevant immune cell population readily amenable to mass spectrometric proteomics. Immunoproteomics with a perspective from biomarker discovery to diagnostic applications was recently reviewed by Tjalsma et al.27 The concept here is to refine, multiplex, and accelerate mass spectrometry- and proteomics-based antibody analytics and diagnostics. De Roos and McArdle presented their view on how to deploy proteomics as a platform for biomarker development in nutrition research.28,29 This paper is probably the most comprehensive summary of (immune-related) nutritional studies as monitored by mass spectrometry. These studies are mostly based on the classical proteomic approach, i.e., protein separation by two-dimensional gel electrophoresis followed by protein spot excision, in-gel protein digestion, and mass spectrometric protein identification, the latter deploying mainly MALDI mass fingerprinting but also LC-MS/MS. Markers to measure immunomodulation in human nutrition intervention studies have been reviewed by Albers et al.30 However, these markers do not descend from proteomic approaches but rather reflect targeted measurements of biomolecules or read-outs from cellular assays, typically performed in (pre)clinical settings. The role of proteomics in the discovery of biomarkers in gastrointestinal diseases has been outlined by Song and Hanash;31 protein microarrays, mass spectrometrybased proteomic tools, and guidelines for biomarker development are described. The authors state that inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) represent diseases for which biomarkers are still pending and that proteomics may help in identifying them. Within IBD, better markers are needed to distinguish between Crohn’s disease and
reviews ulcerative colitis and to improve diagnosis and prediction of therapy. Purcell and Gorman32 have published a review on mass spectrometry-based studies of immune responses and discussed the role of proteomics in (a) the elucidation of the cytotoxic T lymphocytes, (b) the T cell-B cell cooperation and antibody secretion, (c) defining targets of T cell immunity, (d) the discovery of T cell epitopes, (e) analysis of antigen presenting cell (APC) surface proteins, and (f) the sequencing of MHCbound peptides. Addressing a more specific immune context, Weingarten et al. discussed the application of mass spectrometric protein analysis to biomarker and target finding for immunotherapy.33 Their article focuses on regulatory T-cells that play a central role in maintaining the immunological balance and inhibiting T-cell activation both in vivo and in vitro. Inflammation. Inflammation is a basic process whereby tissues of the body respond to injury. It has been described as purposeful, timely, powerful, and as a consequence, also dangerous if resolution is not initiated.34 The normal outcome of the acute inflammatory program is successful resolution and repair of tissue damage, rather than persistence of the inflammatory response.35 Although inflammation is essential for tissue homeostasis, prolonged inflammation is a characteristic feature of many chronic diseases, such as inflammatory bowel disease (IBD) and autoimmunity. Moreover, chronic inflammation has been shown to be implicated in critical conditions such as atherosclerosis, arthritis, cancer, and asthma, all leading to tissue destruction, fibrosis, and impairment or loss of organ function. Celiac disease and Crohn’s disease are prototypic disorders of gastrointestinal mucosal immune function.36 Crohn’s disease is characterized by chronic inflammation of the gastrointestinal tract and associated with multiple genetic mutations, at least one of which has been clearly implicated in innate immunity. Moreover, the disease appears to involve abnormal immune responses to gut microbiota.36 Celiac disease is a disorder of the small intestine characterized by chronic inflammation of the mucosa caused by loss of tolerance to dietary antigens. Among the associated cofactors identified are antigenic peptides in wheat, rye, and barley diets. One of the few mass spectrometry-based in vivo proteomic studies in the context of intestinal inflammation was presented by the Bendixen group.37 The group compared proteome patterns of healthy and inflamed gut tissues harvested from preterm piglets to investigate the effect of inflammation on acquisition of passive immunity. A clear difference in the twodimensional gel electrophoretic protein patterns between healthy and inflamed intestinal tissues was revealed, suggesting that inflamed tissues failed to absorb and transfer Ig from colostrum to epithelial cells. Mass spectrometry identified isoforms of the IgA and IgG heavy chain and Ig κ and λ light chains as being absorbed by healthy intestinal tissues and indicated that colostrum protein uptake in the porcine gut is a selective process deranged in inflamed preterm intestine. Allergy. Most of the to-date identified 20 or more allergyassociated genetic markers are indicators of inflammation rather than of allergy. Kornman et al. described a nutrigenomic strategy to better understand the associations between genetic variations, the susceptibility to inflammation, and the nutritional intervention potential.38 Important allergy markers accepted to date are IL-10,39 TGF-β, TLRs,40 PD-1, and CTLA4.41 Yet, specific IgE levels are successfully used as indicators for an allergic condition. Journal of Proteome Research • Vol. 9, No. 10, 2010 4879
reviews Food allergy is an adverse reaction to food or food additives with an underlying immunological mechanism. An area of immunology and nutrition where proteomics is already a wellestablished working horse is allergen detection, identification, and characterization. Many food allergens have been identified, and these stimuli are often structurally well characterized, typically by mass spectrometry of the implied proteins and peptides. This source of risk necessitates detecting and monitoring (potential) allergens before, during, and after food processing.42 A list of the 10 most sensitizing proteins has been proposed. Although this may vary from country to country, these proteins basically derive from egg, fish, shellfish, milk, soy, wheat, peanuts, tree nuts, citrus fruits and sesame seeds. Most of these food allergens are glycoproteins in the range from 14 to 40 kDa.43 These physicochemical characteristics render them ideal analytes for mass spectrometry and proteomics, with its power to identify, sequence, and quantify proteins and posttranslational modifications such as glycosylation and to differentiate between protein isoforms.13,14 In contrast to the advanced level of understanding about allergen structures, the molecular mechanisms determining a normal or an allergic reaction, i.e., the consequences of allergen exposure for the host, are incompletely understood. Diabesity. The term ”diabesity” is the lingual reflection of the fact that obesity and diabetes are intimately linked disorders. Obesity is a condition resulting from a chronic imbalance between energy intake and energy expenditure.44 Obesity often causes insulin resistance, a decline in the ability of insulin to stimulate glucose uptake in the body, which leads to compensatory oversecretion of this hormone by the pancreatic β-cells and, eventually, to β-cell exhaustion and development of type 2 diabetes mellitus (T2DM).45 Diabetes is a complex metabolic disorder of multiple origins characterized by chronic hyperglycemia with disturbances of carbohydrate, fat, and protein metabolism resulting from defects in insulin secretion and action. Proteomic studies have shed light on diabesity biomarkers and mechanisms.46 A mouse Swiss 2D-PAGE database has been established for T2DM.47 These 2DE reference maps are annotated with protein identifications and were generated from mouse white and brown adipose tissue, pancreatic islets, liver, and skeletal muscle. Skeletal muscle insulin resistance is critical to the pathogenesis of type 2 diabetes. In order to generate a broad view of protein changes in this tissue in relation to obesity and diabetes, biopsies of lean, obese, and type 2 diabetic patients were compared.48 Mass spectrometry revealed 15 proteins as significantly increased or decreased. In addition, a decrease in global abundance of mitochondrial proteins was observed, together with alteration of cytoskeletal structure, chaperone function, and proteasome subunits, indicating changes in muscle structure, protein degradation, and folding. In order to further elucidate molecular alterations associated with insulin resistance in muscle tissue, human skeletal muscle biopsies from patients with T2DM were compared to healthy controls at 2DE level and eight potential markers were identified.49 A series of diabetes-related proteomic studies aimed for the identification of possibly deleterious or protective proteins in the initial cytokine-induced β-cell damage in type-1 diabetes mellitus (T1DM).50–52 The complexity of IL-1β effects on islet protein expression supports the hypothesis that T1DM development is the result of a collective, dynamic instability, rather than the outcome of a single factor.53 4880
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Kussmann et al. Adipose Tissue. Adipose tissue (AT) has been recognized not only as a fat and energy depot but also as an endocrine organ54 secreting a number of hormones. AT has therefore been analyzed in terms of possible links between obesity and insulin resistance.55 Kratchmarova at al. embarked on a proteomic route toward the identification of secreted factors during the differentiation of preadipocytes to adipocytes in vitro.56 Human adipose tissue is divided into subcutaneous and omental fat depots, which have distinct functions. Many epidemiological studies have shown that the size of the omental adipose tissue is associated with a higher risk of obesity-related disorders.57 In this view, a proteome study comparing omental and subcutaneous fat depots has been conducted using 2D-DIGE and LC-MS/MS.58 Forty-three proteins were differentially expressed and involved processes such as glucose and lipid metabolism, lipid transport, protein synthesis, protein folding, and response to stress and inflammation. This result suggests a high metabolic activity and increased cell stress in omental compared to subcutaneous fat. A review by Peral et al.59 summarizes recent work in this domain. Protein Turnover. Nutrition plays a key role in body protein metabolism. Stable-isotope labeled tracers in combination with isotope ratio and atmospheric pressure mass spectrometry (IRMS), allowing precise, accurate, and sensitive determination of isotope enrichments, enabled the study of nutritional influences on protein synthesis and degradation.60,61 Nitrogen balance is negative after a fasting period, also called the postabsorptive phase, when circulating amino acids, glucose, and lipids are derived from endogenous stores rather than from food being absorbed from the gut. The protein balance becomes positive after a meal if an adequate amount of protein is consumed. Information on whole-body protein turnover is valuable, especially in clinical applications, but such data yield only a view on the total protein pool at either whole tissue or organism level. Until the emergence of proteomics, a limited effort has been invested to study protein synthesis and degradation at the level of individual proteins;62 “classical” proteomic approaches, comprehensive protein identification and quantification at a given sampling time point, do not address the dynamics of the proteome in terms of steady-state, synthesis, and degradation of individual protein species but rather yield a “proteomic snapshot”. Some of the discrepancy observed between transcriptome and proteome data63 might be explained by underlying variations of synthesis/degradation rates not captured by traditional proteomic techniques.64 Addressing protein turnover dynamics represent some technical challenges for proteome-wide analysis. Whereas efficient and complete stable-isotope labeling of cells and tissue cultures has been shown to be feasible,64,65 the high isotope enrichment needed for proteome-wide studies is still difficult to achieve in animals and humans. First attempts have been successful in completely labeling rats with 15N by feeding the animals a diet containing algal cells labeled with 15N.66 Long-term metabolic labeling with a diet enriched in 15N did not result in adverse health consequences. A labeling period of 44 days in a male rat resulted in a mean 15N atomic enrichment of >90% in liver and plasma. Proteome dynamics were measured in chicken fed with a semisynthetic diet containing [2H8]-valine at a calculated relative isotope abundance (RIA) of 0.5.67 The RIA was stable over an extended labeling window and enabled calculation of the rates of synthesis and degradation of individual proteins.
Proteomics in Nutrition This study has demonstrated for the first time the possibility to address the analysis of individual protein turnover in whole animals. Measurement of turnover in the human proteome was reported more recently using dynamic incorporation of stable isotopes in cell cultures with amino acids (dynamic SILAC).68 Almost 600 proteins from human adenocarcinoma cells were characterized for time-dependent changes thanks to the incorporation of [13C6]-arginine in a classic label-chase experiment. Although a large number of proteins were analyzed and turnover rates were deduced, the approach depends on cell cultures and thus excludes proteome-wide assessment in whole animals. Nevertheless, it shows the potential of modern proteomic technologies to measure synthesis and degradation rates of individual proteins in the proteome. Incorporation rates of amino acids, liberated during digestion of labeled milk protein and incorporation into plasma proteins, were measured in humans.69 The approach combined proteomic technologies (plasma protein purification, gel separation, LC-MS/MS-based protein identification) with traditional GC-MS analysis for isotope enrichment ([13C6]-phenylalanine) analysis. Twenty-nine individual plasma proteins were identified, and their corresponding postprandial fractional synthesis rates were calculated on the basis of the rate of [13C6]-Phe incorporation, showing a 30-fold difference in synthesis rates. The authors stress the point that concentration of proteins between two study conditions may not change, if synthesis and degradation rates compensate each other, although an increased metabolism of a single protein or a cluster of proteins may have major functional consequences. In a more recent study, the same authors report a methodology to measure synthesis rates of multiple muscle mitochondrial proteins.70 The synthesis rates of 68 mitochondrial and 23 nonmitochondrial proteins isolated from a skeletal muscle mitochondria fraction varied up to 10-fold between the lowest and highest rates. This approach represents a good measurement of the translation rate of gene transcripts and thus offers an opportunity to understand the regulation of specific genes and the correlation of transcriptome and proteome results. Taken together, the ideal proteome turnover approach should combine stable-isotope labeled tracer studies and proteomic techniques for protein isolation, purification, characterization, and quantification. This would enable the largest coverage of protein identities with concurrent synthesis and degradation data for individual proteins. In the future, this information will become even more important as systems biology requires integration and correlation of transcriptome, proteome, and metabolome data, which should also embrace protein metabolism (synthesis and breakdown) information.
Protein and Peptide Bioactives There is evidence that food-derived proteins and peptides provide much more than basic macronutrients and building blocks for protein turnover. Scientists increasingly appreciate proteins and peptides because of their variety of bioactives that function as growth factors, antihypertensive agents, antimicrobials, modifiers of food intake, or immune regulators. Biologically active motifs in the polypeptide chains of proteins are defined as fragments that remain inactive when inserted in the sequences of their own precursors, whereas upon release by proteolytic enzymes, they may interact with appropriate receptors, thus exerting specific bioactivity. The activity of such peptides may be either favorable or detrimental.71,72 Bioactive
reviews peptides may be released during the digestion by host enzymes like trypsin or by microbial enzymes. They can also originate from food processing (industrial processing) or ripening (naturally occurring enzymatic reactions). In order to accurately address questions of bioavailability and bioefficacy, both systemically (i.e., in blood) and locally (e.g., in the gut), bioactive peptides and proteins must be identified and quantified all across from food matrix to the target tissues in the body. Especially in nutrition it is desirable to also generate information on absolute amounts of these peptides and proteins because the basis of proven bioavailability and bioefficacy of a given ingredient are its absolute amounts in the original food matrix as well as in the relevant body fluids or tissues. Bioactive peptides and proteins have been reviewed by Moller et al.,73 and a database of bioactive peptides was developed for determination of potential bioactivity of food proteins and for classification.74 According to present knowledge, bovine milk, cheese, and dairy products seem to be by far the richest sources of bioactive proteins and peptides derived from food. This may be due to the particular purposes to which milk is dedicated beyond nutrition in the first months of life. Bioactive peptides and proteins, however, are also gained from other animal and plant sources. In bovine blood, gelatin, meat, eggs, and various fish species such as tuna, sardine, herring, and salmon, as well as in wheat, maize, soy, rice, mushrooms, pumpkin, and sorghum, bioactive proteins and peptides have been detected either directly or after release by hydrolysis or fermentation.73 Milk. Milk is a rich source of bioactives beneficial for human health. It is the only nutrition that has coevolved with mankind and is therefore particularly relevant and suited to support healthy growth and development of the neonate and infant, including the maturation and maintenance of a balanced immune system.75 Milk bioactives derive from the protein- and peptide-,76 the lipid-77 and the oligosaccharide complement78 of milk from diverse mammalian species. The protein complement of human milk roughly splits into caseins and whey with a 50:50 weight/weight ratio. Bovine milk consists of 80% caseins and 20% whey proteins.76 Reviews of bioactive peptides and proteins present in milk and dairy products have recently been published.76 Caseins serve for example as ion carriers and precursors of bioactive peptides, whereas whey proteins have major functions in immune modulation and defense.79 As a result of its analytical versatility and power for structure elucidation and quantification of large biomolecules, mass spectrometry has developed into the major contributor to comprehensive biomolecule characterization in milk, known under the more recently coined terms milk proteomics/ peptidomics, lipidomics, and glycomics. Casado and Kussmann have recently released a comprehensive review on the protein/ peptide, lipid, and carbohydrate complement of human and animal milk, as assessed by various mass spectrometric approaches.80 Fong et al. presented an update on bovine whey protein fractionation and characterization by proteomic techniques.15 Traditionally, milk and fractions thereof have been assessed in terms of composition, physicochemical properties, and biological functions, and mass spectrometry is largely contributing to various areas of milk and dairy research (reviewed in refs 81 and 82). These studies include identification of milk protein variants and glycoforms, falsification of milk with nondairy ingredients, and identification of peptides in dairy products. Moreover, mass spectrometry has become Journal of Proteome Research • Vol. 9, No. 10, 2010 4881
reviews an indispensable technique for the quality assessment of milkand dairy-based products (reviewed in ref 83). Human milk mainly consists of caseins, R-lactalbumin, lactoferrin, albumin, and various immunoglobulins. These predominant proteins account for >99% of the milk protein mass. However, the remaining
