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Microarrays: A Powerful Tool for Studying the Functions of Food and Its Nutrients
Food protein-derived bioactive peptides are a group of functional food components. Enzymatic hydrolysis of food proteins generates various peptides with physiological functions, such as antihypertensive, opioid, immunostimulating, antimicrobial, antithrombotic, hypocholesterolemic, and antioxidative activities. This chapter includes an overview of bioactive peptides generated from food proteins. Also, utilization of modern nutrigenomics techniques for such peptides is discussed. Nutrigenomics has been rapidly applied to the field of nutrition and health. Although application of this strategy for studying food protein-derived bioactive peptides is still limited, it offers a great possibility for understanding and utilizing bioactive peptides.
The 9-cis-retinoic acid receptors (RXRs), belonging to the members of the steroid/thyroid hormone receptor superfamily, act as auxiliary proteins, heterodimerizing with other nuclear receptors such as retinoic acid receptors (RARs), vitamin D receptor, thyroid hormone receptors, and peroxisome-proliferator activated receptor, thereby transactivating target genes in a ligand-dependent manner. We have previously reported that in the rat, thyroid hormone (TH) positively and negatively regulates the hepatic mRNA levels of RXR beta and RXR gamma, respectively. In the present study, we have tried to elucidate the level at which TH regulates the gene expression of RXR beta and RXR gamma in the rat. A RNA synthesis inhibitor (actinomycin D), but not a protein synthesis inhibitor (cycloheximide), blocked the induction of RXR beta mRNA by TH. On the other hand, none of these drugs inhibited the decrease of RXR gamma mRNA levels caused by TH. Nuclear run-on assays showed that the transcription rate of the RXR beta gene was positively regulated by TH, whereas the transcription of RXR gamma gene was not controlled by TH. Taken together, these results indicate that the gene expression of RXR beta is positively regulated by TH at transcriptional level, while the negative regulation of the RXR gamma gene expression by TH may occur at a post-transcriptional level in intact rat. Thus, the RXR-mediated signal transductions may be modulated in part through TH control of the levels of RXR beta and RXR gamma.
We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.
Osteoclasts modulate bone resorption under physiological and pathological conditions. Previously, we showed that both estrogens and retinoids regulated osteoclastic bone resorption and postulated that such regulation was directly mediated through their cognate receptors expressed in mature osteoclasts. In this study, we searched for expression of other members of the nuclear hormone receptor superfamily in osteoclasts. Using the low stringency homologous hybridization method, we isolated the peroxisome proliferator-activated receptor delta/beta (PPARdelta/beta) cDNA from mature rabbit osteoclasts. Northern blot analysis showed that PPARdelta/beta mRNA was highly expressed in highly enriched rabbit osteoclasts. Carbaprostacyclin, a prostacyclin analogue known to be a ligand for PPARdelta/beta, significantly induced both bone-resorbing activities of isolated mature rabbit osteoclasts and mRNA expression of the cathepsin K, carbonic anhydrase type II, and tartrate-resistant acid phosphatase genes in these cells. Moreover, the carbaprostacyclin-induced bone resorption was completely blocked by an antisense phosphothiorate oligodeoxynucleotide of PPARdelta/beta but not by the sense phosphothiorate oligodeoxynucleotide of the same DNA sequence. Our results suggest that PPARdelta/beta may be involved in direct modulation of osteoclastic bone resorption.
Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel mass spectrometric peptide sequencing and analysis technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.
Proteomics encompasses several disciplines in the quest to unravel the science of life. Mass spectrometry plays a key rôle in proteomics-associated analyses and this review describes that function in terms of the instrumentation used, the results generated, data interpretation and the advances in analytical techniques being made to deal with the production of highly specific, high throughput data. The review has 151 references.
Understanding nutrient-gene interaction requires tools for both the study of nutrigenomics and the characterization of phenotype. Metabolomics or metabolite profiling is a powerful tool for characterizing metabolic phenotype, and tracer-based metabolomics is a subset of metabolomics that focuses on metabolite distribution and flux determination using tracers. In this review, the characterizations of metabolic phenotype by metabolite profiling and by metabolic flux measurements are compared. The rationale and methodologies of tracer-based metabolomics are explained. Tracer-based metabolomics provides a relational database of metabolites linked by the relationship of shared metabolic pathways, common substrates, and cofactors. Such a collection of flux measurements provides precise and accurate information on the operation of the cellular metabolic network and its response to genetic and nutrient environment changes. Nutrient-gene interaction can be studied using the concept of constraint-based modeling, which states that the observed metabolic phenotype is a consequence of constraints from genetic factors and the nutrient environment. Thus, genetic inheritance (genomic constraints) confers a wide range of possible phenotypes whereas selection by metabolic (structural and pathway relationship) and environmental (physical environment and nutrient availability) constraints determines the final observed phenotype. The study of the contribution from nutrient and genetic factors to the survival advantage of cancer cells using flux measurements is a critical first step in our understanding of the relationship between nutrient intake and cancer risk.
To identify patients with medullary thyroid carcinoma (MTC) at a potentially curable stage of the disease, serum concentrations of calcitonin (hCT) were determined in 14,000 patients (including 10,158 patients with thyroid nodules) referred to a thyroid outpatient clinic. Excluding patients in whom elevated basal hCT concentrations had already been known at the time of their referral, 507 patients with thyroid nodules presented basal concentrations of hCT of more than 10 pg/ml. Following stimulation by IV pentagastrin (0.5 microg/kg BW), hCT concentrations of more than 100 pg/ml were seen in 103 patients. This group included 32 new cases of MTC (29 patients with sporadic MTC and 3 new index cases of the familial form) and 43 patients with C cell hyperplasia (CCH). Among the 3,843 patients without thyroid nodules, 2 were found to harbor sporadic MTC while 4 had CCH. As compared to 1.1 cases of MTC per 1,000 patients with nodular thyroid diseases diagnosed in our institution before hCT screening was begun, 3.2 cases of MTC per 1,000 patients were identified when hCT was determined in all patients with thyroid nodules. The determination of hCT in all patients with thyroid nodular disease facilitates the timely diagnosis of MTC, thus providing the chance of curative surgery.
Over the last few years the interest in diagnostic markers for specific diseases has increased continuously. It is expected that they not only improve a patient's medical treatment but also contribute to accelerating the process of drug development. This demand for new biomarkers is caused by a lack of specific and sensitive diagnosis in many diseases. Moreover, diseases usually occur in different types or stages which may need different diagnostic and therapeutic measures. Their differentiation has to be considered in clinical studies as well. Therefore, it is important to translate a macroscopic pathological or physiological finding into a microscopic view of molecular processes and vice versa, though it is a difficult and tedious task. Peptides play a central role in many physiological processes and are of importance in several areas of drug research. Exploration of endogenous peptides in biologically relevant sources may directly lead to new drug substances, serve as key information on a new target and can as well result in relevant biomarker candidates. A comprehensive analysis of peptides and small proteins of a biological system corresponding to the respective genomic information (peptidomics(R)methods) was a missing link in proteomics. A new peptidomic technology platform addressing peptides was recently presented, developed by adaptation of the striving proteomic technologies. Here, concepts of using peptidomics technologies for biomarker discovery are presented and illustrated with examples. It is discussed how the biological hypothesis and sample quality determine the result of the study. A detailed study design, appropriate choice and application of technology as well as thorough data interpretation can lead to significant results which have to be interpreted in the context of the underlying disease. The identified biomarker candidates will be characterised in validation studies before use. This approach for discovery of peptide biomarkes has potential for improving clinical studies.
The major role of matrix metalloproteinases (MMPs) is for homeostatic regulation of the extracellular environment, not simply to degrade matrix as their name suggests. We designed and printed a dedicated, focused DNA microarray, the CLIP-CHIP, that enables the analysis of every human and murine protease, protease homologue and inhibitor on a system-wide basis in cancer. We have also developed novel proteomic approaches to identify cleaved substrates of proteases in complex milieu. Isotope coded affinity tag (ICAT) and iTRAQ labeling of conditioned medium proteins secreted by MDA-MB-231 breast carcinoma cells and Mmp2 -/- murine fibroblasts transfected with protease (MT1-MMP or active MMP-2) or their inactive mutant forms enabled quantitative proteomics to be performed. Comparison of the relative abundance ratios of identical peptides from the two samples identified proteins in the conditioned medium that may have been degraded (low ratios) and those that were shed from the cell membrane (high ratios). MS/MS was used to sequence and identify the potential substrates. These analyses have revealed a plethora of new bioactive substrates and biological roles for MMPs. Biochemical confirmation of cleavage of the potential substrates was performed and the cleavage sites identified by MALDI-TOF. In these studies we discovered and confirmed that CTGF, galectin-1, death receptor-6, HSP90alpha, procollagen C-proteinase enhancer protein, the chemokine fractalkine, and cystatin C were novel MT1-MMP or MMP-2 substrates. These sophisticated cellular control functions highlight new intervention points in multiple pathways to treat early stage cancer.
Osteoarthritis (OA) is a slowly progressing chronic joint disease. Glucosamine (GlcN) is a saccharide that is widely used to relieve symptoms associated with OA. However, the mechanism of the effects of GlcN on articular cartilage remains unclear. We studied the effects of GlcN and its analogues, including chitin derivatives included in health supplements containing GlcN, on a chondrogenic cell line, ATDC5. We examined the effects of these saccharides on the proliferation and differentiation of ATDC5 cells. Glucosamine analogues, such as N-acetyl glucosamine and chitobiose, did not affect the proliferation or differentiation of ATDC5 cells. While GlcN did not affect the proliferation of ATDC5 cells, it inhibited their differentiation. Next, we examined whether GlcN affects mineralization and glycosaminoglycan (GAG) production by ATDC5 cells. Mineralization was markedly inhibited by addition of GlcN to the cell culture medium. Moreover, GlcN induced the formation of sulfated GAG in ATDC5. We also analyzed the mRNA levels in ATDC5 cells. GlcN reduced the mRNA levels of Smad2, Smad4 and MGP. GlcN might inhibit expression of MGP mRNA and induce the production of chondroitin sulfate in ATDC5 cells. The mechanism by which GlcN inhibits mineralization may be by regulating the expression of mRNA for the Smad2 and Smad4 chondrogenic master genes.
Weight control by exercise and dietary calorie restriction (DCR) has been associated with reduced cancer risk, but the underlying mechanisms are not well understood. This study was designed to compare the effects of weight loss by increasing physical activity or decreasing calorie intake on tumor promoter-induced Ras-MAPK and PI3K-Akt pathways. SENCAR mice were randomly assigned to one of the following five groups: ad libitum-fed sedentary control, ad libitum-fed exercise (AL+Exe), exercise but pair-fed at the amount as controls (PF+Exe), 20% DCR, and 20% DCR plus exercise (DCR+Exe). After 10 weeks, body weight and body fat significantly decreased in the groups of DCR, DCR+Exe, and PF+Exe when compared with the controls. AL+Exe did not induce weight loss due to, at least in part, increased food intake. Plasma IGF-1 levels reduced significantly in DCR and DCR+Exe but not PF+Exe. The protein H-Ras and activated Ras-GTP significantly decreased in TPA-induced skin tissues of DCR-fed mice but not exercised mice. PI3K protein, phosphoserine Akt, and p42/p44-MAPK were reduced, however, in both DCR and PF+Exe groups. Immunohistochemistry demonstrated that the significantly reduced H-Ras occurred in subcutaneous fat cells, while the reduced PI3K and PCNA took place only in the epidermis. Plasma leptin decreased in PF+Exe, DCR, and DCR+Exe, while the caspase-3 activity increased in DCR+Exe only. Genomic microarray analysis further indicated that the expression of 34 genes relevant to PI3K and 31 genes to the MAPK pathway were significantly regulated by either DCR or PF+Exe treatments. The reduced PI3K in PF+Exe mice was partially reversed by IGF-1 treatment. The overall results of this study demonstrated that DCR abrogated both Ras and PI3K signaling, which might inhibit TPA-induced proliferation and anti-apoptosis. Selective inhibition of PI3K by PF+Exe but not AL+Exe seems more attributable to the magnitude of the caloric deficit and/or body fat loss than diet versus exercise comparison.
To be able to perform a comprehensive and rigorous benefit-risk analysis of individual food components, and of foods, a number of fundamental questions need to be addressed first. These include whether it is feasible to detect all relevant biological effects of foods and individual food components, how such effects can confidently be categorised into benefits and risks in relation to health and, for that matter, how health can be quantified. This article examines the last of these issues, focusing upon concepts for the development of new biomarkers of health. Clearly, there is scope for refinement of classical biomarkers so that they may be used to detect even earlier signs of disease, but this approach defines health solely as the absence of detectable disease or disease risk. We suggest that the health of a biological system may better be reflected by its ability to withstand and manage relevant physiological challenges so that homeostasis is maintained. We discuss the potential for expanding the range of current challenge tests for use in conjunction with functional genomic technologies to develop new types of biomarkers of health.
Advances in the fabrication of DNA microarrays as well as transformations in detection chemistries have vastly increased the throughput for genotyping, DNA sequencing, and array-based copy number analysis (ABCNA). Rapid changes in technology are not only affecting research but also revolutionizing DNA diagnostics. Here we focus on the application of high-throughput ABCNA and genotyping. Targeted and genome-wide ABCNA has led to the discovery of extensive DNA copy number variation in the population and the delineation of many previously unrecognized submicroscopic chromosomal aberrations (genomic disorders). High-throughput single-nucleotide polymorphism (SNP) genotyping is being widely applied in genome-wide association studies (GWASs) with recent successes in identification of common variants that confer risk for common adult diseases. Future applications of high-throughput genotyping and array-based DNA sequencing technology will undoubtedly involve research and diagnostic analyses of rare mutations and perhaps ultimately enable full individual genome sequencing.
To clarify the dietary function of crude rice glycosphingolipid (RG) extracted from rice bran with ethanol, mice were fed a diet containing 0.5% RG for 2 weeks, and the liver gene expression patterns were analyzed using DNA microarray. Dietary intake of RG did not affect expression of drug metabolism-related genes, showing that RG is unlikely to interact with drugs. Glycolysis-related gene expression was slightly increased, but tricarboxylic acid cycle-related gene expression was slightly decreased. As for lipid metabolism-related genes, expression of carnitine acetyltransferase (Crat), which reversibly catalyzes production of acetylcarnitine from acetyl-CoA, was markedly increased. As for gene involved in energy metabolism, expression of P450 (Cyp) cytochrome 7a1, was increased. These findings suggested that RG, as a functional food material, promotes fatty acid and cholesterol metabolism by altering Crat and Cyp7a1 gene expression.
The present article provides a brief overview of various aspects on neuropeptides, emphasizing their multitude and their wide distribution in both the peripheral and central nervous system. Interestingly, neuropeptides are also expressed in various types of glial cells under normal and experimental conditions. The recent identification of, often multiple, receptor subtypes for each peptide, as well as the development of peptide antagonists, have provided an experimental framework to explore functional roles of neuropeptides. A characteristic of neuropeptides is the plasticity in their expression, reflecting the fact that release has to be compensated by de novo synthesis at the cell body level. In several systems peptides can be expressed at very low levels normally but are upregulated in response to, for example, nerve injury. The fact that neuropeptides virtually always coexist with one or more classic transmitters suggests that they are involved in modulatory processes and probably in many other types of functions, for example exerting trophic effects. Recent studies employing transgene technology have provided some information on their functional role, although compensatory mechanisms in all probability could disguise even a well defined action. It has been recognized that both ‘old’ and newly discovered peptides may be involved in the regulation of food intake. Recently the first disease-related mutation in a peptidergic system has been identified, and clinical efficacy of a substance P antagonist for treatment of depression has been reported. Taken together it seems that peptides may play a role particularly when the nervous system is stressed, challenged or afflicted by disease, and that peptidergic systems may, therefore, be targets for novel therapeutic strategies.
A method for obtaining protein molecular masses with an accuracy of approximately +/- 0.01% by matrix-assisted laser desorption using an internal calibrant is described. The technique allows accurate mass determinations of protein sample sizes as small as 1 pmol. High concentrations of organic and inorganic contaminants (e.g. 1 M urea) do not strongly affect either the signal intensity or the mass assignment. The ability to assign an accurate molecular mass to a protein is contingent on the observation of clearly resolved protonated molecule ions in the mass spectrum.
A multineurotransmitter neuronal system that synthesizes and secretes both acetylated and deacetylated forms of alpha-melantropin and beta-endorphin is present in rat and human brain. The N-acetylated from of alpha-melanotropin had more potent behavioral effects than the deacetylated alpha-melanotropin. In the case of beta-endorphin, however, the deacetylated form has been shown to be more potent than the acetylated form. Enzymatic N-acetylation appears to be an important regulatory process for modulating the behavioral activity of peptides secreted from the opiomelanotropinergic multineurotransmitter neuron.
The adrenal medulla chromaffin vesicle (CV) contains, on a weight basis, as much soluble protein and peptide as catecholamine. The bulk of the protein is accounted for by chromogranins (Cgr) A, B and C. Additionally, a large variety of neuropeptides and their precursor proteins have been found recently within these vesicles. Nevertheless, fractionation of CV lysates indicates the presence of many more peptides than previously reported. In the hope of finding novel bioactive peptides, we initiated a systematic isolation and characterisation of CV peptides. Bovine CV pellets were prepared by sucrose gradient centrifugation and immediately boiled in water to avoid degradation of native proteins and peptides. The water lysates were fractionated through a battery of reversed-phase and ion-exchange high-performance chromatographic steps. We fully or partially characterised a substantial number of novel peptides derived from CgrA and CgrB. A tetradecapeptide and a 13 kDa extended peptide were derived from the bovine homologue of rat secretogranin III. Peptides corresponding to C-terminal fragments of 7B2 and proteoglycan II were also found. Additionally, several sequences had no known precursors. Of the sequences derived from known precursors some corresponded to fragments bracketed by pairs of basic amino acids, but others were preceded or followed by single basic residues or by unusual putative cleavage sites. Some of these peptides were postranslationally modified (pyroglutamylation, glycosylation, phosphorylation, amidation). A significant degree of structural conservation of some of these peptides across species suggests that they may exert biological effects when cosecreted with catecholamines during splanchnic stimulation.
The nanoelectrospray ion source (nanoES) has recently been developed and described theoretically. It is different from conventional electrospray sources and from other miniaturized electrospray sources by (i) its 1-2 microns spraying orifice achieved by pulling the spraying capillary to a fine tip, (ii) its very low flow rate of approximately 20 nL/min and the small size of droplets it generates, and (iii) the absence of solvent pumps and inlet valves. The fabrication and operation of nanoES needles is described in detail. Solutions with up to 0.1 M salt contents could be sprayed without sheath flow or pneumatic assist. Improved desolvation in nanoES led to instrument-limited resolution of the signals of a glycoprotein and the ability to signal average extensively allowed the C-terminal sequencing of a 40 kDa protein. Extensive mass spectrometric and tandem mass spectrometric investigation of the components of an unseparated peptide mixture was demonstrated by verification of 93% of the sequence of carbonic anhydrase. A rapid and robust desalting/concentration step coupled to the nanoES procedure allows the direct analysis of impure samples such as peptide mixtures extracted after in-gel digestion.
The structural determination of circulating human peptides is essential to determine their correct posttranslationally processed form. Human hemofiltrate from patients with end stage renal disease is accessible in large quantities and is used as a source for the preparation of circulating peptides. After complete peptide extraction from hemofiltrate, a systematic separation with different chromatographic techniques is achieved. Single peptides are selected according to their mass and chromatographic elution position. Following chromatographic purification, amino acid sequence analysis is performed in combination with data base search. The identification of circulating peptides leads to numerous fragments resulting from cleavage of larger plasma proteins as well as to the discovery of new peptide hormones. The results obtained so far give insight into the degradation of plasma proteins such as fibrinogen, which results in the generation of fragments with biological activity themselves and in the identification of a novel cytokine HCC-1, the first member of beta-defensins in humans, hBD-1, and different peptides not present in any data base.
Mass spectrometry has become an important technique to correlate proteins to their genes. This has been achieved, in part, by improvements in ionization and mass analysis techniques concurrently with large-scale DNA sequencing of whole genomes. Genome sequence information has provided a convenient and powerful resource for protein identification using data produced by matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) and tandem mass spectrometers. Both of these approaches have been applied to the identification of electrophoretically separated protein mixtures. New methods for the direct identification of proteins in mixtures using a combination of enzymatic proteolysis, liquid chromatographic separation, tandem mass spectrometry and computer algorithms which match peptide tandem mass spectra to sequences in the database are also emerging. This tutorial review describes the principles of ionization and mass analysis for peptide and protein analysis and then focuses on current methods employing MALDI and electrospray ionization for protein identification and sequencing. Database searching approaches to identify proteins using data produced by MALDI/TOF and tandem mass spectrometry are also discussed.
A structurally unusual member of the adipokinetic hormone/red pigment-concentrating hormone peptide family was isolated from corpora cardiaca of the painted lady butterfly, Vanessa cardui. Its primary structure was assigned by Edman degradation and nano-electrospray-time-of-flight mass spectrometry as pQLTFTSSWGGK (Vac-AKH). Vac-AKH represents the first 11mer and the first nonamidated peptide in this family.
The peptide shows significant adipokinetic activity in adult specimens of V. cardui. Injection of 10 pmol of synthetic Vac-AKH into 4-day-old decapitated males resulted in an ≈ 150% increase of hemolymph lipids after 90 min. Half maximal adipokinetic activity was achieved with about 0.1 pmol of Vac-AKH. During a 2-h incubation of corpora cardiaca/corpora allata complexes in medium containing 50 mm KCl, significant amounts of Vac-AKH were released from the glands.
The pars intercerebralis-corpora cardiaca system (PI-CC) of insects is the endocrinological equivalent of the hypothalamus-pituitary system of vertebrates. Peptide profiles of the pars intercerebralis and the corpora cardiaca were characterized using simple sampling protocols in combination with MALDI-TOF and electrospray ionization double quadrupole time of flight (ESI-Qq-TOF) mass spectrometric technologies. The results were compared with earlier results of conventional sequencing methods and immunocytochemical methods. In addition to many known peptides, several m/z signals corresponding to putative novel peptides were observed in the corpora cardiaca and/or pars intercerebralis. Furthermore, for a number of peptides evidence was provided about their localization and MALDI-TOF analysis of the released material from the corpora cardiaca yielded information on the hormonal status of particular brain peptides.
Progress in the sequencing of genomes has resulted in an increasing demand for a functional analysis of gene products in order to understand the underlying physiology. Proteomics has established itself as a highly valuable technology for producing functionally related data in an unparalleled fashion, but is methodologically restricted to the analysis of proteins with higher molecular masses (>10 kDa). The development of a technology which covers peptides with low molecular weight and small proteins (0.5 to 15 kDa) was necessary, since peptides, amongst them families of hormones, cytokines and growth factors, play a central role in many biological processes. To summarise the technologies used for this approach the term "peptidomics" is introduced. In this article, we present the rationale and first results of a novel, universal peptide display approach for the analysis and visualisation of peptides and small proteins from biological samples. Special attention is given to samples derived from extracellular fluids such as blood plasma and cerebrospinal fluid. Additionally, a high throughput identification procedure for the analysis of peptides in their native and processed molecular form is outlined.
A Matrix-assisted laser desorption/ionization hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer was employed to acquire neuropeptide mass spectra, directly from neuropeptide secreting tissue deposited on the sample target, in the presence of dihydroxybenzoic acid as matrix. The cockroach corpus cardiacum served as model neuroendocrine tissue. Twelve neuropeptide ion peaks, with mass-to-charge ratio values ranging between 800 and 3,000 Da were selected for tandem mass spectrometry. All peptides below 1,600 Da could be fully sequenced; tandem mass spectrometry analysis of the remaining (three) largest peptides resulted in (limited) sequence tags, which, also due to unavailability of an appropriate neuropeptide structure database, did not allow complete structure elucidation.
Mass spectrometry has become a primary tool for proteomics because of its capabilities for rapid and sensitive protein identification and quantitation. It is now possible to identify thousands of proteins from microgram sample quantities in a single day and to quantify relative protein abundances. However, the need for increased capabilities for proteome measurements is immense and is now driving both new strategies and instrument advances. These developments include those based on integration with multi-dimensional liquid separations and high accuracy mass measurements and promise more than order of magnitude improvements in sensitivity, dynamic range and throughput for proteomic analyses in the near future.
In 2002, the Nobel Prize for chemistry was awarded to the inventors of two novel ionization techniques in mass spectrometry, MALDI and ESI. These techniques, often in combination with data from genomic databases, represent an extremely powerful tool in analytical (bio)chemistry, with many applications, e.g., in the field of proteomics. Peptides, which are small proteins, have, despite their importance as controlling agents in numerous physiological processes, as yet been much less intensively studied by these novel techniques than larger proteins. The term peptidomics, i.e., the study of all peptides expressed by a certain cell, organ or organism was only introduced in 2001. In neuroendocrinology, spectacular progress could already be realized and the future looks bright. In this minireview we discuss the different methodologies that are used in peptidomics and give an overview of the wide range of applications.
The DNA microarray technique has been increasingly utilized in various fields of life sciences. It allows us to analyze the expression levels of thousands of genes simultaneously. The high productivity will facilitate the evaluation of changes in amino acid metabolism and their consequences in response to dietary proteins and amino acids. We compared the expression profiles by the GeneChip system in the liver and other tissues among three groups of rats fed with a 12% casein, a 12% gluten or a protein-free diet. Feeding the gluten or the protein-free diet up- or down-regulated a few hundred genes in the liver compared to the casein diet. Although some of the genes were already known to respond to changes in the protein nutritional state, the majority was newly identified responders. This paper also discusses the possibility of a use this technology for safety evaluation of excessive intake of dietary components, especially of amino acids.
The field of proteomics holds promise for the discovery of new biomarkers for the early detection and diagnosis of disease, molecular targets for therapy and markers for therapeutic efficacy and toxicity. A variety of proteomics approaches may be used to address these goals. Two-dimensional gel electrophoresis (2D-PAGE) is the cornerstone of many discovery-based proteomics studies. Technologies such as laser capture microdissection (LCM) and highly sensitive MS methods are currently being used together to identify greater numbers of lower abundance proteins that are differentially expressed between defined cell populations. Newer technologies such as reverse phase protein arrays will enable the identification and profiling of target pathways in small biopsy specimens. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) analysis enables the high throughput characterization of lysates from very few tumor cells or body fluids and may be best suited for diagnosis and monitoring of disease. Such technologies are expected to supplement our arsenal of mRNA-based assays, and we believe that in the future, entire cellular networks and not just a single deregulated protein will be the target of therapeutics and that we will soon be able to monitor the status of these pathways in diseased cells before, during and after therapy.
Knowledge of the human genome is helping us better understand nutrition. The nutrition-health relationship depends on the adaptive capacity of genes and their functioning with the diet consumed. The greater the efficiency of the system, the lower the metabolic wear suffered. Several epidemiological studies have reported that early-life metabolic imprinting occurs in Man. Both in the uterus and during the first years of life, under and overfed mother-child units imprint gene changes that lead to chronic metabolic problems in later life. Many other mechanisms can modify gene performance, such as gene silencing, gene compensation by a vitamin or a bioactive phytochemical, or simply DNA multiplication during cell reproduction and polyploidisation. The new area of science related to the lateral transfer of recombinant genes is opening new horizons to the nutritional sciences changing the nutritional value or the organoleptic characteristics of food, or even changing genes in persons at risk of chronic disease. Many recent studies provide details about the kinds of diet, nutrients and other compounds that are the best for Man; biotechnology is becoming an instrument enabling food to be offered in the best of conditions.
During the course of evolution, nature has developed a vast number of peptides in all living and past species that display an exceeding diversity of structure and biological effects, such as hormonal and enzyme-controlling activity, communication between cells, and participation in host defence. Sensitive mass spectrometric technologies have been introduced and facilitate access to new natural peptides, even in trace amounts, and allow the quantitative determination of the peptide status of cells, organs and whole organisms (peptidomics). Among the large number of new biologically active peptides identified from an increasing variety of natural sources, regulators of ion channels, chemoattractants, protease inhibitors, metabolism-related hormones, cytotoxins, and antimicrobials have been found. These novel peptides serve as research tools and have potential as diagnostic biomarkers and for the development of peptide and peptidometic drugs.
Current solution based proteomic analysis methods are generally based on enzymatic digestion of a protein mixture followed by separation using multidimensional liquid chromatography and/or electrophoresis where peptide identification is typically accomplished by tandem mass spectrometry (MS/MS). It is generally accepted that no single chromatographic or electrophoretic procedure is capable of resolving the complex mixture of peptides that results from a global proteolytic digest of a proteome. Therefore, combining two or more orthogonal (multimodal) separation procedures dramatically improves the overall resolution and results in a larger number of peptides being identified from complex proteome digests. Separation of a proteome digest is a particularly challenging analytical problem due to the large number of peptides and the wide concentration dynamic range. While it has been demonstrated that increasing the number of dimensions of separation prior to MS analysis increases the number of peptides that may be identified, a balance between the time invested and the overall results obtained must be carefully considered. This manuscript provides a review of two- and three-dimensional peptide separation strategies combined with MS for the analysis of complex peptide mixtures.
Determining the relative levels of neuropeptides in two samples is important for many biological studies. An efficient, sensitive and accurate technique for relative quantitative analysis involves tagging the peptides in the two samples with isotopically distinct labels, pooling the samples and analyzing them using liquid chromatography/mass spectrometry (LC/MS). In this study, we compared two different sets of isotopic tags for analysis of endogenous mouse pituitary peptides: succinic anhydride with either four hydrogens or deuteriums and [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propyl]trimethylammonium chloride with either nine hydrogens or deuteriums. These two labels react with amines and impart either a negative charge (succinyl) or a positive charge (4-trimethylammoniumbutyryl (TMAB)). Every endogenous mouse pituitary peptide labeled with the light TMAB reagent eluted from the C18 reversed-phase column at essentially the same time as the corresponding peptide labeled with the heavy reagent. Most of the peptides labeled with succinyl groups also showed co-elution of the heavy- and light-labeled forms on LC/MS. The mass difference between the heavy and light TMAB reagents (9 Da per label) was larger than that of the heavy and light succinyl labels (4 Da per label), and for some peptides the larger mass difference provided more accurate determination of the relative abundance of each form. Altogether, using both labels, 82 peptides were detected in Cpe(fat/fat) mouse pituitary extracts. Of these, only 16 were detected with both labels, 41 were detected only with the TMAB label and 25 were detected only with the succinyl label. A number of these peptides were de novo sequenced using low-energy collisional tandem mass spectrometry. Whereas the succinyl group was stable to the collision-induced dissociation of the peptide, the TMAB-labeled peptides lost 59 Da per H9 TMAB group. Several peptides identified in this analysis represent previously undescribed post-translational processing products of known pituitary prohormones. In conclusion, both succinyl and TMAB isotopic labels are useful for quantitative peptidomics, and together these two labels provide more complete coverage of the endogenous peptides.
Advances in biological mass spectrometry have resulted in the development of numerous strategies for the large-scale quantification of protein expression levels within cells. These measurements of protein expression are most commonly accomplished through differential incorporation of stable isotopes into cellular proteins. Several variations of the stable isotope quantification method have been demonstrated, differing in isotope composition and incorporation strategy. In general, the majority of these methods establish only relative quantification of expressed proteins. To address this, the absolute quantification (AQUA) strategy was developed for the precise determination of protein expression and post-translational modification levels. The AQUA method relies on the use of a synthetic internal standard peptide that is introduced at a known concentration to cell lysates during digestion. This AQUA peptide precisely mimics a peptide produced during proteolysis of the target protein, except that it is enriched in certain stable isotopes. Analysis of the proteolyzed sample by a selected reaction monitoring (SRM) experiment in a tandem mass spectrometer results in the direct detection and quantification of both the native peptide and isotope labeled AQUA internal standard peptide. As an example, the development and application of a method to measure a tryptic peptide representing the amount of polyubiquitin chain formation through lysine 48 (K48) is presented. The simplicity and sensitivity of the method, coupled with the widespread availability of tandem mass spectrometers, make the AQUA strategy a highly useful procedure for measuring the levels of proteins and post-translational modifications directly from cell lysates.
Meal feeding after a period of food deprivation results in a subsequent increase in the protein and RNA content of the liver. To gain insight into the mechanisms involved in the response to food intake, changes in the association of selected mRNAs with polysomes were examined. On the day of the study, rat livers were collected at 0, 15, 60, and 180 min after the start of feeding and analyzed for biomarkers of the translational control of protein synthesis. Protein synthesis was increased within 60 min and was sustained for 180 min. Assembly of the active eukaryotic initiation factor (eIF) 4F complex was elevated within 15 min, as indicated by the relative association of eIF4E . eIF4G, but returned to the basal value within 180 min. Phosphorylation of the ribosomal protein (rp) S6 kinase S6K1 and its substrate rpS6 was increased within 15 min and was sustained for at least 180 min. Both eIF4F assembly and activation of S6K1 have been linked to upregulated translation of a subset of mRNAs. To identify translationally regulated mRNAs, polysomal (i.e., actively translated) and nonpolysomal (nontranslated) fractions were isolated and subjected to microarray analysis. The mRNAs encoding 78 proteins, including 42 proteins involved in protein synthesis, exhibited increased abundance in polysomes in response to feeding. Overall, the results demonstrate that protein synthesis as well as ribosomal protein mRNA translation undergo rapid and sustained stimulation in the liver after meal feeding and thus contribute to the previously observed increases in protein and RNA content.
There is abundant epidemiological evidence that vegetable consumption decreases colorectal cancer (CRC) risk. However, the molecular targets in the genome are mostly unknown. The present study investigated the effects of vegetable consumption on gene expression in the colon mucosa of female C57Bl/6 mice using cDNA microarray technology. Mice were fed one of 8 diets: a control diet containing no vegetables (diet 1); a diet containing 100 g/kg (diet 2, 10% dose), 200 g/kg (diet 3, 20% dose), or 400 g/kg (diet 4, 40% dose) of a vegetable mixture; or a diet containing 70 g/kg of cauliflower (diet 5, 7% dose), 73 g/kg of carrots (diet 6, 7.3% dose), 226 g/kg of peas (diet 7, 22.6% dose); or 31 g/kg of onions (diet 8, 3.1% dose). The vegetable mixture used in diets 2 to 4 consisted of the 4 individual vegetables used in diets 5 to 8: cauliflower (30% wet wt), carrots (30% wet wt), peas (30% wet wt), and onions (10% wet wt). To assess gene expression changes, colonic mucosal cells were collected after the mice were killed. Total RNA was isolated and microarray technology was used to measure the expression levels of 602 genes simultaneously. For 39 genes, significant dose-dependent effects were found, although in general the relations were not linear. For 15 genes, the altered expression could indeed explain reduced cancer risk at various stages of CRC development. Eleven genes were modulated by the vegetable mixture as well as by one or more of the individual vegetables. For 7 of the genes, the modulation by the mixture was due to the effect of a particular vegetable. These genes are of particular interest because they were consistently affected and could be involved in the prevention of CRC by vegetable consumption.
The direct analysis of tissue sections by MALDI mass spectrometry holds tremendous potential for biomarker discovery. This technology routinely allows many hundreds of proteins to be detected over a mass range of approximately 2000-70 000 Da while maintaining the spatial localization of the proteins detected. This technology has been applied to a wide range of tissue samples, including human glioma tissue and human lung tumor tissue. In many cases, biostatistical analyses of the resulting protein profiles revealed patterns that correlated with disease state and/or clinical endpoints. This work serves as a review of recent applications and summarizes the current state of technology.
Evidence continues to implicate dietary components and genetic susceptibilities as important determinants of cancer risk and tumor behavior. Variation in cancer incidence among and within populations with similar dietary patterns suggests that an individual's response may reflect interactions with genetic factors, which may modify gene, protein, and metabolite expression patterns. Nutrigenomics, defined as the interaction between nutrition and an individual's genome, will likely provide important clues about responders and nonresponders. In this symposium, the role of bioactive food components in colon cancer susceptibility was used to exemplify the application of "omic" technologies for cancer prevention. Topics that were addressed included dietary changes and gene polymorphisms (nutrigenetics), DNA methylation (nutritional epigenomics), gene expression (nutritional transcriptomics), altered formation or bioactivation of proteins (proteomics), and characterizing how the quantity and timing of exposure influence small molecular weight cellular constituents (metabolomics). The final presentation focused on exfoliated cells as a surrogate sample for the evaluation of bioactive food components in cancer prevention. The goal of the symposium was to provide an example of each of the "omic" technologies as they relate to nutrition, cancer risk, and tumor behavior, and to help the participants understand that an integrated framework that simultaneously examines all of the "omic" technologies is needed.
Worldwide, lung cancer is the most prevalent and lethal malignant disease. In addition to avoidance of the most predominant risk factor, i.e., tobacco use, consumption of high amounts of vegetables and fruits could be an effective means of preventing lung cancer. However, the molecular mechanisms underlying lung cancer risk reduction by vegetables are not clear. In the present study, the effect of vegetables on gene expression changes in the lungs of female C57Bl/6 mice was investigated using cDNA microarray technology. The mice were fed 1 of 8 diets for 2 wk: a control diet containing no vegetables (diet 1); a diet containing a vegetable mixture at 100 (diet 2, 10% dose), 200 (diet 3, 20% dose), or 400 (diet 4, 40% dose) g/kg; or a diet containing cauliflower at 70 (diet 5, 7% dose); carrots at 73 (diet 6, 7.3% dose); peas at 226 (diet 7, 22.6% dose); or onions at 31 (diet 8, 3.1% dose) g/kg. The vegetable mixture consisted of these 4 individual vegetables. After the mice were killed, the lungs were removed and total RNA was isolated from the lungs for expression analysis of 602 genes involved in pathways of (anti)-carcinogenesis. The results of this study suggest that individual vegetables have a higher potential of modulating genes (5 from the 8 modulated genes) in favor of lung cancer risk prevention, in comparison with the vegetable mixture (2 from the 7 modulated genes); the other gene modulations are expected to enhance lung cancer risk. The pathways involved were miscellaneous and included cell growth, apoptosis, biotransformation, and immune response. Furthermore, carrots were able to modulate most gene expressions, and most of these effects occurred in processes that favored lung cancer risk prevention. The current study provides more insight into the genetic mechanisms by which vegetables, in particular carrots, can prevent lung cancer risk.
Although clinical manifestations of cognitive dysfunction and impairments of activities of daily living are the current standard measures for the diagnosis of Alzheimer's disease, biomarkers are receiving increasing attention in research centers as possible early diagnostic measures or as surrogate measures of the ongoing pathology. In preparation for the upcoming development of the Diagnostic and Statistical Manual of Mental Disorders (5th ed; DSM-V) nosology, the American Psychiatric Association has sponsored an effort to reassess the current approaches to diagnosis in dementia in general and Alzheimer's disease in particular. This article focuses on the potential use of biomarkers in the diagnosis of Alzheimer's disease, in the monitoring of mild cognitive impairment, and as possible prognostic markers in normal controls at risk for dementia. Most advanced information is available with the biomarkers found in the cerebrospinal fluid, but there are many other potential biomarkers using blood, brain imaging, or a combination. The current biomarker approaches to diagnosis are reviewed along with a special emphasis on near-term recommendations and further research directions.
Prohormone convertase (PC) 1/3 and 2 are involved in the generation of neuropeptides from their precursors. A quantitative peptidomic approach was used to explore the role PC2 plays in the processing of hypothalamic peptides. In this approach, extracts from mice lacking PC2 activity and from wild-type littermates were labeled with isotopic tags, combined, fractionated on a reverse phase HPLC column, and analyzed by electrospray ionization mass spectrometry. Altogether, 53 neuropeptides or other peptides derived from secretory pathway proteins were identified and sequenced using tandem mass spectrometry. These peptides arise from 21 distinct proteins: proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, promelanin-concentrating hormone, proneurotensin, proneuropeptide Y, provasopressin, pronociceptin/orphanin, prothyrotropin-releasing hormone, cocaine- and amphetamine-regulated transcript, chromogranin A and B, secretogranin II, prohormone convertase 1 and 2, propeptidyl-amidating monooxygenase, and proteins designated proSAAS and VGF. Approximately one third of the peptides found in wild-type mice were not detectable in PC2 knock-out mice, and another third were present at levels ranging from 25 to 75% of wild-type levels. Comparison of the cleavage sites suggests that sequences with a Trp, Tyr and/or Pro in the P1' or P2' position, or a basic residue in the P3 position, are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.
Neuropeptidomics is the analysis of the neuropeptides present in a tissue extract. Most neuropeptidomic studies use mass spectrometry to detect and identify the peptides, which provides information on the precise posttranslationally modified form of each peptide. Quantitative peptidomics uses isotopic labels to compare the levels of peptides in extracts from two different samples. This technique is ideal for examining neuropeptide levels in a variety of systems and is especially suited for studies of mice lacking peptide-processing enzymes. This review is focused on the neuropeptidomics technique and its application to the analysis of mice with a mutation that inactivates carboxypeptidase E, a critical enzyme in the biosynthesis of many neuroendocrine peptides. Mice without carboxypeptidase E activity are overweight, and a key question is the identification of the peptide or peptides responsible. The quantitative peptidomics approach has provided some insights toward the answer to this question.
Recent studies using single-nucleotide polymorphism arrays have pinpointed novel oncogenes and tumor suppressors involved in specific types of human cancers.
As cancer incidence is projected to increase for decades there is a need for effective preventive strategies. Fortunately, evidence continues to mount that altering dietary habits is an effective and cost-efficient approach for reducing cancer risk and for modifying the biological behavior of tumors. Predictive, validated and sensitive biomarkers, including those that reliably evaluate "intake" or exposure to a specific food or bioactive component, that assess one or more specific biological "effects" that are linked to cancer, and that effectively predict individual "susceptibility" as a function of nutrient-nutrient interactions and genetics, are fundamental to evaluating who will benefit most from dietary interventions. These biomarkers must be readily accessible, easily and reliably assayed, and predictive of a key process(es) involved in cancer. The response to a food is determined not only by the effective concentration of the bioactive food component(s) reaching the target tissue, but also by the amount of the target requiring modification. Thus, this threshold response to foods and their components will vary from individual to individual. The key to understanding a personalized response is a greater knowledge of nutrigenomics, proteomics and metabolomics.
The general characteristics of neuropeptides are discussed as a background for the understanding of their role in regulation of physiological systems. The extent of those systems that are crucially affected by neuropeptides is vast and the complexity of their interactions makes the clinical focus on a specific neuropeptide unsatisfactory. The clinical potential of neuropeptides affecting eating disorders, CNS behavioral disorders and the neuroregenerative and neuroprotective action of neuropeptides is discussed. It is probable that successful neuropeptide therapeutics will depend upon the application of translational and combinational research using various ingenious combinations of neuropeptides, their agonists and antagonists, neuropeptide receptor agonists and antagonists, improved methods of delivery and the development of peptides targeted to the genetic profile of individual patients.
This article reviews the works published since 2001 (included) on the micro/nano-HPLC analysis of bioactive and biomarker peptides. The main achievements related to the improvement of the detection sensitivity, quantitation repeatability and reproducibility, and separation selectivity are highlighted. A wide attention is paid to the application of micro/nano-HPLC to the analysis of bioactive peptides in biological matrices. The uses of micro/nano-HPLC in peptidomics to discover new endogenous bioactive peptides and to develop quantitation procedures to compare the levels of peptides of interest in two different biological samples are also considered. Finally, the application of micro/nano-HPLC to the analysis of biomarker peptides for various diseases is also included in this review.
MS is currently one of the most important analytical techniques in biological and medical research. ESI and MALDI launched the field of MS into biology. The performance of mass spectrometers increased tremendously over the past decades. Other technological advances increased the analytical power of biological MS even more. First, the advent of the genome projects allowed an automated analysis of mass spectrometric data. Second, improved separation techniques, like nanoscale HPLC, are essential for MS analysis of biomolecules. The recent progress in bioinformatics is the third factor that accelerated the biochemical analysis of macromolecules. The first part of this review will introduce the basics of these techniques. The field that integrates all these techniques to identify endogenous peptides is called peptidomics and will be discussed in the last section. This integrated approach aims at identifying all the present peptides in a cell, organ or organism (the peptidome). Today, peptidomics is used by several fields of research. Special emphasis will be given to the identification of neuropeptides, a class of short proteins that fulfil several important intercellular signalling functions in every animal. MS imaging techniques and biomarker discovery will also be discussed briefly.
In the present work, an exhaustive review of the main developments and applications of CE-MS for peptide analysis is given. This review includes the use of different CE separation modes, MS analyzers, capillary coatings, preconcentration techniques, on-chip applications as well as other different multidimensional strategies for peptide analysis. Key applications are critically discussed and relevant works published from January 2000 to May 2007 are summarized including information concerning the type of sample, CE-MS parameters as well as some figures of merit of the different CE-MS procedures developed for peptide analysis and peptidomics.