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PubMed2006

Shotgun proteomics using the iTRAQ isobaric tags.

Shotgun proteomic methods involving isobaric tagging of peptides enable high-throughput proteomic analysis. iTRAQ reagents allow simultaneous identification and quantitation of proteins in four different samples using tandem mass spectrometry (MS). In this article, we provide a brief description of proteome analysis using iTRAQ reagents and review the current applications of these reagents in proteomic studies. We also compare different aspects of protein identification including protein sequence coverage and proteome coverage obtained using iTRAQ reagents with those using other shotgun proteomic techniques. We briefly discuss the issue of isotope purity correction in measured peak areas during protein quantitation using iTRAQ reagents. Finally, we conclude with some of the current challenges in MS-based proteomic analysis that are limiting protein identifications obtained by different shotgun proteomic methods.

Animals↗

PubMed2001

Cardiovascular proteomics: evolution and potential.

The development of proteomics is a timely one for cardiovascular research. Analyses at the organ, subcellular, and molecular levels have revealed dynamic, complex, and subtle intracellular processes associated with heart and vascular disease. The power and flexibility of proteomic analyses, which facilitate protein separation, identification, and characterization, should hasten our understanding of these processes at the protein level. Properly applied, proteomics provides researchers with cellular protein "inventories" at specific moments in time, making it ideal for documenting protein modification due to a particular disease, condition, or treatment. This is accomplished through the establishment of species- and tissue-specific protein databases, providing a foundation for subsequent proteomic studies. Evolution of proteomic techniques has permitted more thorough investigation into molecular mechanisms underlying cardiovascular disease, facilitating identification not only of modified proteins but also of the nature of their modification. Continued development should lead to functional proteomic studies, in which identification of protein modification, in conjunction with functional data from established biochemical and physiological methods, has the ability to further our understanding of the interplay between proteome change and cardiovascular disease.

Animals↗

PubMed2004

Proteomics in platelet science.

Proteomics, the rapid large-scale analysis of cellular proteins, is becoming an important tool in the study of human platelet biology. A number of investigators have used proteomics techniques to study platelets including two-dimensional gel electrophoresis, protein separation, and mass spectrometry protein identification. Most efforts have focused on: 1) cataloging the spectrum of proteins that comprise the normal platelet "proteome", 2) characterizing proteins released from activated platelets, and 3) identifying phosphoproteins generated upon platelet stimulation. The incorporation of modern proteomics technology to platelet studies will further our understanding of platelet activation, adhesion, and aggregation at the protein level. Moreover, proteomics offers hope that novel biomarkers can be discovered that diagnose inherited and/or acquired platelet disorders, or predict the susceptibility of such individuals to bleeding or thrombosis. Finally, proteomics could lead to identification of novel agents that have therapeutic value. This review discusses the current status and future opportunities of proteomics technologies in platelet science.

Blood Platelets↗

PubMed2005

Phosphoprotein profiling of erythropoietin receptor- dependent pathways using different proteomic strategies.

Proteomic techniques provide new tools for the global analysis of protein profiles but also for the investigation of specific protein functions. The analysis of signaling cascades has traditionally been performed by the determination of enzymatic or transcription factor activities representing a certain pathway. Functional proteomics now allows more comprehensive approaches to study cellular responses induced during ligand/receptor interactions. In this study we evaluated proteomic strategies for the investigation of structure-function relationships in the erythropoietin receptor signalling complex. After expression of epidermal growth factor/erythropoietin receptor mutant molecules in an identical cellular background we characterized their potential to induce cellular activities. Using this system we focused our efforts on post-translational modifications of signalling proteins reflecting a substantial part of receptor-dependent signaling events. Although tyrosine phosphorylated proteins were enriched by immunoprecipitation the analysis using the classical approach combining two-dimensional gel electrophoresis and identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry revealed that low expressed signaling proteins cannot be detected by this technique. An alternative strategy using one-dimensional gel separation of phosphoproteins and liquid chromatography-tandem mass spectrometry, however, allowed us to identify multiple proteins involved in intracellular signalling representing already established pathways but also proteins which have not been linked to EPO-induced signaling so far. This approach offers the potential to extend functional proteomic studies to complex signaling processes.

Animals↗

PubMed2005

Proteomic evaluation of milk from different mammalian species as a substitute for breast milk.

UNLABELLED: As milk represents the main source of nutrition for infants, the question of an effective human milk substitute becomes mandatory when a formula-fed baby is allergic to cows' milk proteins. In this case, formulas containing extensively hydrolysed milk proteins should be preferred, but even such a formula may cause allergic reactions in highly sensitive patients. If there is evidence of cows' milk allergy with IgE-associated symptoms, after 6 mo of age, a soy bean formula may be recommended only when tolerance to soy protein has been established by clinical challenge. In infants with allergic reactions to cows' milk proteins, even after extensive hydrolyzation, proteomic techniques coupled to immunological methods may make it possible to select other milk products that do not contain the same allergens as ordinary cow's milk. In this paper, evidence will be presented that proteomic evaluation of proteins from different mammalian species may be a suitable method of testing whether proteins from the milk of different mammalian species may be used as a substitute for untreated bovine milk. CONCLUSION: Proteomic evaluation of milk from different mammalian species may not only be of help when recommending suitable feeding in cases of cows' milk allergy but also gives new insight into the background to allergic reactions caused by milk proteins.

Animals↗

PubMed2005

An emerging role for comprehensive proteome analysis in human pregnancy research.

Elucidation of underlying cellular and molecular mechanisms is pivotal to the comprehension of biological systems. The successful progression of processes such as pregnancy and parturition depends on the complex interactions between numerous biological molecules especially within the uterine microenvironment. The tissue- and stage-specific expression of these bio-molecules is intricately linked to and modulated by several endogenous and exogenous factors. Malfunctions may manifest as pregnancy disorders such as preterm labour, pre-eclampsia and fetal growth restriction that are major contributors to maternal and perinatal morbidity and mortality. Despite the immense amount of information available, our understanding of several aspects of these physiological processes remains incomplete. This translates into significant difficulties in the timely diagnosis and effective treatment of pregnancy-related complications. However, the emergence of powerful mass spectrometry-based proteomic techniques capable of identifying and characterizing multiple proteins simultaneously has added a new dimension to the field of biomedical research. Application of these high throughput methodologies with more conventional techniques in pregnancy-related research has begun to provide a novel perspective on the biochemical blueprint of pregnancy and its related disorders. Further, by enabling the identification of proteins specific to a disease process, proteomics is likely to contribute, not only to the comprehension of the underlying pathophysiologies, but also to the clinical diagnosis of multifactorial pregnancy disorders. Although the application of this technology to pregnancy research is in its infancy, characterization of the cellular proteome, unearthing of functional networks and the identification of disease biomarkers can be expected to significantly improve maternal healthcare in the future.

Female↗

PubMed2005

The clinical application of proteomics.

BACKGROUND: Proteomics is defined as a scientific approach used to elucidate all protein species within a cell or tissue, and many researchers are taking advantage of proteomic technology to elucidate protein changes between healthy and diseased states. METHODS: The application of proteomic techniques and strategies to the field of medicine is slowly transforming the way biomarker discovery is conducted. However, the complexity of serum is the source of both its promise to clinical applications and its challenge to proteomic analysis. Like any new technology when it is first introduced, proteomics has been touted with much hope and promise. RESULTS AND CONCLUSIONS: We provide a review of the clinical application of proteomics with the emphasis on current practical issues and challenges facing proteomic research.

Humans↗

PubMed2004

Tissue-print and print-phoresis as platform technologies for the molecular analysis of human surgical specimens: mapping tumor invasion of the prostate capsule.

Molecular profiling of human biopsies and surgical specimens is frequently complicated by their inherent biological heterogeneity and by the need to conserve tissue for clinical diagnosis. We have developed a set of novel 'tissue print' and 'print-phoresis' technologies to facilitate tissue and tumor-marker profiling under these circumstances. Tissue printing transfers cells and extracellular matrix components from a tissue surface onto nitrocellulose membranes, generating a two-dimensional anatomical image on which molecular markers can be visualized by specific protein and RNA- and DNA-detection techniques. Print-phoresis is a complementary new electrophoresis method in which thin strips from the print are subjected to polyacrylamide gel electrophoresis, providing a straightforward interface between the tissue-print image and gel-based proteomic techniques. Here we have utilized these technologies to identify and characterize markers of tumor invasion of the prostate capsule, an event generally not apparent to the naked eye that may result in tumor at the surgical margins ('positive margins'). We have also shown that tissue-print technologies can provide a general platform for the generation of marker maps that can be superimposed directly onto histopathological and radiological images, permitting molecular identification and classification of individual malignant lesions.

Biomarkers↗

PubMed2005

Peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display.

The human Plasma Proteome Project pilot phase aims to analyze serum and plasma specimens to elucidate specimen characteristics by various proteomic techniques to ensure sufficient sample quality for the HUPO main phase. We used our proprietary peptidomics technologies to analyze the samples distributed by HUPO. Peptidomics summarizes technologies for visualization, quantitation, and identification of the low-molecular-weight proteome (<15 kDa), the "peptidome." We analyzed all four HUPO specimens (EDTA plasma, citrate plasma, heparin plasma, and serum) from African- and Asian-American donors and compared them to in-house collected Caucasian specimens. One main finding focuses on the most suitable method of plasma specimen collection. Gentle platelet removal from plasma samples is beneficial for improved specificity. Platelet contamination or activation of platelets by low temperature prior to their removal leads to distinct and multiple peptide signals in plasma samples. Two different specimen collection protocols for platelet-poor plasma are recommended. Further emphasis is placed on the differences between plasma and serum on a peptidomic level. A large number of peptides, many of them in rather high abundance, are only present in serum and not detectable in plasma. This ex vivo generation of multiple peptides hampers discovery efforts and is caused by a variety of factors: the release of platelet-derived peptides, other peptides derived from cellular components or the clot, enzymatic activities of coagulation cascades, and other proteases. We conclude that specimen collection is a crucial step for successful peptide biomarker discovery in human blood samples. For analysis of the low-molecular-weight proteome, we recommend the use of platelet-depleted EDTA or citrate plasma.

Anticoagulants↗

PubMed2006

Sub-speciating Campylobacter jejuni by proteomic analysis of its protein biomarkers and their post-translational modifications.

We have identified several protein biomarkers of three Campylobacter jejuni strains (RM1221, RM1859, and RM3782) by proteomic techniques. The protein biomarkers identified are prominently observed in the time-of-flight mass spectra (TOF MS) of bacterial cell lysate supernatants ionized by matrix-assisted laser desorption/ionization (MALDI). The protein biomarkers identified were: DNA-binding protein HU, translation initiation factor IF-1, cytochrome c553, a transthyretin-like periplasmic protein, chaperonin GroES, thioredoxin Trx, and ribosomal proteins: L7/L12 (50S), L24 (50S), S16 (30S), L29 (50S), and S15 (30S), and conserved proteins similar to strain NCTC 11168 proteins Cj1164 and Cj1225. The protein biomarkers identified appear to represent high copy, intact proteins. The significant findings are as follows: (1) Biomarker mass shifts between these strains were due to amino acid substitutions of the primary polypeptide sequence and not due to changes in post-translational modifications (PTMs). (2) If present, a PTM of a protein biomarker appeared consistently for all three strains, which supported that the biomarker mass shifts observed between strains were not due to PTM variability. (3) The PTMs observed included N-terminal methionine (N-Met) cleavage as well as a number of other PTMs. (4) It was discovered that protein biomarkers of C. jejuni (as well as other thermophilic Campylobacters) appear to violate the N-Met cleavage rule of bacterial proteins, which predicts N-Met cleavage if the penultimate residue is threonine. Two protein biomarkers (HU and 30S ribosomal protein S16) that have a penultimate threonine residue do not show N-Met cleavage. In all other cases, the rule correctly predicted N-Met cleavage among the biomarkers analyzed. This exception to the N-Met cleavage rule has implications for the development of bioinformatics algorithms for protein/pathogen identification. (5) There were fewer biomarker mass shifts between strains RM1221 and RM1859 compared to strain RM3782. As the mass shifts were due to the frequency of amino acid substitutions (and thus underlying genetic variations), this suggested that strains RM1221 and RM1859 were phylogenetically closer to one another than to strain RM3782 (in addition, a protein biomarker prominent in the spectra of RM1221 and RM1859 was absent from the RM3782 spectrum due to a nonsense mutation in the gene of the biomarker). These observations were confirmed by a nitrate reduction test, which showed that RM1221 and RM1859 were C. jejuni subsp. jejuni whereas RM3782 was C. jejuni subsp. doylei. This result suggests that detection/identification of protein biomarkers by pattern recognition and/or bioinformatics algorithms may easily subspeciate bacterial microorganisms. (6) Finally, the number and variation of PTMs detected in this relatively small number of protein biomarkers suggest that bioinformatics algorithms for pathogen identification may need to incorporate many more possible PTMs than suggested previously in the literature.

Amino Acid Sequence↗

PubMed2006

Plasma proteome analysis: 2D gels and chips.

The knowledge of concentration, modification and interaction of proteins is fundamental in determining the phenotype of living organisms. Plasma, the primary clinical specimen, contains numerous and diverse proteins. The functions of these proteins are as manifold as the diversity of the protein themselves. Many of them have been largely used for many years as biomarkers of diseases and indicators of the physiological functions. The study of plasma proteome promises to be a significant advance in various areas of biological and clinical research. Two-dimensional polyacrylamide gel electrophoresis is considered as a primary tool in separating thousand of plasma proteins. This approach enables comparing normal and diseased samples revealing differently expressed proteins. Other proteomic techniques suitable for plasma analysis such as protein microarrays are now either established or are still being improved. This article briefly reviews the application of two-dimensional electrophoresis and the current status of technical aspects for plasma proteome.

Blood Proteins↗

PubMed2005

High-resolution functional proteomics by active-site peptide profiling.

Characterization and functional annotation of the large number of proteins predicted from genome sequencing projects poses a major scientific challenge. Whereas several proteomics techniques have been developed to quantify the abundance of proteins, these methods provide little information regarding protein function. Here, we present a gel-free platform that permits ultrasensitive, quantitative, and high-resolution analyses of protein activities in proteomes, including highly problematic samples such as undiluted plasma. We demonstrate the value of this platform for the discovery of both disease-related enzyme activities and specific inhibitors that target these proteins.

Animals↗

PubMed2004

Proteomic analysis of kinase inhibitor selectivity and function.

Small molecule inhibitors of protein kinases have become highly popular tools in signal transduction research, despite the fact that rather limited data about their respective selectivities have been available. We established an efficient chemical proteomics method to characterize the cellular targets of the widely used inhibitor SB 203580, which was deemed to be rather specific for p38 kinase. Our results revealed several protein kinases as high affinity targets of SB 203580 and therefore imply a far more complicated cellular mode of action of this inhibitor than previously assumed. This raises the important question whether a lack of selectivity is inherent to many other "specific" inhibitors of protein kinases and warrants their evaluation employing experimental approaches adapted from our described proteomic technique.

Animals↗

PubMed2001

Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all-trans retinoic acid in vitro.

Embryonic stem (ES) cells are totipotent stem cells, which can differentiate into various kinds of cell types, including neurons. They are widely used as a model system for investigating mechanisms of differentiation events during early mouse development. In this study, proteomic techniques were used to approach the protein profile associated with the early-stage differentiation of ES cells into neuronal cells induced by all-trans retinoic acid (ATRA) in vitro. In comparison of the protein profile of parent ES cells with that of ES-derived neural-committed cells, which was induced by ATRA for four days, 24 differentially displayed protein spots were selected from two-dimensional electrophoresis (2-DE) gels for further protein identification by pepide mass fingerprinting (PMF). Nine proteins were known to being involved in the process of neural differentiation and/or neural survival. Of those, alpha-3/alpha-7 tubulin and vimentin were down-regulated, while cytokeratin 8, cytokeratin 18, G1/S-special cyclin D2, follistatin-related protein, NEL protein, platelet-activating factor acetylhydrolase IB alpha-subunit, and thioredoxin peroxidase 2 were upregulated during differentiation of ES cells to neural cells. Additionally, other 12 protein (five upregulated and seven downregulated) spots associated with ES cell differentiation into neuronal cells were not matched to known proteins so far, implicating that they might be novel proteins. The results above indicated that the molecular mechanisms of differentiation of ES cells to neural cells in vitro might be similar to those of other neural systems in vitro and identified that proteomic analysis is an effective strategy to comprehensively unravel the regulatory network of differentiation.

Animals↗

PubMed2007

Anabolic effects of a non-myotoxic dose of the beta2-adrenergic receptor agonist clenbuterol on rat plantaris muscle.

Previous investigations of the effects of clenbuterol have used suprapharmacological doses that induce myocyte death, alter muscle phenotype, and do not approximate the proposed therapeutic dose for humans. Recently, we reported that smaller doses of clenbuterol induce muscle growth without causing myocyte death. In the present study we used histochemical and proteomic techniques to investigate the molecular effects of this dose. Male Wistar rats (n = 6, per group) were infused with saline or 10 microg/kg/day clenbuterol via subcutaneously implanted osmotic pumps. After 14 days the animals' plantaris muscles were isolated for histochemical and proteomic analyses. Clenbuterol induced significant muscle growth with concomitant protein accretion and preferential hypertrophy of fast oxidative glycolytic fibers. Clenbuterol reduced the optical density of mitochondrial staining in fast fibers by 20% and the glycogen content of the muscle by 30%. Differential analysis of two-dimensional gels showed that heat shock protein 72 and beta-enolase increased, whereas aldolase A, phosphogylcerate mutase, and adenylate kinase decreased. Only heat shock protein 72 has previously been investigated in clenbuterol-treated muscles. The clenbuterol-induced increase in muscle growth was concomitant with qualitative changes in the muscle's proteome that need to be considered when proposing therapeutic uses for this agent.

Adrenergic beta-Agonists↗

PubMed2004

Gender-specific proteomic alterations in glycolytic and mitochondrial pathways in aging monkey hearts.

We utilized proteomic techniques in a primate model (Macaca fascicularis) of aging to determine potential mechanisms to explain gender differences in protection of the aging heart. The majority of prior work in this field utilized rodent models, and importantly no prior study utilized a proteomic approach in the aging heart. We studied changes in proteins in seven monkeys in each group (young and old males and females (YMs, OMs, YFs, and OFs, respectively)), and used two-dimensional gel electrophoresis in combination with mass spectrometry in five monkeys in each group. We found decreases (P < 0.05) in the expression of key enzymes in glycolysis (e.g. pyruvate kinase, alpha-enolase, triosephosphate isomerase), glucose oxidation (e.g. pyruvate dehydrogenase E1 beta-subunit), and the tricarboxylic acid (TCA) cycle (2-oxoglutarate dehydrogenase) in left ventricular (LV) samples from OM monkeys; these changes in glycolytic, glucose oxidation, and TCA enzymes were not observed either in YMs, YFs or OFs. We found additional gender differences in the reduced expression and function of proteins that are responsible for electron transport and oxidative phosphorylation in mitochondria only in hearts from OM monkeys, with corresponding decreased oxidation rates with NADH and ascorbate-N,N,N',N' ''-tetramethyl-p-phenylenediamine substrates. The changes in glycolytic and mitochondrial metabolic pathways in OM monkey hearts are similar to changes observed in hearts affected by diabetes or LV dysfunction, and could be involved in the mechanism for the cardiomyopathy of aging. The sparing of these changes in OF hearts could be involved in the mechanism mediating delayed cardiovascular risk in OFs.

Aging↗

PubMed2003

Functional genomics and proteomics in control of breathing.

New genomic and proteomic techniques offer remarkable promise as tools to address longstanding questions regarding molecular mechanisms involved in the control of breathing. Here, these techniques are described. Additionally, recent examples of the application of these and related molecular techniques are provided-particularly including observations regarding the role of S-nitrosylation signaling reactions in regulating ventilatory responses.

Animals↗

PubMed2005

Proteins associated with Cisplatin resistance in ovarian cancer cells identified by quantitative proteomic technology and integrated with mRNA expression levels.

Nearly all women diagnosed with ovarian cancer receive combination chemotherapy including cis- or carboplatin. Despite high initial response rates, resistance to cisplatin develops in roughly one-third of women during primary treatment and in all women treated for recurrent disease. ICAT coupled with tandem MS is a quantitative proteomic technique for high throughput protein expression profiling of complex protein mixtures. Using ICAT/MS/MS we profiled the nuclear, cytosolic, and microsomal fractions obtained from IGROV-1 [corrected] (cisplatin-sensitive) and IGROV-1/CP [corrected] (cisplatin-resistant) ovarian cancer cell lines. The proteomes of cisplatin-sensitive and -resistant ovarian cancer cells were compared, and protein expression was correlated with mRNA expression profiles. A total of 1117 proteins were identified and quantified. The relative expression of 121 of these varied between the two cell lines. Sixty-three proteins were overexpressed in cisplatin-sensitive, and 58 were over expressed in cisplatin-resistant cells. Examples of proteins at least 5-fold overexpressed in resistant cells and with biological relevance to cancer include cell recognition molecule CASPR3 (13.3-fold), S100 protein family members (8.7-fold), junction adhesion molecule Claudin 4 (7.2-fold), and CDC42-binding protein kinase beta (5.4-fold). Examples of cancer-related proteins at least 5-fold overexpressed in sensitive cells include hepatocyte growth factor inhibitor 1B (13.3-fold) and programmed cell death 6-interacting protein (12.7-fold). The direction of changes in expression levels between proteins and mRNAs were not always in the same direction, possibly reflecting posttranscriptional control of protein expression. We identified proteins whose expression profiles correlate with cisplatin resistance in ovarian cancer cells. Several proteins may be involved in modulating response to cisplatin and have potential as markers of treatment response or treatment targets.

Cell Line, Tumor↗