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PubMed2006

Antigen-epitope retrieval to facilitate proteomic analysis of formalin-fixed archival brain tissue.

Formalin is a routine fixative facilitating tissue preservation and histopathology. Proteomic techniques require freshly frozen specimens, which are often difficult to procure, and methods facilitating proteomic analysis of archival formalin-fixed brain tissue are lacking. We employed antigen-epitope-retrieval principles to facilitate proteomic analysis of brain tissue that had been fixed and stored in formalin for 3-7 years. Twenty-micrometer-thick cryopreserved OCT-embedded sections from inferior temporal cortex of human (7 years in formalin) or mouse brain specimens (3 years in formalin) were hematoxylin-/eosin-stained. Approximately 16-64-mm2 areas of the tissue sections were manually scraped off slides, or approximately 2 mm2 of human brain cortex was captured off membrane-coated slides using laser microdissection. Tissue was treated using various pH and temperature conditions prior to trypsin digestion and nano-LC-MS/MS. The largest number of proteins were retrieved by solubilization at pH 9 at 95 degrees C for 1 h; treatments at pH 4 or 6 at 25 or 65 degrees C were generally ineffective. Three-year formalin-fixed murine tissue did not yield more proteins compared to human tissue. Use of formalin-fixed tissue for proteomics is an invaluable tool for medical research. The combination of proteomics and microdissection enables selective enrichment and identification of novel, unique, or abundant proteins that may be important in pathogenesis.

Animals↗
PubMed1999

Large scale characterization of plant plasma membrane proteins.

After a brief review of the strategies used to date to identify systematically plasma membrane (PM) proteins, emphasis was given to the proteomic approach of PM proteins from the model plant Arabidopsis thaliana. Comparative analysis of two-dimensional gels from PM and cytosolic fractions was used to assess the cellular origin of proteins found in PM fraction. The classification obtained was confirmed by protein sequencing that showed, in addition, that most analyzed proteins were peripheral proteins. A large proportion of these appeared to correspond to PM-constitutive proteins that were present in the PM from different plant organs, but were not uniquely located at the PM depending on the organ. In addition, the presence of organ-specific sets of PM-specific proteins was also demonstrated. Additional procedures were developed to identify integral PM proteins. The combined use of PM washes with alkaline carbonate buffer or Triton X-100/KBr, and of a new detergent to solubilize protein, resulted in improved recovery of hydrophobic proteins on gels. Results are discussed in terms of construction of comprehensive proteomes for PM and other membranes and organelles.

Animals↗
PubMed2005

Global protein expression analysis in apicomplexan parasites: current status.

Members of the phylum Apicomplexa are important protozoan parasites that cause some of the most serious, and in some cases, deadly diseases in humans and animals. They include species from the genus Plasmodium, Toxoplasma, Eimeria, Neospora, Cryptosporidium, Babesia and Theileria. The medical, veterinary and economic impact of these pathogens on a global scale is enormous. Although chemo- and immuno-prophylactic strategies are available to control some of these parasites, they are inadequate. Currently, there is an urgent need to design new vaccines or chemotherapeutics for apicomplexan diseases. High-throughput global protein expression analyses using gel or non-gel based protein separation technologies coupled with mass spectrometry and bioinformatics provide a means to identify new drug and vaccine targets in these pathogens. Protein identification based proteomic projects in apicomplexan parasites is currently underway, with the most significant progress made in the malaria parasite, Plasmodium falciparum. More recently, preliminary two-dimensional gel electrophoresis maps of Toxoplasma gondii and Neospora caninum tachyzoites and Eimeria tenella sporozoites, have been produced, as well as for micronemes in E. tenella. In this review, the status of proteomics in the analysis of global protein expression in apicomplexan parasites will be compared and the challenges associated with these investigations discussed.

Animals↗
PubMed2004

Comparative analysis of complete genomes reveals gene loss, acquisition and acceleration of evolutionary rates in Metazoa, suggests a prevalence of evolution via gene acquisition and indicates that the evolutionary rates in animals tend to be conserved.

In this study we systematically examined the differences between the proteomes of Metazoa and other eukaryotes. Metazoans (Homo sapiens, Ceanorhabditis elegans and Drosophila melanogaster) were compared with a plant (Arabidopsis thaliana), fungi (Saccharomyces cerevisiae and Schizosaccaromyces pombe) and Encephalitozoan cuniculi. We identified 159 gene families that were probably lost in the Metazoan branch and 1263 orthologous families that were specific to Metazoa and were likely to have originated in their last common ancestor (LCA). We analyzed the evolutionary rates of pan-eukaryotic protein families and identified those with higher rates in animals. The acceleration was shown to occur in: (i) the LCA of Metazoa or (ii) independently in the Metazoan phyla. A high proportion of the accelerated Metazoan protein families was found to participate in translation and ribosome biogenesis, particularly mitochondrial. By functional analysis we show that no metabolic pathway in animals evolved faster than in other organisms. We conclude that evolution in the LCA of Metazoa was extensive and proceeded largely by gene duplication and/or invention rather than by modification of extant proteins. Finally, we show that the rate of evolution of a gene family in animals has a clear, but not absolute, tendency to be conserved.

Animals↗
PubMed2004

Bioinformatic analysis of the urine proteome of acute allograft rejection.

The urinary proteome in health and disease attracts increasing attention because of the potential diagnostic and pathophysiologic biomarker information carried by specific excreted proteins or their constellations. This cross-sectional study aimed to analyze the urinary proteome in patients with biopsy-proven acute rejection (n = 23) compared with transplant recipients with stable graft function (n = 22) and healthy volunteers (n = 20) and to correlate this with clinical, morphologic, and laboratory data. Urine samples were preadsorbed on four different protein chip surfaces, and the protein composition was analyzed using a surface-enhanced laser desorption/ionization time-of-flight mass spectrometer platform. The data were analyzed using two independent approaches to sample classification. Patients who experienced acute rejection could be distinguished from stable patients with a sensitivity of 90.5 to 91.3% and a specificity of 77.2 to 83.3%, depending on the classifier used. Protein masses that were important in constructing the classification algorithms included those of mass 2003.0, 2802.6, 4756.3, 5872.4, 6990.6, 19,018.8, and 25,665.7 Da. Normal urine was distinguished from transplant urine using a protein marker of mass 78,531.2 Da with both a sensitivity and a specificity of 100%. In conclusion, (1) urine proteome in transplant recipients with stable graft function was significantly different from healthy control subjects, and (2) acute rejections were characterized by a constellation of excreted proteins. Analysis of the urinary proteome may expedite the noninvasive prediction of acute graft rejection, thus importantly assisting in establishing the diagnosis.

Acute Disease↗
PubMed2006

Molecular phenotype of zebrafish ovarian follicle by serial analysis of gene expression and proteomic profiling, and comparison with the transcriptomes of other animals.

BACKGROUND: The ability of an oocyte to develop into a viable embryo depends on the accumulation of specific maternal information and molecules, such as RNAs and proteins. A serial analysis of gene expression (SAGE) was carried out in parallel with proteomic analysis on fully-grown ovarian follicles from zebrafish (Danio rerio). The data obtained were compared with ovary/follicle/egg molecular phenotypes of other animals, published or available in public sequence databases. RESULTS: Sequencing of 27,486 SAGE tags identified 11,399 different ones, including 3,329 tags with an occurrence superior to one. Fifty-eight genes were expressed at over 0.15% of the total population and represented 17.34% of the mRNA population identified. The three most expressed transcripts were a rhamnose-binding lectin, beta-actin 2, and a transcribed locus similar to the H2B histone family. Comparison with the large-scale expressed sequence tags sequencing approach revealed highly expressed transcripts that were not previously known to be expressed at high levels in fish ovaries, like the short-sized polarized metallothionein 2 transcript. A higher sensitivity for the detection of transcripts with a characterized maternal genetic contribution was also demonstrated compared to large-scale sequencing of cDNA libraries. Ferritin heavy polypeptide 1, heat shock protein 90-beta, lactate dehydrogenase B4, beta-actin isoforms, tubulin beta 2, ATP synthase subunit 9, together with 40 S ribosomal protein S27a, were common highly-expressed transcripts of vertebrate ovary/unfertilized egg. Comparison of transcriptome and proteome data revealed that transcript levels provide little predictive value with respect to the extent of protein abundance. All the proteins identified by proteomic analysis of fully-grown zebrafish follicles had at least one transcript counterpart, with two exceptions: eosinophil chemotactic cytokine and nothepsin. CONCLUSION: This study provides a complete sequence data set of maternal mRNA stored in zebrafish germ cells at the end of oogenesis. This catalogue contains highly-expressed transcripts that are part of a vertebrate ovarian expressed gene signature. Comparison of transcriptome and proteome data identified downregulated transcripts or proteins potentially incorporated in the oocyte by endocytosis. The molecular phenotype described provides groundwork for future experimental approaches aimed at identifying functionally important stored maternal transcripts and proteins involved in oogenesis and early stages of embryo development.

Animals↗
PubMed2002

Comparative genomics of Arabidopsis and maize: prospects and limitations.

The completed Arabidopsis genome seems to be of limited value as a model for maize genomics. In addition to the expansion of repetitive sequences in maize and the lack of genomic micro-colinearity, maize-specific or highly-diverged proteins contribute to a predicted maize proteome of about 50,000 proteins, twice the size of that of Arabidopsis.

Arabidopsis↗
PubMed2004

Troponin T as a marker of differentiation revealed by proteomic analysis in renal arterioles.

Renovascular hypertension is characterized by stenosis of the renal artery and high plasma renin levels. The renal phenotype is characterized by high levels of renin in the hypoperfused kidney due to the recruitment of renin-producing cells along the afferent arterioles. This increase in myoepithelioïd cells is due mainly to the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype. To understand the molecular mechanisms involved in this recruitment, we used the established rat model of renovascular hypertension known as the two-kidney, one-clip model in the Lewis rat. Renal arterioles were isolated using magnetized iron suspension. Differential proteomic analysis was performed using 2-D polyacrylamide gel electrophoresis followed by mass spectrometry. Comparative analysis of soluble proteins extracted from afferent arterioles of clipped and contralateral kidneys showed 14 proteins significantly differentially expressed by at least a factor of 2. These proteins were identified by mass spectrometry. The most striking protein revealed by proteomics is troponin T, which is down-regulated in the afferent arterioles of the clipped kidney. Confocal microscopy showed that troponin T is specific of the smooth muscle phenotype and absent in the myoepithelioïd phenotype. Our data suggest that troponin T is only present in renal smooth muscle cells.

Animals↗
PubMed2004

Protein patterns of pig oocytes during in vitro maturation.

In vitro maturation (IVM) of fully grown mammalian oocytes is characterized by initial germinal vesicle (GV) breakdown and rearrangement of microtubule network during the first meiosis (MI), followed by extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (MII). Only fully matured oocytes are capable of undergoing fertilization and the initiation of zygotic development. These observations are mostly based on morphological evaluation; however, the molecular events responsible for these processes are not known. In this study, we have launched the analysis of pig oocytes during in vitro maturation using a proteomics approach. First, oocyte proteins have been separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Remarkably, several proteins, including peroxiredoxins, ubiquitin carboxyl-terminal hydrolase isozyme L1, and spermine synthase, are even more abundant than actin, usually the most abundant protein in somatic cells. Furthermore, we have initiated comparative analysis of the oocytes at different stages of maturation to characterize candidate proteins, which are differentially expressed during in vitro maturation. To date, we have identified antiquitin (D7A1), the member of aldehyde dehydrogenase family7 that has been significantly increased in MI and MII stages compared with GV oocytes. To our knowledge, this is the first pig oocyte proteome available so far that may be used as a reference map. The proteins that are differentially regulated during IVM may present potential biomarkers of oocyte maturation and quality. It is a useful inventory toward a deeper understanding of the mechanisms underlying reproduction and development.

Animals↗
PubMed2004

Proteome-wide identification of cellular targets affected by bisindolylmaleimide-type protein kinase C inhibitors.

Bisindolylmaleimide compounds such as GF109203X are potent inhibitors of protein kinase C (PKC) activity. Although bisindolylmaleimides are not entirely selective for PKC and are known to inhibit a few other protein kinases, these reagents have been extensively used to study the functional roles of PKC family enzymes in cellular signal transduction for more than a decade. Here, we establish a proteomics approach to gain further insights into the cellular effects of this compound class. Functional immobilization of suitable bisindolylmaleimide analogues in combination with the specific purification of cellular binding proteins by affinity chromatography led to the identification of several known and previously unknown enzyme targets. Subsequent in vitro binding and activity assays confirmed the protein kinases Ste20-related kinase and cyclin-dependent kinase 2 (CDK2) and the non-protein kinases adenosine kinase and quinone reductase type 2 as novel targets of bisindolylmaleimide inhibitors. As observed specifically for CDK2, minor chemical variation of the ligand by immobilizing the closely related bisindolylmaleimides III, VIII, and X dramatically affected target binding. These observed changes in affinity correlated with both the measured IC(50) values for in vitro CDK2 inhibition and results from molecular docking into the CDK2 crystal structure. Moreover, the conditions for affinity purification could be adapted in a way that immobilized bisindolylmaleimide III selectively interacted with either PKC alpha or ribosomal S6 protein kinase 1 only after activation of these kinases. Thus, we have established an efficient technique for the rapid identification of cellular bisindolylmaleimide targets and further demonstrate the comparative selectivity profiling of closely related kinase inhibitors within a cellular proteome.

Animals↗
PubMed2025

Multi-Omics Analysis of the Potential Mechanisms of Skin Albinism in Edangered Percocypris pingi: Abnormal Ubiquitination and Calcium Signal Inhibition.

Percocypris pingi is an endangered protected fish species in China. Its albino variants exhibit growth retardation and physiological abnormalities. Understanding its albinism mechanism holds significant scientific importance for molecular breeding programs and disease model development. This study integrated transcriptomic and proteomic analyses, combined with histopathological and molecular biological techniques, to systematically compare molecular differences in skin tissues between albino and wild-type P. pingi, with a focus on elucidating the multidimensional regulatory mechanisms underlying skin albinism. Our findings suggest that albinism in P. pingi is synergistically driven by hyperactivation of ubiquitin-mediated proteolysis (which suppressed TYR/TYRP1 enzymatic activity and disrupted the pH homeostasis of melanosomes), and inhibition of calcium signaling (which impeded melanin transport). This discovery provides novel insights into the mechanisms of pigment loss in fish species and offers a valuable reference for molecular breeding of endangered species as well as research on pigmentation-related disorders.

Animals↗
PubMed2006

Plasma proteome analysis reveals the geographical origin and liver tumor status of Dab (Limanda limanda) from UK marine waters.

The flatfish species dab (Limanda limanda) is the sentinel for offshore marine monitoring in the United Kingdom National Marine Monitoring Programme (NMMP). At certain sites in the North and Irish Seas, the prevalence of macroscopic liver tumors can exceed 10%. The plasma proteome of these fish potentially contains reporter proteins or "biomarkers" that may enable development of diagnostic tests for liver cancer and further our understanding of the disease. Following selection of sample groups by quality-assured histopathology ("phenotype anchoring"), we used surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry to produce proteomic profiles of plasma from 213 dab collected during the 2004 UK NMMP. The resulting protein profiles were compared between fish from the North and Irish Seas and between fish with liver neoplasia or nondiseased liver. Significant differences were found between the plasma proteomes of dab from the North Sea and Irish Sea, which in conjunction with artificial neural networks can correctly determine from which sea dab were captured in 85% of the cases. In addition, the presence of liver tumors is associated with significant changes in the plasma proteome. We conclude that SELDI-based plasma profiling is potentially of use in nonlethal marine monitoring using wild sentinels such as dab. Furthermore, accurate selection of sample groups is critical for avoiding effects of confounding factors such as age, gender, and geographic origin of samples.

Adenoma, Liver Cell↗
PubMed2004

AT1 receptor blockade alters metabolic, functional and structural proteins after reperfused myocardial infarction: detection using proteomics.

Angiotensin II (AngII) type 1 receptor (AT1R) blockers (ARBs) limit left ventricular (LV) dysfunction and necrosis after reperfused myocardial infarction (RMI) and proteomics can detect changes in protein levels after injury. We applied proteomics to detect changes in levels of specific protein in the ischemic zone (IZ) and non-ischemic zone (NIZ) of dog hearts after in vivo RMI (90 min of anterior ischemia; 120 min of reperfusion) and treatment with intravenous vehicle (control) and the ARBs valsartan or irbesartan (10 mg/kg) over 30 min before RMI. We also assessed LV function, infarction and apoptosis. Both ARBs limited the RMI-induced LV dysfunction, infarct size and apoptosis. Proteomics detected differential expression of 5 randomly selected proteins in the IZ compared to the NIZ after RMI: decrease in a subunit of ATP synthase isoform precursor (consistent with increased conversion to a subunit under metabolic stress), M chain creatine kinase (consistent with cellular damage) and ventricular myosin light chain-1 (consistent with structural damage and decreased contractility); and increase in NAD+ -isocitrate dehydrogenase (ICDH) and alpha subunit and ATP synthase D chain (mitochondrial, consistent with metabolic dysfunction). Importantly, changes in NAD+ -ICDH and ATP synthase D chain were reversed by ARB therapy. Thus, proteomics can detect regional changes in metabolic, contractile, and structural proteins after RMI and several of these proteins are favorably modified by ARBs, suggesting that they may be novel therapeutic targets.

ATP Synthetase Complexes↗
PubMed2001

Proteome map of the chloroplast lumen of Arabidopsis thaliana.

The thylakoid membrane of the chloroplast is the center of oxygenic photosynthesis. To better understand the function of the luminal compartment within the thylakoid network, we have carried out a systematic characterization of the luminal thylakoid proteins from the model organism Arabidopsis thaliana. Our data show that the thylakoid lumen has its own specific proteome, of which 36 proteins were identified. Besides a large group of peptidyl-prolyl cis-trans isomerases and proteases, a family of novel PsbP domain proteins was found. An analysis of the luminal signal peptides showed that 19 of 36 luminal precursors were marked by a twin-arginine motif for import via the Tat pathway. To compare the model organism Arabidopsis with another typical higher plant, we investigated the proteome from the thylakoid lumen of spinach and found that the luminal proteins from both plants corresponded well. As a complement to our experimental investigation, we made a theoretical prediction of the luminal proteins from the whole Arabidopsis genome and estimated that the thylakoid lumen of the chloroplast contains approximately 80 proteins.

Amino Acid Motifs↗
PubMed2004

Proteomics: a novel tool to unravel the patho-physiology of uraemia.

BACKGROUND: Uraemic toxicity results in the dysfunction of many organ systems, provoking an increase in morbidity and mortality. To date, only approximately 90 uraemic retention solutes have been described. To examine unknown uraemic substances thoroughly, the identification of as many compounds as possible in the ultrafiltrate and/or plasma of patients would lead to a less biased definition of the uraemic retention process compared with what is proposed today. METHODS: We describe the application of a novel proteomic tool for the identification of a large number of molecules present in ultrafiltrate from uraemic and normal plasma obtained with high- or low-flux membranes. Separation by capillary electrophoresis was coupled on-line to a mass spectrometer, yielding identification of polypeptides based on their molecular weight. RESULTS: Between 500 and >1000 polypeptides with a molecular weight ranging from 800 to 10,000 Da could be detected in individual samples, and were identified via their mass and their particular migration time in capillary electrophoresis. In ultrafiltrate from uraemic plasma, 1394 polypeptides were detected in the high-flux vs 1046 in the low-flux samples, while in ultrafiltrate from normal plasma, 544 polypeptides vs 490 were found in ultrafiltrate from normal plasma obtained from membranes with comparable cut-off. In addition, polypeptides >5 kDa were virtually only detected in the uraemic ultrafiltrate from the high-flux membrane (n = 28 vs n = 5 with the low-flux membrane). To demonstrate the feasibility of further characterizing the detected molecules, polypeptides present exclusively in uraemic ultrafiltrate were chosen for sequencing analyses. A 950.6 Da polypeptide was identified as a fragment of the salivary proline-rich protein. A 1291.8 Da fragment was derived from alpha-fibrinogen. CONCLUSION: The data presented here strongly suggest that the application of proteomic approaches such as capillary electrophoresis and mass spectrometry will result in the identification of many more uraemic solutes than those known at present. This could enable the introduction of more direct elimination strategies, since it is possible to obtain an extended appreciation of the removal capacities of particular dialyser membranes.

Female↗
PubMed2006

Proteomic analysis of experimentally induced azole resistance in Candida glabrata.

OBJECTIVES: The aim of the present study was to identify changes in the proteome of a laboratory-derived azole-resistant strain of Candida glabrata compared with its susceptible parent strain in an effort to identify proteins that are differentially expressed in association with azole resistance. METHODS: Soluble and membrane protein fractions were isolated from mutant strain F15 (fluconazole MIC>128 mg/L) and parent strain 66032 (fluconazole MIC=16 mg/L) grown to mid-log phase. Soluble proteins were resolved by both two-dimensional (2D) and one-dimensional (1D) polyacrylamide gel electrophoresis (GE) whereas membrane proteins were resolved by 1D GE. Spots or bands representing differentially expressed proteins were identified by matrix-assisted desorption ionization-time of flight mass spectroscopy (MALDI-TOF MS) and peptide mass fingerprinting. RESULTS: A total of 22 proteins were found to be more abundantly represented, and 3 proteins were found to be less abundantly represented, in strain F15 compared with strain 66032. These included up-regulation of the ATP-binding cassette transporter Cdr1p, the ergosterol biosynthesis enzyme Erg11p, proteins involved in glycolysis and glycerol metabolism, and proteins involved in the response to oxidative stress and cadmium exposure. CONCLUSIONS: In addition to transcriptional regulation of Cdr1p, this study identified the differential expression of several proteins that may contribute to azole resistance and suggests the possibility for a post-transcriptional mechanism for increased expression of Erg11p.

Antifungal Agents↗
PubMed2001

Identification of putative exported/secreted proteins in prokaryotic proteomes.

The increasing number of bacterial genomes being sequenced fuels an equal demand for methods to rapidly analyze the proteomes of these organisms. One group of proteins of pressing importance is the exported/secreted proteins, given their dominant immunogenicity and role in pathogenesis. With this in mind, a weight matrix algorithm and two artificial neural networks, one based on amino acid position within the N-terminus and the other on amino acid frequency, were developed for identification of such proteins. The neural networks and a hybrid method, combining the weight matrix algorithm and the amino acid frequency neural network, were tested independently against a standard data set of secreted and cytoplasmic proteins to determine their accuracy in predicting secreted prokaryotic proteins. The results of these analyses demonstrated that the amino acid position neural network provided the highest accuracy (Mathews correlation coefficient of 0.93) in predicting secreted proteins of Gram-negative bacteria, whereas the hybrid method was best (Mathews correlation coefficient of 0.97) for prediction of Gram-positive secreted proteins. These two methods were integrated into a single program (ExProt) designed to analyze whole proteomes. In addition to protein localization, ExProt also contains a neural network trained to identify the most probable signal peptidase I cleavage site of secreted proteins. When tested against the standard protein data set ExProt correctly predicted 73.5 and 84.5% of the cleavage sites in Gram-positive and Gram-negative secreted proteins, respectively. Comparative analysis of Gram-negative, Gram-positive, Mycobacterium tuberculosis, and Archaea proteomes with ExProt revealed that the fraction of putative exported/secreted proteins encoded by bacterial genomes ranged from 8% for Methanococcus jannaschii to 37% for Mycoplasma pneumoniae.

Algorithms↗
PubMed2006

Biomarker candidate identification in Yersinia pestis using organism-wide semiquantitative proteomics.

The accurate mass and time tag mass spectrometry method and clustering analysis were used to compare the abundance change of 992 Yersinia pestis proteins under four contrasting growth conditions (26 and 37 degrees C, with or without Ca2+) that mimicked growth states in either a flea vector or mammalian host. Eighty-nine proteins were observed to have similar abundance change profiles to 29 known virulence associated proteins, providing identification of additional biomarker candidates. Eighty-seven hypothetical proteins, which clustered into 5 distinct clusters of like-protein abundance change, were identified as unique biomarkers related specifically to growth condition.

Bacterial Proteins↗