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PubMed2004

In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database.

An extensive analysis of the Arabidopsis thaliana peripheral and integral thylakoid membrane proteome was performed by sequential extractions with salt, detergent, and organic solvents, followed by multidimensional protein separation steps (reverse-phase HPLC and one- and two-dimensional electrophoresis gels), different enzymatic and nonenzymatic protein cleavage techniques, mass spectrometry, and bioinformatics. Altogether, 154 proteins were identified, of which 76 (49%) were alpha-helical integral membrane proteins. Twenty-seven new proteins without known function but with predicted chloroplast transit peptides were identified, of which 17 (63%) are integral membrane proteins. These new proteins, likely important in thylakoid biogenesis, include two rubredoxins, a potential metallochaperone, and a new DnaJ-like protein. The data were integrated with our analysis of the lumenal-enriched proteome. We identified 83 out of 100 known proteins of the thylakoid localized photosynthetic apparatus, including several new paralogues and some 20 proteins involved in protein insertion, assembly, folding, or proteolysis. An additional 16 proteins are involved in translation, demonstrating that the thylakoid membrane surface is an important site for protein synthesis. The high coverage of the photosynthetic apparatus and the identification of known hydrophobic proteins with low expression levels, such as cpSecE, Ohp1, and Ohp2, indicate an excellent dynamic resolution of the analysis. The sequential extraction process proved very helpful to validate transmembrane prediction. Our data also were cross-correlated to chloroplast subproteome analyses by other laboratories. All data are deposited in a new curated plastid proteome database (PPDB) with multiple search functions (http://cbsusrv01.tc.cornell.edu/users/ppdb/). This PPDB will serve as an expandable resource for the plant community.

Amino Acid Sequence↗

PubMed2006

Proteomic profiling identifies cyclooxygenase-2-independent global proteomic changes by celecoxib in colorectal cancer cells.

Celecoxib, a selective inhibitor of the enzyme cyclooxygenase-2 (COX-2), has been shown to be a promising chemoprevention agent. The chemopreventive efficacy of celecoxib is believed to be a consequence of its COX-2-dependent and COX-2-independent effects on a variety of cellular processes including proliferation, apoptosis, angiogenesis, and immunosurveillance. In an attempt to identify proteomic markers modulated by celecoxib that are independent of its inhibitory effect on COX-2, the colorectal cancer cell line HCT-116, a nonexpresser of COX-2, was treated with celecoxib. We used the powerful, state-of-the-art two-dimensional difference gel electrophoresis technology coupled with mass spectrometric sequencing to compare global proteomic profiles of HCT-116 cells before and after treatment with celecoxib. Among the differentially expressed proteins identified following celecoxib treatment were proteins involved in diverse cellular functions including glycolysis, protein biosynthesis, DNA synthesis, mRNA processing, protein folding, phosphorylation, redox regulation, and molecular chaperon activities. Our study presents a comprehensive analysis of large-scale celecoxib-modulated proteomic alterations, at least some of which may be mechanistically related to the COX-2-independent chemopreventive effect of celecoxib.

Blotting, Western↗

PubMed2001

[Identification of proteome molecules by proteomics using two-dimensional gel electrophoresis and MALDI-TOF MS].

Genomic technologies have enabled rapid accumulation of information from complex biological systems over the last two decades. The complete DNA sequence is now known for many organisms and the informational database obtained from genome sequencing projects has provided the base for the specification of proteome - the protein complement of genome. Genomic functions can be inferred from the analysis of gene structure and gene expression profiles because proteins are the functional molecules of an organism. Integrated technologies including protein separation, identification, characterization and information manage system are essential to analyze the proteins in complex cellular matrix. This study is focusing on the strategies of proteome analysis using sample preparation, 2-dimensional gel electrophoresis, processing of protein spots and identification of proteins, protein-protein interaction and posttranslational modification using MALDI-TOF-MS. 2-D gel electrophoresis is currently the most powerful protein separation technique and MALDI-TOF MS is powerful identification technique for protein and peptides as a sensitive, rapid, and high resolution analytical method. The developed integrated proteome technologies are very useful to understand the biological phenomena at molecular level by identifying the new molecules and their modifications in various cellular processes, and can be applied for biotechnology including medical science.

Databases, Protein↗

PubMed2025

Hepatocyte proteome destabilization and novel targets for PFASs unveiled through combined thermal proteome profiling and deep transfer learning.

Identifying protein targets for per- and polyfluoroalkyl substances (PFASs) is essential to understand their toxicity and health risks. However, knowledge about their interacting proteins is limited since reliable identification methods are lacking. We developed an integrated approach combining thermal proteome profiling (TPP) and deep transfer learning (DTL) modeling to efficiently identify cellular targets of PFAS. TPP measured PFAS binding proteins and the affinities by nanospray liquid chromatography tandem mass spectrometry, while DTL models were constructed to predict PFAS-protein affinities using neural network algorithms. TPP results revealed that PFASs uniquely destabilized the proteome of HepG2 cells, unlike the stabilizing effects by other xenobiotics. Key protein targets for three representative PFASs (PFOA, GenX and Novec 649) were identified, which exhibited weak binding affinities (median EC50 ≈ 30 μM). The number of protein targets increased with molecular weights among the three PFASs. The DTL model achieved a higher Pearson correlation coefficient of 0.89, and reduced mean squared errors by 54 % over previous models for drug-protein interactions. Notably, TPP and DTL jointly pinpointed ribosomal proteins as novel targets of GenX, potentially linking it to cell apoptosis through disrupted protein synthesis. Biolayer interferometry validated GenX binding to RPL4 protein, driven by electrostatic interactions and halogen bonds. This integrated approach effectively uncovers novel PFASs targets, advancing insights into their adverse health effects.

Humans↗

PubMed2001

New Frontiers in the Proteome Research Quantitative Proteomics.

In the past years, proteome research has achieved very exciting advances. The automation of 2DE-MS approach and the use of multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) have overcome many disadvantages of two-dimensional gel electrophoresis. The stable isotope labeling and the isotope-coded affinity tags (ICAT) strategy provided an ideal method for accurately quantitating the low copy number proteins which play a key role in cells or tissues. At the same time, the research of the quantitative proteomics has been improved greatly with the development of the protein chip technology. Recent advances of the research in the quantitative proteomics and the related technologies are reviewed.

Journal Article↗

PubMed2001

From proteomics to genomics.

Presently, science is moving from genomics to proteomics in order to get insight into the functional network of gene expression. Actually however, proteomics is much older than genomics and dates back to the introduction of the two-dimensional gel electrophoresis technique (2-DE) independently by Klose and O'Farrell. Based on this approach almost all cellular proteins can be separated. New developments in mass spectrometry allowed identification of single spots in the 2-DE protein pattern, including the underlying genes. Joachim Klose has focused his pioneering 2-DE studies on mouse models with special emphasis on quantitative protein variants. According to him, proteins are living molecules exhibiting a characteristic protein phenotype.

Animals↗

PubMed2004

Two-dimensional electrophoresis database of Listeria monocytogenes EGDe proteome and proteomic analysis of mid-log and stationary growth phase cells.

Listeria monocytogenes is the causative agent of listeriosis, one of the most significant foodborne diseases in industrialized countries. The complete genome of the L. monocytogenes EGDe strain, belonging to the serogroup 1/2a, has been sequenced and is comprised of 2853 open reading frames. The objective of the current study was to construct a two-dimensional (2-D) database of the proteome of this strain. The soluble protein fractions of the microorganism were recovered either in the mid-log or in the stationary phase of growth at 37 degrees C. These fractions were analyzed by 2-D electrophoresis (2-DE), using immobilized pH gradient strips of various pH values (3-10, 3-6, and 5-8) for the first-dimensional separations and 12.5% acrylamide gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 201 protein spots corresponding to 126 different proteins were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The 2-DE maps presented here provide a first basis for further investigations of protein expression in L. monocytogenes. In this way, the comparison of proteome between cells in the exponential or stationary phase of growth at 37 degrees C allowed us to characterize 161 variations in protein spot intensity, of which 38 were identified. Among the differentially expressed proteins were ribosomal proteins (RpsF, RplJ, and RpmE), proteins involved in cellular metabolism (GlpD, PdhD, Pgm, Lmo1372, Lmo2696, and Lmo2743) or in stress adaptation (GroES and ferritin), a fructose-specific phosphotransferase enzyme IIB (Lmo0399) and different post-translational modified forms of listeriolysin (LLO).

Bacterial Proteins↗

PubMed2005

Great mood in proteomics: Beijing and the HUPO Human Brain Proteome Project.

More than 1200 attendees came together at the 3(rd) HUPO World Congess in Beijing, October 25-27, 2004. In numerous different sessions the wide range of proteomic areas became visible. The HUPO Brain Proteome Project (HUPO BPP) organized an evening session on October 23, presenting the first results of two pilot studies as well as the newest, very positive international development in this field. The rising importance became even more apparent in the plenary presentation of all HUPO initiatives and the following congress activities.

Animals↗

PubMed2006

Top-down proteomic analysis of the soluble sub-proteome of the obligate thermophile, Geobacillus thermoleovorans T80: insights into its cellular processes.

We report the first analysis of the soluble sub-proteome of the obligate thermophile, Geobacillus thermoleovorans T80, utilizing a robust multidimensional protein identification protocol. A total of 1,336 proteins were initially identified utilizing automated MS/MS identification software. Intensive manual curation resulted in a final list containing a total of 294 unique proteins. Physiochemical characterization and functional classification of the soluble sub-proteome was carried out. The strategy has allowed us to gain an insight into the cellular processes of this obligate thermophile, identifying a variety of proteins known to play a role in stress response. Included within these were a number of sigma factors such as sigma(A) that initiate transcription of the heat shock operons controlled by the HrcA-CIRCE complex within gram positive bacteria. In addition, it has enabled us to assign a degree of functionality to 29 out of 36 gene products detected in this study that were hitherto described as being only hypothetical conserved proteins.

Bacillaceae↗

PubMed2006

Proteomics of acute myeloid leukaemia: Cytogenetic risk groups differ specifically in their proteome, interactome and post-translational protein modifications.

Acute myeloid leukaemia (AML) is characterized by specific cytogenetic aberrations that are strong determinants of prognostic outcome and therapeutic response. Because the pathological outcome of AML patients with cytogenetic abnormalities differs considerably, we hypothesized that their proteome may also differ specifically in their expression pattern, protein interaction pathways and post-translational modifications (PTM). We performed this study using 42 AML patients diagnosed for various cytogenetic abnormalities based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MS) and MSMS tandem MS. We could identify significant differences in the proteome and PTM of peptides, later confirmed by other methods, between cytogenetic groups. The interactome analysis based on computational bioinformatics reveals major regulating networks: MAPK8 and MYC for complex aberrant karyotype, TP53 for t(8;21), TP53-MYC-PRKAC for 11q23 and JUN and MYC for Inv(16). Further, we analysed 42 MS spectra representative of hnRNPH1, calreticulin and hnRNPA2/B1 in a peak explorer, which reveals a cytogenetic-specific PTM of beta-O-linked N-acetyl glucosamine (O-GlcNAc) of hnRNPH1 in AML patients with 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation and methylation of hnRNPA2/B1 in patients with translocations of t(8;21) and inv(16). This report may lead to a new thinking about AML pathogenesis, as differences at PTM level could be used to distinguish different subtypes of AML.

Cell Shape↗

PubMed2006

The work of the Human Proteome Organisation's Proteomics Standards Initiative (HUPO PSI).

This article describes the origins, working practices and various development projects of the HUman Proteome Organisation's Proteomics Standards Initiative (HUPO PSI), specifically, our work on reporting requirements, data exchange formats and controlled vocabulary terms. We also offer our view of the two functional genomics projects in which the PSI plays a role (FuGE and FuGO), discussing their impact on our process and laying out the benefits we see as accruing, both to the PSI and to biomedical science as a whole as a result of their widespread acceptance.

Humans↗

PubMed2004

Proteome Analyst: custom predictions with explanations in a web-based tool for high-throughput proteome annotations.

Proteome Analyst (PA) (http://www.cs.ualberta.ca/~bioinfo/PA/) is a publicly available, high-throughput, web-based system for predicting various properties of each protein in an entire proteome. Using machine-learned classifiers, PA can predict, for example, the GeneQuiz general function and Gene Ontology (GO) molecular function of a protein. In addition, PA is currently the most accurate and most comprehensive system for predicting subcellular localization, the location within a cell where a protein performs its main function. Two other capabilities of PA are notable. First, PA can create a custom classifier to predict a new property, without requiring any programming, based on labeled training data (i.e. a set of examples, each with the correct classification label) provided by a user. PA has been used to create custom classifiers for potassium-ion channel proteins and other general function ontologies. Second, PA provides a sophisticated explanation feature that shows why one prediction is chosen over another. The PA system produces a Naïve Bayes classifier, which is amenable to a graphical and interactive approach to explanations for its predictions; transparent predictions increase the user's confidence in, and understanding of, PA.

Internet↗

PubMed2005

A systematic proteomic study of seed filling in soybean. Establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database.

A high-throughput proteomic approach was employed to determine the expression profile and identity of hundreds of proteins during seed filling in soybean (Glycine max) cv Maverick. Soybean seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This led to the establishment of high-resolution proteome reference maps, expression profiles of 679 spots, and corresponding matrix-assisted laser desorption ionization time-of-flight mass spectrometry spectra for each spot. Database searching with these spectra resulted in the identification of 422 proteins representing 216 nonredundant proteins. These proteins were classified into 14 major functional categories. Proteins involved in metabolism, protein destination and storage, metabolite transport, and disease/defense were the most abundant. For each functional category, a composite expression profile is presented to gain insight into legume seed physiology and the general regulation of proteins associated with each functional class. Using this approach, an overall decrease in metabolism-related proteins versus an increase in proteins associated with destination and storage was observed during seed filling. The accumulation of unknown proteins, sucrose transport and cleavage enzymes, cysteine and methionine biosynthesis enzymes, 14-3-3-like proteins, lipoxygenases, storage proteins, and allergenic proteins during seed filling is also discussed. A user-intuitive database (http://oilseedproteomics.missouri.edu) was developed to access these data for soybean and other oilseeds currently being investigated.

Databases, Protein↗

PubMed2006

[Advances in plant proteomics. II. Application of proteome techniques to plant biology research].

Proteome techniques have widely been applied to the fields of plant genetics, plant development, and plant physiology and ecology to investigate plant genetic diversity, plant development such as seed maturation and germination processes, differentiation of plant tissue and organ, separation and functional identification of novel component of various organells, mechanisms of plant adapted to abiotic or biotic stresses including high temperature, low temperature, high salt, drought, and pathogens and insects, and interaction of plant with microbe. The prospects of plant proteomics are discussed.

Biology↗

PubMed2003

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session.

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.

Mass Spectrometry↗

PubMed2004

A detergent- and cyanogen bromide-free method for integral membrane proteomics: application to Halobacterium purple membranes and the human epidermal membrane proteome.

A simple and rapid method for characterizing hydrophobic integral membrane proteins and its utility for membrane proteomics using microcapillary liquid chromatography coupled on-line with tandem mass spectrometry (microLC-MS/MS) is described. The present technique does not rely on the use of detergents, strong organic acids or cyanogen bromide-mediated proteolysis. A buffered solution of 60% methanol was used to extract, solubilize, and tryptically digest proteins within a preparation of Halobacterium (H.) halobium purple membranes. Analysis of the digested purple membrane proteins by microLC-MS/MS resulted in the identification of all the predicted tryptic peptides of bacteriorhodopsin, including those that are known to be post-translationally modified. In addition, 40 proteins from the purple membrane preparation were also identified, of which 80% are predicted to contain between 1 and 16 transmembrane domains. To evaluate the general applicability of the method, the same extraction, solubilization, and digestion conditions were applied to a plasma membrane fraction prepared from human epidermal sheets. A total of 117 proteins was identified in a single microLC-MS/MS analysis, of which 55% are known to be integral or associated with the plasma membrane. Due to its simplicity, efficiency, and absence of MS interfering compounds, this technique can be used for the characterization of other integral membrane proteins and may be concomitantly applied for the analysis of membrane protein complexes or large-scale proteomic studies of different membrane samples.

Bacteriorhodopsins↗

PubMed2004

The Human Proteome Organization Plasma Proteome Project pilot phase: reference specimens, technology platform comparisons, and standardized data submissions and analyses.

A comprehensive, systematic characterization of cirolating proteins in health and disease will greatly facilitate development of biomarkers for prevention, diagnosis, and therapy of cancers and other diseases. The Human Proteome Organization Plasma Proteome Project pilot phase aims to (1) compare the advantages and limitations of many technology platforms; (2) contrast reference specimens of human plasma (ethylenediaminetetra acetic acid, heparin, citrate-anticoagulated) and serum, in terms of numbers of proteins identified and any interferences with various technology platforms; and (3) create a global knowledge base/data repository.

Biomarkers↗

PubMed2004

Towards a standardized human proteome database: quantitative proteome profiling of living cells.

Comparative proteome profiling, performed by two-dimensional polyacrylamide gel electrophoresis or multidimensional protein identification technology, usually relies on the relative comparison of samples of interest with respect to a reference. Currently, no standardized quantitative protein expression database of human cells, facilitating data comparisons between different laboratories, exists. Recently, we have published two-dimensional polyacrylamide gel electrophoresis-based techniques to assess absolute protein data comprising protein amounts, synthesis rates and biological half-lives (Mol. Cell. Proteomics 2002, 1, 528-537). Determination of protein amounts by fluorography of two-dimensional gels was followed by the exact quantification of the amount of incorporated (35)S radiolabel. Here we demonstrate an application of this highly standardized method to quiescent human T cells, phythaemagglutinin-stimulated T cells and Jurkat cells, a human T lymphoblast cell line. While the protein composition of quiescent T cells differed significantly compared to that of Jurkat cells, it was only slightly different compared to the activated T cells. Synthesis profile analyses demonstrated that activated T cells clearly differed from the quiescent cells, performing apparently almost like lymphoblast cells. The great sensitivity of this approach was further demonstrated with human umbilical vein endothelial cells treated for six hours with vascular endothelial growth factor. While no significant alteration of protein amounts was detected at all upon activation, the synthesis rate of several proteins was found to be more than doubled.

Autoradiography↗