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PubMed2004

Functional proteomics mapping of a human signaling pathway.

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Proteome mapping of mature pollen of Arabidopsis thaliana.

The male gametophyte of Arabidopsis is a three-celled pollen grain that is thought to contain almost all the mRNAs needed for germination and rapid pollen tube growth. We generated a reference map of the Arabidopsis mature pollen proteome by using multiple protein extraction techniques followed by 2-DE and ESI-MS/MS. We identified 135 distinct proteins from a total of 179 protein spots. We found that half of the identified proteins are involved in metabolism (20%), energy generation (17%), or cell structure (12%); these percentages are similar to those determined for the pollen transcriptome and this similarity is consistent with the idea that in addition to the mRNAs, the mature pollen grain contains proteins necessary for germination and rapid pollen tube growth. We identified ten proteins of unknown function, three of which are flower- or pollen-specific, and we identified nine proteins whose RNAs were absent from the transcriptome, seven of which are involved in metabolism, energy generation, or cell wall structure. Our work complements and extends recent analyses of the pollen transcriptome.

Amino Acid Sequence↗
PubMed2006

A proteome reference map and proteomic analysis of Bifidobacterium longum NCC2705.

A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF-MS and/or ESI-MS/MS. Isoelectric point values estimated by gel electrophoresis matched closely with their predicted ones, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose as determined by semiquantitative RT-PCR. BL0033 time course and concentration experiments showed that the induction time and fructose concentration correlates to increased expression of BL0033. At the same time, an ABC (ATP-binding cassette) transporter ATP-binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared with glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system in which BL0033 might play an important role.

ATP-Binding Cassette Transporters↗
PubMed2003

Using standard positions and image fusion to create proteome maps from collections of two-dimensional gel electrophoresis images.

Databases for two-dimensional protein gels pose new challenges in extracting meaningful information from large numbers of experiments. In order to create expression profiles, positions of corresponding protein spots across all gel images have to be established. In larger gel sets errors may accumulate rapidly during this spot matching process, effectively limiting the number of samples available for data mining. Here we present a novel approach for organizing spot data based on the concept of a standard position for a protein species. Standard positions are meaningful average positions that are determined using all occurrences of a protein species. They can be extended to spots that are not annotated via interpolation. The standard position of a spot can serve as a unifying index across all gels in a database, thus allowing creation and analysis of expression profiles that span the whole collection. The standard position gives a much more accurate estimation of a spot's position on a gel than can be obtained using theoretical isoelectric point and molecular weight. Positional indexing is a complement to a priori identifications (e.g. by mass spectrometry or Edman degradation). Moreover it can be used in advance to select spots that are worth identifying because they show relevant expression profiles. Furthermore, we show how to combine all spots that occur on any of the gels into one synthetic but nevertheless realistic-looking image. This composite image is produced such that all spots have their standard positions. It can serve as a proteome reference map for an organism. As an application, we have computed a reference map from 23 gel images of Bacillus subtilis, using an enhanced prerelease version of the gel analysis software Delta2D (DECODON, Greifswald, Germany).

Bacterial Proteins↗
PubMed2006

Array-based proteomics: mapping of protein circuitries for diagnostics, prognostics, and therapy guidance in cancer.

The human proteome, due to the enormity of post-translational permutations that result in large numbers of isoforms, is much more complex than the genome and alterations in cancer can occur in ways that are not predictable by translational analysis alone. Proteomic analysis therefore represents a more direct way of investigating disease at the individual patient level. Furthermore, since most novel therapeutic targets are proteins, proteomic analysis potentially has a central role in patient care. At the same time, it is becoming clear that mapping entire networks rather than individual markers may be necessary for robust diagnostics as well as tailoring of therapy. Consequently, there is a need for high-throughput multiplexed proteomic techniques, with the capability of scanning multiple cases and analysing large numbers of endpoints. New types of protein arrays combined with advanced bioinformatics are currently being used to identify molecular signatures of individual tumours based on protein pathways and signalling cascades. It is envisaged that analysing the cellular 'circuitry' of ongoing molecular networks will become a powerful clinical tool in patient management.

Biomarkers, Tumor↗
PubMed2002

Functional proteomics: mapping protein-protein interactions and pathways.

The function of a protein is defined by its interactions with other proteins and molecules. Mapping of protein interactions can highlight new functionalities for a known protein or can even define the function of novel proteins. With the draft sequence of the human genome now available, it is possible to perform high-throughput mapping of protein-protein interactions in humans, which is termed as functional proteomics. The developments in functional proteomics are particularly timely since pharmaceutical companies are searching for technologies that will strengthen their genomic efforts and prioritize their drug discovery pipeline. In this article we review recent developments in functional proteomics.

Drug Design↗
PubMedDate not supplied

Direct proteomic mapping of Streptomyces Luteogriseus Strain 103 and cnn1 and insights into antibiotic biosynthesis.

Global proteome of the antibiotic-production strain 103 and the nonantibiotic-production mutant cnn1 were directly analyzed using 2D LC-MS/MS. A total of 726 and 809 proteins have been identified, respectively. Physical and chemical characterization, subcellular location and functional classification of the global protein were carried out. By searching the key enzymes of several probable antibiotic biosynthesis pathways in the identified proteins of strain 103, only polyketide synthase was found, which suggested that Maituolaimysin be synthesized through polyketide pathway. The same searching result was obtained in strain cnn1, which confirmed the conclusion drew from strain 103. Other proteins associated with the polyketide pathway of the two strains were searched according to the protein classification scheme of Streptomyces coelicolor (available at http://www.sanger.ac.uk/Projects/S_coelicolor/) and most of them were found. The activity inhibition of beta-ketoacyl ACP synthase, a key enzyme in the polyketide pathway, directly resulted in the decrease of Maituolaimysin production, which proved the conclusion obtained in the proteomic research.

Anti-Bacterial Agents↗
PubMed2026

Spatial proteomic mapping of the human and mouse retina using IBEX.

We generated a comparative spatial proteomic atlas of the human and mouse retina using a highly multiplexed immunohistochemistry technique called iterative bleaching extends multiplexity (IBEX). We refined the IBEX workflow by integrating an antibody dissociation option alongside chemical bleaching. This dual strategy enabled removal of the entire antibody complex, permitting the flexible use of antibodies from the same host species across iterative cycles. We coupled this workflow with super-resolution imaging via deconvolution and applied it to the retina of healthy humans and WT mice and the Crb1rd8 mouse model. We successfully imaged over 25 protein markers on human and mouse tissue sections, generating spatial atlases of the major retinal cell populations. Cross-species protein expression was compared to scRNA-seq datasets to identify protein and transcript disparities. Super-resolution IBEX delineated the ultrastructural features of the outer limiting membrane (OLM), identifying CD44 as a core structural component tightly colocalized with a highly organized F-actin belt within Müller glial endfeet. Using the Crb1rd8 mouse model, disruption of this complex was spatially associated with rosette formation and OLM structural failure. In summary, spatial proteomic atlases of the human and mouse retina were used to reveal insights into the arrangement of major retinal cell populations and OLM structure.

Animals↗
PubMed2003

Fungal proteomics: mapping the mitochondrial proteins of a Trichoderma harzianum strain applied for biological control.

Mitochondria are essential for cellular functions across organisms. A proteomic approach was taken to separate and identify mitochondrial proteins from a strain of Trichoderma harzianum with well established biocontrol properties. We optimized a method for the preparation of a sample enriched with mitochondria by ensuring efficient cell lysis and including several washing steps to minimize cytoplasmic contamination. We separated hundreds of proteins, using two-dimensional gel electrophoresis and identified 31 protein spots from T. harzianum, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and liquid chromatrography with mass spectrometry. Over 50% of the identified proteins were known to localize in the mitochondria. Three protein spots were identified from T. harzianum and a further five protein spots from the genera Trichoderma and Hypocrea. The remaining protein spots were identified by cross-species identification from other filamentous fungi, including Neurospora crassa and Aspergillus spp, and yeasts, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. In total, we identified 25 protein spots from T. harzianum, representing proteins that have not been characterized in existing Trichoderma protein databases. To our knowledge, this is the first two-dimensional mitochondrial protein map of a filamentous fungus.

Chromatography, Liquid↗
PubMed2026

Nuclear Proteome Map of Mouse Heart Chambers.

Heart specialization involves nuclear programs; however, chamber-specific regulation of the nuclear proteome landscape remains unknown. In this study, we isolated the nucleus from four major anatomical regions of healthy mouse heart (fresh) and employed quantitative mass spectrometry-based proteomics to construct a comprehensive nuclear proteome landscape of left ventricle (LV, 2403 proteins), right ventricle (RV, 2242 proteins), left atrium (LA, 2368 proteins), and right atrium (RA, 1816 proteins). This led to the discovery of nuclear regional proteome signatures (ventricular signature, 297 proteins; atrial signature, 183 proteins) associated with oxidative metabolism and redox regulation, ferroptosis, extracellular-matrix remodeling, SUMO- and stress-responsive control and transcriptional regulation. Chamber-level analyses further identify distinct nuclear features in LV (120 proteins), LA (188 proteins), and RA (72 proteins). In addition, we defined conserved core nuclear proteome (230 proteins) shared across all anatomical regions, enriched for transcription-regulator complexes, nucleolar/ribosome-associated, RNA-processing, and chromatin-organization components. Within this core network, we report 78 transcription factors/co-factors and select nuclear, chromatin and RNA export-associated proteins, including 29 specific factors (e.g., Alpk3, Rbm14, Arglu1, Hmgb1, Myef2, Sf1) associated with the heart. Regionally, we verified spatial localization in heart of H2ac21 and Sun2 in LA and Ptbp2 in LV by immunofluorescence. This study provides insights into the chamber-resolved view of the nuclear proteome in the heart, establishes a framework for linking nuclear proteomic signatures to atrial and ventricular biology, unique features of the heart nuclear proteome landscape relative to other organs, and a baseline for studying nuclear remodeling in cardiac pathophysiology.

Animals↗
PubMed2006

Mild protease treatment as a small-scale biochemical method for mitochondria purification and proteomic mapping of cytoplasm-exposed mitochondrial proteins.

Because of its importance in basic biology and medicine, great efforts are being devoted to unraveling of the genuine mitochondrial proteome, which is the dynamic protein complement that the organelle uses to maintain its structure and functionality. Several proteomic investigations have now clearly shown that all the purification approaches we have at our disposal suffer from the problem of co-purification; therefore, it is very difficult to distinguish novel mitochondrial proteins from those that are just contaminants of the preparation. The question is further complicated by the fact that the mitochondrial proteome depends on the tissue source. Density gradient centrifugation is the most widespread purification method for obtaining highly pure mitochondrial fractions. The main disadvantage of these methods is the low yield of purified mitochondria that precludes their use in low-scale purifications. Here, we have treated small aliquots of crude mitochondria from mouse liver and from cultured hepatocytes (HEPA1-6) with trypsin under mild proteolysis conditions and have evaluated the suitability of this reaction as a small-scale purification approach. The protease removed several cytoplasmic and endoplasmic reticulum proteins, together with a fraction of mitochondrial proteins that we hypothesize to be associated with the cytosolic face of the outer mitochondrial membrane. The peculiar topology of these mitochondrial proteins could be indicative of their functional roles. Finally, our study represents an application of advanced mass spectrometry technology to the evaluation of biochemical approaches for the treatment of mitochondria.

Animals↗
PubMed2004

High-resolution proteomic mapping in the vertebrate central nervous system: close proximity of connexin35 to NMDA glutamate receptor clusters and co-localization of connexin36 with immunoreactivity for zonula occludens protein-1 (ZO-1).

Combined confocal microscopy and freeze-fracture replica immunogold labeling (FRIL) were used to examine the connexin identity at electrical synapses in goldfish brain and rat retina, and to test for "co-localization" vs. "close proximity" of connexins to other functionally interacting proteins in synapses of goldfish and mouse brain and rat retina. In goldfish brain, confocal microscopy revealed immunofluorescence for connexin35 (Cx35) and NMDA-R1 (NR1) glutamate receptor protein in Mauthner Cell/Club Ending synapses. By FRIL double labeling, NR1 glutamate receptors were found in clusters of intramembrane particles in the postsynaptic membrane extraplasmic leaflets, and these distinctive postsynaptic densities were in close proximity (0.1-0.3 microm) to neuronal gap junctions labeled for Cx35, which is the fish ortholog of connexin36 (Cx36) found at neuronal gap junctions in mammals. Immunogold labeling for Cx36 in adult rat retina revealed abundant gap junctions, including several previously unrecognized morphological types. As in goldfish hindbrain, immunogold double labeling revealed NR1-containing postsynaptic densities localized near Cx36-labeled gap junction in rat inferior olive. Confocal immunofluorescence microscopy revealed widespread co-localization of Cx36 and ZO-1, particularly in the reticular thalamic nucleus and amygdala of mouse brain. By FRIL, ZO-1 immunoreactivity was co-localized with Cx36 at individual gap junction plaques in rat retinal neurons. As cytoplasmic accessory proteins, ZO-1 and possibly related members of the membrane-associated guanylate kinase (MAGUK) family represent scaffolding proteins that may bind to and regulate the activity of many neuronal gap junctions. These data document the power of combining immunofluorescence confocal microscopy with FRIL ultrastructural imaging and immunogold labeling to determine the relative proximities of proteins that are involved in short- vs. intermediate-range molecular interactions in the complex membrane appositions at synapses between neurons.

Animals↗
PubMed2003

Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance mechanisms.

Leishmania is a protozoan parasite responsible for significant morbidity and mortality worldwide. Few parasites have been subjected to proteomic analysis to date, but a genome sequencing project for Leishmania major is currently underway, making these studies possible. Here we present a high resolution proteome for L. major comprising almost 3700 spots, making it the most complete two-dimensional gel representation of a parasite proteome generated to date. We have identified a number of landmark proteins by mass spectrometry and show that several of these are valid for the related species Leishmania donovani infantum. We have also observed several forms and fragments of alpha- and beta-tubulins and show that the number and amount of these fragments increase with the age of the parasite culture. Trypanothione reductase (TRYR), which replaces glutathione reductase in trypanosomatid parasites, is an essential protein specific to these parasites and as such is under considerable scrutiny as a drug target. Two-dimensional gel analysis of a L. major strain overexpressing TRYR revealed increased amounts of five spots, all at the predicted molecular weight for TRYR and differing by 0.08 pH units in pI. Mass spectrometry identified four of these as TRYR, leading to the novel suggestion that it could be post-translationally modified. Finally quantitative comparative analysis of a methotrexate-resistant mutant of L. major generated in vitro found that a known primary resistance mediator, the pteridine reductase PTR1, was overexpressed. This constitutes the first proteomic analysis of drug resistance in a parasite and also the clearest identification of a primary drug resistance mechanism using this approach. Together these results provide a framework for further proteomic studies of Leishmania species and demonstrate that these tools are valuable for the essential study of potential drug targets and drug resistance mechanisms.

Animals↗
PubMed2000

Mining genomes and mapping proteomes: identification and characterization of protein subunit vaccines.

Currently, there is an extensive and unprecedented effort to obtain the complete nucleotide sequence of the complex genomes of many micro-organisms. In this post-genomic era, based on the availability of the entire genome sequence of an organism, three new disciplines of molecular biology have emerged: genomics, transcriptional profiling and proteomics. All these technologies have the potential to accelerate the process of identifying protective protein antigens as subunit vaccine targets as well as validating and extending the range of available candidate antigens. The progress of these technologies has led to the origination of the science of bioinformatics for management and critical evaluation of the large amount of information generated. Although genomics, transcriptional profiling and proteomics are each based on different principles, there is considerable synergy between them. Appropriate application of any one, or a combination of two or more of these approaches, coupled with bioinformatics, would allow identification of a short-list of vaccine candidates from the entire list of several hundreds to thousands of proteins encoded by the genome. These candidates would then require usual channelling through the subsequent process involving recombinant expression, purification and testing for immunogenicity and protective efficacy.

Animals↗
PubMed2004

Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy.

The molecular complexity of tissues and the inaccessibility of most cells within a tissue limit the discovery of key targets for tissue-specific delivery of therapeutic and imaging agents in vivo. Here, we describe a hypothesis-driven, systems biology approach to identifying a small subset of proteins induced at the tissue-blood interface that are inherently accessible to antibodies injected intravenously. We use subcellular fractionation, subtractive proteomics and bioinformatics to identify endothelial cell surface proteins exhibiting restricted tissue distribution and apparent tissue modulation. Expression profiling and gamma-scintigraphic imaging with antibodies establishes two of these proteins, aminopeptidase-P and annexin A1, as selective in vivo targets for antibodies in lungs and solid tumours, respectively. Radio-immunotherapy to annexin A1 destroys tumours and increases animal survival. This analytical strategy can map tissue- and disease-specific expression of endothelial cell surface proteins to uncover novel accessible targets useful for imaging and therapy.

Aminopeptidases↗
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↗
PubMed1999

Proteome mapping, mass spectrometric sequencing and reverse transcription-PCR for characterization of the sulfate starvation-induced response in Pseudomonas aeruginosa PAO1.

A set of proteins induced in Pseudomonas aeruginosa PAO1 during growth in the absence of sulfate was characterized by differential two-dimensional electrophoresis and MS. Thirteen proteins were found to be induced de novo or upregulated in P. aeruginosa grown in a succinate/salts medium with sodium cyclohexylsulfamate as the sole sulfur source. Protein spots excised from the two-dimensional gels were analysed by N-terminal Edman sequencing and MS sequencing (MS/MS) of internal protein fragments. The coding sequences for 11 of these proteins were unambiguously identified in the P. aeruginosa genome sequence. Expression of these genes was investigated by reverse transcription-PCR, which confirmed that repression in the presence of sulfate was acting at a transcriptional level. Three classes of sulfur-regulated proteins were found. The first class (five proteins) were high-affinity periplasmic solute-binding proteins with apparent specificity for sulfate and sulfonates. A second class included enzymes involved in sulfonate and sulfate ester metabolism (three proteins). The remaining three proteins appeared to be part of a more general stress response, and included two antioxidant proteins and a putative lipoprotein. This study demonstrates the power of the proteomics approach for direct correlation of the responses of an organism to an environmental stimulus with the genetic structures responsible for that response, and the application of reverse transcription-PCR significantly increases the conclusions that can be drawn from the proteomic study.

Amino Acid Sequence↗