Catalog Of Proteins Research Articles

Schistosome infections induce significant changes in the host biliary proteome.

To identify the changes induced by the schistosome infection in bile protein composition, and therefore in bile function, we compared the proteome of the gallbladders collected from non-infected healthy mice and from mice infected with S. bovis during 4months. For this, gallbladders from both groups of mice were homogenized and these homogenates were fractionated by serial centrifugation and acrylamide gel electrophoresis. The proteins were in gel digested and analysed by LC-MS/MS for identification. The present work reports the first data on the proteome of the mouse gallbladder and provides a comprehensive catalogue of biliary proteins that may be of great use in other studies addressing biliary physiology and pathology. We observed that there were significant differences in the biliary proteome of mice infected and non-infected with S. bovis and that chronic schistosome infections may produce important physiological alterations in the bile function.

A purification strategy for analysis of the DNA/RNA-associated sub-proteome from chloroplasts of mustard cotyledons.

Plant cotyledons are a tissue that is particularly active in plastid gene expression in order to develop functional chloroplasts from pro-plastids, the plastid precursor stage in plant embryos. Cotyledons, therefore, represent a material being ideal for the study of composition, function and regulation of protein complexes involved in plastid gene expression. Here, we present a pilot study that uses heparin-Sepharose and phospho-cellulose chromatography in combination with isoelectric focussing and denaturing SDS gel electrophoresis (two-dimensional gel electrophoresis) for investigating the nucleic acids binding sub-proteome of mustard chloroplasts purified from cotyledons. We describe the technical requirements for a highly resolved biochemical purification of several hundreds of protein spots obtained from such samples. Subsequent mass spectrometry of peptides isolated out of cut spots that had been treated with trypsin identified 58 different proteins within 180 distinct spots. Our analyses indicate a high enrichment of proteins involved in transcription and translation and, in addition, the presence of massive post-translational modification of this plastid protein sub-fraction. The study provides an extended catalog of plastid proteins from mustard being involved in gene expression and its regulation and describes a suitable purification strategy for further analysis of low abundant gene expression related proteins.

In-depth proteomic analysis of carp (Cyprinus carpio L) spermatozoa

Using a combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography-electrospray ionization tandem mass spectrometry, we identified 348 proteins in carp spermatozoa, most of which were for the first time identified in fish. Dynein, tubulin, HSP90, HSP70, HSP60, adenosylhomocysteinase, NKEF-B, brain type creatine kinase, mitochondrial ATP synthase, and valosin containing enzyme represent high abundance proteins in carp spermatozoa. These proteins are functionally related to sperm motility and energy production as well as the protection of sperm against oxidative injury and stress. Moreover, carp spermatozoa are equipped with functionally diverse proteins involved in signal transduction, transcription, translation, protein turnover and transport. About 15% of proteins from carp spermatozoa identified here were also detected in seminal plasma which may be a result of leakage from spermatozoa into seminal plasma, adsorption of seminal plasma proteins on spermatozoa surface, and expression in both spermatozoa and cells secreting seminal plasma proteins. The availability of a catalog of carp sperm proteins provides substantial advances for an understanding of sperm function and for future development of molecular diagnostic tests of carp sperm quality, the evaluation of which is currently limited to certain parameters such as sperm count, morphology and motility or viability. The mass spectrometry data are available at ProteomeXchange with the dataset identifier PXD000877 (DOI: http://dx.doi.org/10.6019/PXD000877).

Transition state mimetics of the Plasmodium export element are potent inhibitors of Plasmepsin V from P. falciparum and P. vivax.

Following erythrocyte invasion, malaria parasites export a catalogue of remodeling proteins into the infected cell that enable parasite development in the human host. Export is dependent on the activity of the aspartyl protease, plasmepsin V (PMV), which cleaves proteins within the Plasmodium export element (PEXEL; RxL↓xE/Q/D) in the parasite's endoplasmic reticulum. Here, we generated transition state mimetics of the native PEXEL substrate that potently inhibit PMV isolated from Plasmodium falciparum and Plasmodium vivax. Through optimization, we identified that the activity of the mimetics was completely dependent on the presence of P1 Leu and P3 Arg. Treatment of P. falciparum-infected erythrocytes with a set of optimized mimetics impaired PEXEL processing and killed the parasites. The striking effect of the compounds provides a clearer understanding of the accessibility of the PMV active site and reaffirms the enzyme as an attractive target for the design of future antimalarials.

Proteomic analysis of human vitreous humor.

BackgroundThe vitreous humor is a transparent, gelatinous mass whose main constituent is water. It plays an important role in providing metabolic nutrient requirements of the lens, coordinating eye growth and providing support to the retina. It is in close proximity to the retina and reflects many of the changes occurring in this tissue. The biochemical changes occurring in the vitreous could provide a better understanding about the pathophysiological processes that occur in vitreoretinopathy. In this study, we investigated the proteome of normal human vitreous humor using high resolution Fourier transform mass spectrometry.ResultsThe vitreous humor was subjected to multiple fractionation techniques followed by LC-MS/MS analysis. We identified 1,205 proteins, 682 of which have not been described previously in the vitreous humor. Most proteins were localized to the extracellular space (24%), cytoplasm (20%) or plasma membrane (14%). Classification based on molecular function showed that 27% had catalytic activity, 10% structural activity, 10% binding activity, 4% cell and 4% transporter activity. Categorization for biological processes showed 28% participate in metabolism, 20% in cell communication and 13% in cell growth. The data have been deposited to the ProteomeXchange with identifier PXD000957.ConclusionThis large catalog of vitreous proteins should facilitate biomedical research into pathological conditions of the eye including diabetic retinopathy, retinal detachment and cataract.

Proteomic Analyses of Human Gingival and Periodontal Ligament Fibroblasts

Although human gingival fibroblasts (hGFs) and human periodontal ligament fibroblasts (hPDLFs) exhibit numerous phenotypic similarities, it has been suggested that the secretory and behavioral differences, which exist between these cell types, are a result of the membrane protein composition of these cells. Four matched pairs of hGFs and hPDLFs were cultured. Before confluence, membrane-bound and -associated proteins from cells of the fourth passage were extracted. The processed protein samples were evaluated using capillary-liquid chromatography-nanospray tandem mass spectrometry. Global protein identification was performed on an orbitrap mass spectrometer equipped with a microspray source operated in positive ion mode. Proteome software was used to validate protein identifications derived from tandem mass spectrometry sequencing results. Four hundred fifty proteins were common to both hGFs and hPDLFs. Of the proteins identified, 214 were known membrane-bound or -associated proteins, and 165 proteins were known nuclear-associated proteins. Twenty-seven proteins, identified from the 450 proteins, common to both hGFs and hPDLFs, were detected in statistically significant greater quantities in either hGFs or hPDLFs. More specifically, 13 proteins were detected in significantly greater quantities in hGFs, whereas 14 proteins were detected in significantly greater quantities in hPDLFs. Distinct differences in the cellular protein catalog may reflect the dynamic role and high energy requirements of hGFs in extracellular matrix remodeling and response to inflammatory challenge as well as the role of hPDLFs in monitoring mechanical stress and maintaining tissue homeostasis during regeneration and remineralization.

Proteomic identification of rainbow trout sperm proteins

Proteomics represents a powerful tool for the analysis of fish spermatozoa, since these cells are transcriptionally inactive. The aim of the present study was to generate an inventory of the most prominent rainbow trout sperm proteins by SDS-PAGE prefractionation combined with nano-LC-MS/MS based identification. This study provides the first in-depth analysis of the rainbow trout sperm proteome, with a total of 206 identified proteins. We found that rainbow trout spermatozoa are equipped with functionally diverse proteins related to energetic metabolism, signal transduction, protein turnover, transport, cytoskeleton, oxidative injuries, and stress and reproduction. The availability of a catalog of rainbow trout sperm proteins provides a crucial tool for the understanding of fundamental molecular processes in fish spermatozoa, for the ongoing development of novel markers of sperm quality and for the optimization of short- and long-term sperm preservation procedures. The MS data are available at ProteomeXchange with the dataset identifier PXD000355 and DOI 10.6019/PXD000355.

Comparative proteomic profiles of the normal and aganglionic hindgut in human Hirschsprung disease

Hirschsprung disease (HSCR) is the third most common congenital disorder of the gastrointestinal tract. This study aims to elucidate changes in protein expression between the normal and aganglionic hindgut in human HSCR. The biopsies were obtained from the normal and aganglionic hindgut in human HSCR, and the comparative proteomics were analyzed by mass spectrometry (MS)-based two-dimensional gel electrophoresis (2DE). A total of 932-986 protein spots were identified in each of the gut segments, among which 30 spots had at least an eightfold difference in volume (%). Of the 30 differentially expressed spots, 15 proteins were identified via sequence analysis. Among these 15 proteins, eight were upregulated and seven were downregulated in the aganglionic group. The well-represented classes included biomarkers of enteric ganglions, extracellular matrix proteins, LIM domain proteins, serum proteins, and other pleiotropic proteins. Five proteins were selected and verified by western blotting and real-time PCR, and the results were consistent with the results of 2DE. MS-based 2DE can help to identify pathological relevant proteins in HSCR; it defines an extensive protein catalog of the normal and aganglionic hindgut and may constitute the basis to understand pathophysiological mechanisms related to the HSCR.

Computational protein design as an optimization problem

Proteins are chains of simple molecules called amino acids. The three-dimensional shape of a protein and its amino acid composition define its biological function. Over millions of years, living organisms have evolved a large catalog of proteins. By exploring the space of possible amino acid sequences, protein engineering aims at similarly designing tailored proteins with specific desirable properties. In Computational Protein Design (CPD), the challenge of identifying a protein that performs a given task is defined as the combinatorial optimization of a complex energy function over amino acid sequences.In this paper, we introduce the CPD problem and some of the main approaches that have been used by structural biologists to solve it, with an emphasis on the exact method embodied in the dead-end elimination/A⁎ algorithm (DEE/A⁎). The CPD problem is a specific form of binary Cost Function Network (CFN, aka Weighted CSP). We show how DEE algorithms can be incorporated and suitably modified to be maintained during search, at reasonable computational cost.We then evaluate the efficiency of CFN algorithms as implemented in our solver toulbar2, on a set of real CPD instances built in collaboration with structural biologists. The CPD problem can be easily reduced to 0/1 Linear Programming, 0/1 Quadratic Programming, 0/1 Quadratic Optimization, Weighted Partial MaxSAT and Graphical Model optimization problems. We compare toulbar2 with these different approaches using a variety of solvers. We observe tremendous differences in the difficulty that each approach has on these instances.Overall, the CFN approach shows the best efficiency on these problems, improving by several orders of magnitude against the exact DEE/A⁎ approach. The introduction of dead-end elimination before or during search allows to further improve these results.

Proteins Involved in Motility and Sperm-Egg Interaction Evolve More Rapidly in Mouse Spermatozoa

Proteomic studies of spermatozoa have identified a large catalog of integral sperm proteins. Rapid evolution of these proteins may underlie adaptive changes of sperm traits involved in different events leading to fertilization, although the selective forces underlying such rapid evolution are not well understood. A variety of selective forces may differentially affect several steps ending in fertilization, thus resulting in a compartmentalized adaptation of sperm proteins. Here we analyzed the evolution of genes associated to various events in the sperm’s life, from sperm formation to sperm-egg interaction. Evolutionary analyses were performed on gene sequences from 17 mouse strains whose genomes have been sequenced. Four of these are derived from wild Mus musculus, M. domesticus, M. castaneus and M. spretus. We found a higher proportion of genes exhibiting a signature of positive selection among those related to sperm motility and sperm-egg interaction. Furthermore, sperm proteins involved in sperm-egg interaction exhibited accelerated evolution in comparison to those involved in other events. Thus, we identified a large set of candidate proteins for future comparative analyses of genotype-phenotype associations in spermatozoa of species subjected to different sexual selection pressures. Adaptive evolution of proteins involved in motility could be driven by sperm competition, since this selective force is known to increase the proportion of motile sperm and their swimming velocity. On the other hand, sperm proteins involved in gamete interaction could be coevolving with their egg partners through episodes of sexual selection or sexual conflict resulting in species-specific sperm-egg interactions and barriers preventing interspecies fertilization.

Brevibacillus as a biological tool: a short review

The significance of Brevibacillus has been documented scientifically in the published literature and commercially in heterologous recombinant protein catalogs. Brevibacillus is one of the most widespread genera of Gram-positive bacteria, recorded from the diverse environmental habitats. The high growth rate, better transformation efficiency by electroporation, availability of shuttle vectors, production of negligible amount of extracellular protease, and the constitutive expression of heterologous proteins make some strains of this genus excellent laboratory models. Regarding biotechnological applications, this genus continues to be a source of various enzymes of great biotechnological interest due to their ability to biodegrade low density polyethylene, ability to act as a candidate bio-control agent, and more recently acknowledged as a tool for the overexpression. This article reviews the properties of Brevibacillus spp. as better biological tools with varied applications.

Proteomic analysis of human substantia nigra identifies novel candidates involved in Parkinson's disease pathogenesis

Parkinson's disease (PD) pathology spreads throughout the brain following a region-specific process predominantly affecting the substantia nigra (SN) pars compacta. SN exhibits a progressive loss of dopaminergic neurons responsible for the major cardinal motor symptoms, along with the occurrence of Lewy bodies in the surviving neurons. To gain new insights into the underlying pathogenic mechanisms in PD, we studied postmortem nigral tissues dissected from pathologically confirmed PD cases (n = 5) and neurologically intact controls (n = 8). Using a high-throughput shotgun proteomic strategy, we simultaneously identified 1795 proteins with concomitant quantitative data. To date, this represents the most extensive catalog of nigral proteins. Of them, 204 proteins displayed significant expression level changes in PD patients versus controls. These were involved in novel or known pathogenic processes including mitochondrial dysfunction, oxidative stress, or cytoskeleton impairment. We further characterized four candidates that might be relevant to PD pathogenesis. We confirmed the differential expression of ferritin-L and seipin by Western blot and demonstrated the neuronal localization of gamma glutamyl hydrolase and nebulette by immunohistochemistry. Our preliminary findings suggest a role for nebulette overexpression in PD neurodegeneration, through mechanisms that may involve cytoskeleton dynamics disruption. All MS data have been deposited in the ProteomeXchange with identifier PXD000427 (http://proteomecentral.proteomexchange.org/dataset/PXD000427).

Proteomic analysis of human osteoarthritis synovial fluid

BackgroundOsteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography.ResultsWe identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples.ConclusionsWe present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis.

Evolutionary conservation of the mature oocyte proteome

The proteome profiles of mature ovulated oocytes of the Cnidaria basal eumetazoan, the starlet sea anemone Nematostella vectensis was compared with published data of mammalian mouse mature oocytes. We identified 1837 proteins in N. vectensis oocytes including known oocyte- and germ-cell-specific markers, proteins associated with RNPs and vitellogenin, a major component of egg yolk proteins. Our findings suggest highly conserved enriched functional pathways in N. vectensis and the mouse mature oocytes. This study provides the first catalog of cnidarian oocyte proteins, revealing highly conserved ancient organization of life processes for over 500 million years of evolution. SignificanceThe current study provides the first proteomic profile of an oocyte of a cnidarian organism the starlet sea anemone N. vectensis and gives new insights on the ancient origin of an oocyte proteome template. The comparative analysis with a chordate oocyte suggests that the oocyte proteome predates the divergence of the cnidarian and bilaterian lineages. In addition, the data generated in the study will serve as a valuable resource for further developmental and evolutional studies.

Proteomic identification of rainbow trout seminal plasma proteins

In the study, the combination of protein fractionation by 1DE and HPLC-ESI-MS/MS was used to characterize the rainbow trout seminal plasma proteome. Our results led to the creation of a catalogue of rainbow trout seminal plasma proteins (152 proteins) and significantly contributed to the current knowledge regarding the protein composition of fish seminal plasma. The major proteins of rainbow trout seminal plasma, such as transferrin, apolipoproteins, complement C3, serum albumin, and hemopexin-, alpha-1-antiproteinase-, and precerebellin-like protein, were recognized as acute-phase proteins (proteins that plasma concentration changes in response to inflammation). This study provides the basis for further functional studies of fish seminal plasma proteins, as well as for the identification of novel biomarkers for sperm quality. The MS data have been deposited in the ProteomeXchange with identifier PXD000306 (http://proteomecentral.proteomexchange.org/dataset/PXD000306).

A deep insight into the sialotranscriptome of the mosquito, Psorophora albipes

BackgroundPsorophora mosquitoes are exclusively found in the Americas and have been associated with transmission of encephalitis and West Nile fever viruses, among other arboviruses. Mosquito salivary glands represent the final route of differentiation and transmission of many parasites. They also secrete molecules with powerful pharmacologic actions that modulate host hemostasis, inflammation, and immune response. Here, we employed next generation sequencing and proteome approaches to investigate for the first time the salivary composition of a mosquito member of the Psorophora genus. We additionally discuss the evolutionary position of this mosquito genus into the Culicidae family by comparing the identity of its secreted salivary compounds to other mosquito salivary proteins identified so far.ResultsIllumina sequencing resulted in 13,535,229 sequence reads, which were assembled into 3,247 contigs. All families were classified according to their in silico-predicted function/ activity. Annotation of these sequences allowed classification of their products into 83 salivary protein families, twenty (24.39%) of which were confirmed by our subsequent proteome analysis. Two protein families were deorphanized from Aedes and one from Ochlerotatus, while four protein families were described as novel to Psorophora genus because they had no match with any other known mosquito salivary sequence. Several protein families described as exclusive to Culicines were present in Psorophora mosquitoes, while we did not identify any member of the protein families already known as unique to Anophelines. Also, the Psorophora salivary proteins had better identity to homologs in Aedes (69.23%), followed by Ochlerotatus (8.15%), Culex (6.52%), and Anopheles (4.66%), respectively.ConclusionsThis is the first sialome (from the Greek sialo = saliva) catalog of salivary proteins from a Psorophora mosquito, which may be useful for better understanding the lifecycle of this mosquito and the role of its salivary secretion in arboviral transmission.

Diversity and evolution of ABC proteins in basidiomycetes

ABC proteins constitute one of the largest families of proteins. They are implicated in wide variety of cellular processes ranging from ribosome biogenesis to multidrug resistance. With the advance of fungal genomics, the number of known fungal ABC proteins increases rapidly but the information on their biological functions remains scarce. In this work we extended the previous analysis of fungal ABC proteins to include recently sequenced species of basidiomycetes. We performed an identification and initial cataloging of ABC proteins from 23 fungal species representing 10 orders within class Agaricomycotina. We identified more than 1000 genes coding for ABC proteins. Comparison of sets of ABC proteins present in basidiomycetes and ascomycetes revealed the existence of two groups of ABC proteins specific for basidiomycetes. Results of survey should contribute to the better understanding of evolution of ABC proteins in fungi and support further experimental work on their characterization.

Proteomic analysis of pleural effusion from lung adenocarcinoma patients by shotgun strategy

To construct a protein catalogue of malignant pleural effusion from lung adenocarcinoma patients and to screen the potential candidates of biomarkers for diagnostic value in human lung adenocarcinoma. Five malignant pleural effusion samples of lung adenocarcinoma patients were collected from January 2009 to September. A composite sample was analyzed using shotgun strategy. Pleural effusion samples were separated by means of SDS-PAGE. Proteomic analysis was performed by 1D-LC-MS/MS, and then the proteins were identified using SEQUEST software and protein database search. Among 230 unique proteins, 123 proteins were identified with higher confidence levels (at least two unique peptide sequences matched). Most of these proteins have been reported in plasma. However, there are 7 proteins, including JUP protein, suprabasin, annexin A2, transforming growth factor-beta-induced protein ig-h3 (βig-h3), V-set and immunoglobulin domain-containing protein 4 precursor, ifapsoriasin 2 and actin, cytoplasmic 1 have not been reported in serum. Seven proteins may represent potential candidates of biomarkers. Annexin A2 is of special interest since it may play a role in the regulation of intercellular adhesion and cell proliferation.

Characterizing the normal proteome of human ciliary body

BackgroundThe ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.ResultsIn this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.ConclusionsMore than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.

Overexpression of Aldo-keto reductase family 1 B10 protein in ductal carcinoma in situ of the breast correlates with HER2 positivity

Ductal carcinoma in situ of the breast constitutes the early stage of breast cancer when cancer cells are confined by the intact myoepithelial cell layer. Transition from DCIS to invasive carcinoma is a process yet poorly understood. By liquid chromatography (LC) and mass spectrometry (MS/MS) methods, we analyzed this early event using paired samples of micro-dissected cells overlaid with focally disrupted myoepithelial layers and their adjacent counterparts within the intact duct from formalin-fixed paraffin-embedded blocks. AKR1B10, a member of Aldo-keto reductase family, was shown to be abundantly located in the filtering cells among a catalog of proteins. Moreover, strong correlation between AKR1B10 and HER2 positivity was found in an independent cohort of DCIS samples. AKR1B10 could become a potential diagnosis and therapeutic marker for early breast cancers with HER2 overexpression and poor prognosis.