A Draft Map of Rhesus Monkey Tissue Proteome for Biomedical Research

Though the rhesus monkey is one of the most valuable non-human primate animal models for various human diseases because of its manageable size and genetic and proteomic similarities with humans, proteomic research using rhesus monkeys still remains challenging due to the lack of a complete protein sequence database and effective strategy. To investigate the most effective and high-throughput proteomic strategy, comparative data analysis was performed employing various protein databases and search engines. The UniProt databases of monkey, human, bovine, rat and mouse were used for the comparative analysis and also a universal database with all protein sequences from all available species was tested. At the same time, de novo sequencing was compared to the SEQUEST search algorithm to identify an optimal work flow for monkey proteomics. Employing the most effective strategy, proteomic profiling of monkey organs identified 3,481 proteins at 0.5% FDR from 9 male and 10 female tissues in an automated, high-throughput manner. Data are available via ProteomeXchange with identifier PXD001972. Based on the success of this alternative interpretation of MS data, the list of proteins identified from 12 organs of male and female subjects will benefit future rhesus monkey proteome research.     

Profiling human brain proteome by multi-dimensional separations coupled with MS

In our initial attempt to analyze the human brain proteome, we applied multi-dimensional protein separation and identification techniques using a combination of sample fractionation, 1-D SDS-PAGE, and MS analysis. The complexity of human brain proteome requires multiple fractionation strategies to extend the range and total number of proteins identified. According to the method of Klose (Methods Mol. Biol. 1999, 112, 67), proteins of the temporal lobe of human brain were fractionated into (i) cytoplasmic and nucleoplasmic, (ii) membrane and other structural, and (iii) DNA-binding proteins. Each fraction was then separated by SDS-PAGE, and the resulting gel line was cut into approximately 50 bands. After trypsin digestion, the resulting peptides from each band were analyzed by RP-LC/ESI-MS/MS using an LTQ spectrometer. The SEQUEST search program, which searched against the IPI database, was used for peptide sequence identification, and peptide sequences were validated by reversed sequence database search and filtered by the Protein Hit Score. Ultimately, 1533 proteins could be detected from the human brain. We classified the identified proteins according to their distribution on cellular components. Among these proteins, 24% were membrane proteins. Our results show that the multiple separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.     

Optimized proteomic analysis on gels of cell-cell adhering junctional membrane proteins

A high level of structural organization of functional membrane domains in very narrow regions of a plasma membrane is crucial for the functions of plasma membranes and various other cellular functions. Conventional proteomic analyses are based on total soluble cellular proteins. Thus, because of insolubility problems, they have major drawbacks for use in analyses of low-abundance proteins enriched in very limited and specific areas of cells, as well as in analyses of the membrane proteins in two-dimensional gels. We optimized proteomic analyses of cell-cell adhering junctional membrane proteins on gels. First, we increased the purity of cell-cell junctions, which are very limited and specific areas for cell-cell adhesion, from hepatic bile canaliculi. We then enriched junctional membrane proteins via a guanidine treatment; these became selectively detectable on two- dimensionally electrophoresed gels after treatment with an extremely high concentration of NP-40. The framework of major junctional integral membrane proteins was shown on gels. These included six novel junctional membrane proteins of type I, type II, and tetraspanin, which were identified by mass spectrometry and by a database sequence homology search, as well as 12 previously identified junctional membrane proteins, such as cadherins and claudins.     

Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosis

We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.     

Possible target‐related proteins and signal network of bufalin in A549 cells suggested by both iTRAQ‐based and label‐free proteomic analysis

Bufalin (BF) exhibited antiproliferation and antimigration effects on human A549 lung cancer cells. To search its target‐related proteins, protein expression profiles of BF‐treated and control cells were compared using two quantitative proteomic methods, iTRAQ‐based and label‐free proteomic analysis. A total of 5428 proteins were identified in iTRAQ‐based analysis while 6632 proteins were identified in label‐free analysis. The number of common identified proteins of both methods was 4799 proteins. By application of 1.20‐fold for upregulated and 0.83‐fold for downregulated cutoff values, 273 and 802 differentially expressed proteins were found in iTRAQ‐based and label‐free analysis, respectively. The number of common differentially expressed proteins of both methods was 45 proteins. Results of bioinformational analysis using Metacore^TM showed that the two proteomic methods were complementary and both suggested the involvement of oxidative stress and regulation of gene expression in the effects of BF, and fibronectin‐related pathway was suggested to be an important pathway affected by BF. Western blotting assay results confirmed BF‐induced change in levels of fibronectin and other related proteins. Overexpression of fibronectin by plasmid transfection ameliorated antimigration effects of BF. Results of the present study provided information about possible target‐related proteins and signal network of BF.     

Overcoming species boundaries in peptide identification with Bayesian information criterion-driven error-tolerant peptide search (BICEPS)

Currently, the reliable identification of peptides and proteins is only feasible when thoroughly annotated sequence databases are available. Although sequencing capacities continue to grow, many organisms remain without reliable, fully annotated reference genomes required for proteomic analyses. Standard database search algorithms fail to identify peptides that are not exactly contained in a protein database. De novo searches are generally hindered by their restricted reliability, and current error-tolerant search strategies are limited by global, heuristic tradeoffs between database and spectral information. We propose a Bayesian information criterion-driven error-tolerant peptide search (BICEPS) and offer an open source implementation based on this statistical criterion to automatically balance the information of each single spectrum and the database, while limiting the run time. We show that BICEPS performs as well as current database search algorithms when such algorithms are applied to sequenced organisms, whereas BICEPS only uses a remotely related organism database. For instance, we use a chicken instead of a human database corresponding to an evolutionary distance of more than 300 million years (International Chicken Genome Sequencing Consortium (2004) Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432, 695-716). We demonstrate the successful application to cross-species proteomics with a 33% increase in the number of identified proteins for a filarial nematode sample of Litomosoides sigmodontis.     

Proteomic analysis of the pinworm Syphacia muris (Nematoda: Oxyuridae), a parasite of laboratory rats

Syphacia muris (Nematoda: Oxyuridae) is a ubiquitous nematode that commonly infects rats in the laboratory which can interfere in the development of biological assays. The somatic extract of S. muris adults collected from infected rats was investigated using a proteomic approach. A shot-gun liquid chromatography/tandem mass spectrometry procedure was used. We used the MASCOT search engine (Matrix-Science) and ProteinPilot software v2.0 (Applied Biosystems) for the database search. A total of 359 proteins were accurately identified from the worms. The largest protein families consisted of metabolic enzymes and those involved in the nucleic metabolism and cell cycle. Proteins of transmembrane receptors and those involved in protein metabolism, chaperones, structural and motor, signalling and calcium-binding proteins also were identified in the proteome of S. muris. Proteome array of S. muris may contribute to further elucidation of biological system of S. muris as well as host-parasite relationships.     

Proteomic analysis of the neutrophil proteins of the tammar wallaby (Macropus eugenii)

A proteomic analysis of neutrophils from the tammar wallaby, Macropus eugenii, has been performed. Neutrophils were isolated from peripheral blood using density gradient centrifugation with Histopaque-1077, followed by treatment with ammonium chloride to lyse residual erythrocytes. Two-dimensional gel electrophoresis (2-DE) of lysed neutrophils was undertaken followed by in-gel trypsin digest and nanoliquid chromatography coupled tandem mass spectrometry (LC-MS) analysis and database searches. Seventy-seven proteins were isolated, 53 of which could be identified with high confidence as primarily of cytosolic origin. Protein identifications were only possible by matching identical peptide sequences within the NCBInr mammalian database with the Mascot search program. Sequence identities were only deemed acceptable if more than three peptides were identified, the precursor/protein ion tolerances were less than ± 0.25 Da and the total Mowse scores were greater than 100. The validity of this approach was tested using a scrambled database where no single identified peptide showed Mowse scores greater than 55. This is the first report of the neutrophil proteins of any marsupial and represents a first step in examining the identity of proteins involved in innate defence in this marsupial.     

Microchip reactor packed with metal-ion chelated magnetic silica microspheres for highly efficient proteolysis

An easily replaceable and regenerable protease microreactor with metal-ion chelated adsorption of enzyme has been fabricated on chip. Magnetic microspheres with small size (approximately 200 nm in diameter) and strong magnetism were synthesized and were modified with tetraethyl orthosilicate. The metal chelating agent of iminodiacetic acid was then reacted with glycidoxypropyltrimethoxysilane before its immobilization onto the surface of magnetic silica microspheres (MS microspheres). The metal ion of copper and enzyme were subsequently adsorbed onto the surface. The prepared MS microspheres were then locally packed into the microchannel by the application of a strong magnetic field using a magnet to form an on-chip enzymatic microreactor. Capability of the proteolytic microreactor was demonstrated by cytochrome c and bovine serum albumin as model proteins. The digestion products were characterized using MALDI-TOF/TOF MS with sequence coverage of 77% and 21% observed, respectively. This microreactor was also applied to the analysis of one RPLC fraction of rat liver extract. After a database search, 23 unique peptides corresponding to 7 proteins were identified when one RPLC fraction of rat liver extract was digested by the microreactor. This opens a route for its future application in top-down proteomic analysis.     

A differential proteomic approach identifies structural and functional components that contribute to the differentiation of brain capillary endothelial cells

When in the vicinity of astrocytes, brain capillary endothelial cells (BCECs) develop the characteristic structural and functional features of the blood-brain barrier (BBB). The latter has low cellular permeability and restricts various compounds from entering the brain. We recently reported that the cytoskeleton-related proteins actin, gelsolin and filamin-A undergo the largest quantitative changes in bovine BCECs after re-induction of BBB functions by co-culture with glial cells. In the present study, we used an in-depth, proteomic approach to quantitatively compare differences in Triton-X-100-solubilized proteins from bovine BCECs with limited or re-induced BBB functions (i.e. cultured in the absence or presence of glial cells, respectively). The 81 protein spots of differing abundance were linked to 55 distinct genes. According to the Protein ANalysis THrough Evolutionary Relationships classification system and an Ingenuity Pathway Analysis, these quantitative changes mainly affected proteins involved in (i) cell structure and motility and (ii) protein metabolism and modification. The fold-changes affecting HSPB1, moesin and ANXA5 protein levels were confirmed by western blot analysis but were not accompanied by changes in the corresponding mRNA expression levels. Our results reveal that the bovine BCECs' phenotype adaptation to variations in their environment involves the reorganization of the actin cytoskeleton.     

Application of both high-performance liquid chromatography combined with tandem mass spectrometry shotgun and 2-D polyacrylamide gel electrophoresis for streptococcal exoproteins gave reliable proteomic data

Streptococci secrete a large number of exoproteins including virulence-associated toxins and enzymes. To construct a reliable database of streptococcal exoproteins, we integrated the results that were derived from two approaches: LC-based shotgun proteomic analysis and 2-D PAGE-based proteomic analysis. We identified 74 and 82 proteins by LC-based and gel-based analysis, respectively. Forty-five proteins were identified by both methods. In addition, two proteins, one identified by both methods and the other only by LC-based shotgun analysis, were newly annotated. We therefore found the importance of combinational analysis by the two methods for the construction of a more reliable database.     

Comparative secretome analysis of human follicular dermal papilla cells and fibroblasts using shotgun proteomics

The dermal papilla cells (DPCs) of hair follicles are known to secrete paracrine factors for follicular cells. Shotgun proteomic analysis was performed to compare the expression profiles of the secretomes of human DPCs and dermal fibroblasts (DFs). In this study, the proteins secreted by DPCs and matched DFs were analyzed by 1DE/LTQ FTICR MS/MS, semi-quantitatively determined using emPAI mole percent values and then characterized using protein interaction network analysis. Among the 1,271 and 1,188 proteins identified in DFs and DPCs, respectively, 1,529 were further analyzed using the Ingenuity Pathway Analysis tool. We identified 28 DPC-specific extracellular matrix proteins including transporters (ECM1, A2M), enzymes (LOX, PON2), and peptidases (C3, C1R). The biochemically- validated DPC-specific proteins included thrombospondin 1 (THBS1), an insulin-like growth factor binding protein3 (IGFBP3), and, of particular interest, an integrin beta1 subunit (ITGB1) as a key network core protein. Using the shotgun proteomic technique and network analysis, we selected ITGB1, IGFBP3, and THBS1 as being possible hair-growth modulating protein biomarkers.     

Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions

The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which we have identified 217 proteins. This allows a direct comparison of the proteomes of an important nuclear structure between two widely divergent species: human and Arabidopsis. The comparison identified many common proteins, plant-specific proteins, proteins of unknown function found in both proteomes, and proteins that were nucleolar in plants but nonnucleolar in human. Seventy-two proteins were expressed as GFP fusions and 87% showed nucleolar or nucleolar-associated localization. In a striking and unexpected finding, we have identified six components of the postsplicing exon-junction complex (EJC) involved in mRNA export and nonsense-mediated decay (NMD)/mRNA surveillance. This association was confirmed by GFP-fusion protein localization. These results raise the possibility that in plants, nucleoli may have additional functions in mRNA export or surveillance.     

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.     

Insights from the molecular characterization of mercury stress proteins identified by proteomics in E.coli nissle 1917

Differently expressed proteins in probiotic Escherichia coli nissle 1917 under mercury stress identified by using a proteomic approach. We applied to separate proteins by using two-dimensional gel electrophoresis and proteins were identified using MALDI-TOF-MS using PMF, by mascot database search using the NCBI database. we identified six proteins after exposure to mercury stress with respect to different functional classes. It is useful to understand the molecular insights into mercury stress in probiotic E. coli. Next we describe a structure generated by homology modelling and functional domain identification; it is interesting to study the impact of stress on protein structures. MS characterization and computational methods together provide the opportunity to examine the impact of stress arising from mercury. The role of these proteins in metal tolerance and structure relation is discussed. To the best of our knowledge, proteomics of E. coli nissle 1917 overview of mercury stress has been reported for the first time.     

Identification of new Golgi complex specific proteins by direct organelle proteomic analysis

The Golgi complex is in the crossroad of the endocytic and secretory pathways. Its function is to post-translationally modify and sort proteins and lipids, and regulate the membrane balance in the cell. To understand the structure-function relationship of the Golgi complex the Golgi proteome has to be identified first. We have used a direct organelle proteomic analysis to identify new Golgi complex proteins. Enriched stacked Golgi membrane fractions from rat livers were isolated, and the proteins from these membranes were subsequently digested into peptides. The peptides were fractionated by cation-exchange chromatography followed by protein identification by automated capillary-LC/ESI-MS/MS analysis and database searches. Two different search programs, ProID and MASCOT were used. This resulted in a total of 1125 protein identifications in two experiments. In addition to the known Golgi resident proteins, a significant number of unknown proteins were identified. Some of these were further characterized in silico using different programs to provide insight into their structure, intracellular localization and biological functions. The Golgi localization of two of these newly identified proteins was also confirmed by indirect immunofluorescence.