Combination Of SDS-PAGE Research Articles

LIMITED PROTEOLYSIS OF FIBRINOGEN αC-REGION REVEALS ITS STRUCTURE

Aim. The purpose of our study was to compare hydrolytic action of proteases from Gloydius halys halys, Agkistrodon contortrix contortrix and Calloselasma rhodostoma rhodostoma snake venoms and from Bacillus thuringiensis вар. israelensis IMV B-7465 culture medium on αC-regions of fibrinogen molecule. Methods. Products of hydrolysis were characterized by SDS-PAGE under reducing conditions with following Western-Blot using the mouse monoclonal 1-5А (anti-Aα509-610) and ІІ-5С (anti-Aα20-78) antibody. MALDI-TOF analysis of fibrinogen hydrolysis products was performed using a Voyager-DE. Results. Combination of SDS-PAGE, FPLC and MALDI-TOF analysis enabled to detect the peptide bonds cleaved by studied proteases. In particular proteases from Gloydius halys halys and Agkistrodon contortrix contortrix snake venoms cleaved peptide bond Aα413-414. Action of protease from Calloselasma rhodostoma rhodostoma on fibrinogen led to the formation of hydrolytic product generated from C-terminal portion of Aα-chain that corresponded to fragments generated by enzymes from two other snakes. On the other hand protease from Bacillus thuringiensis вар. israelensis IMV B-7465 culture medium cleaved peptide bond Aα504-505. Conclusions. Use of limited proteolysis technique as the source of additional information for computer modeling allowed us to propose an improved model of 3D-structure of fibrinogen αC-regions. This model takes into account the behavior of αC-regions in the physiological condition and contributes to the general knowledge about fibrinogen structure.

Prognostic accuracy of MALDI-TOF mass spectrometric analysis of plasma in COVID-19.

SARS-CoV-2 infection poses a global health crisis. In parallel with the ongoing world effort to identify therapeutic solutions, there is a critical need for improvement in the prognosis of COVID-19. Here, we report plasma proteome fingerprinting that predict high (hospitalized) and low-risk (outpatients) cases of COVID-19 identified by a platform that combines machine learning with matrix-assisted laser desorption ionization mass spectrometry analysis. Sample preparation, MS, and data analysis parameters were optimized to achieve an overall accuracy of 92%, sensitivity of 93%, and specificity of 92% in dataset without feature selection. We identified two distinct regions in the MALDI-TOF profile belonging to the same proteoforms. A combination of SDS-PAGE and quantitative bottom-up proteomic analysis allowed the identification of intact and truncated forms of serum amyloid A-1 and A-2 proteins, both already described as biomarkers for viral infections in the acute phase. Unbiased discrimination of high- and low-risk COVID-19 patients using a technology that is currently in clinical use may have a prompt application in the noninvasive prognosis of COVID-19. Further validation will consolidate its clinical utility.

A Unique Relative of Rotifer Birnavirus Isolated from Australian Mosquitoes.

The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. Using a broad-spectrum system based on detection of long double-stranded RNA, we have discovered and isolated a birnavirus from Aedes notoscriptus mosquitoes collected in northern New South Wales, Australia. Phylogenetic analysis of Aedes birnavirus (ABV) showed that it is related to Rotifer birnavirus, a pathogen of microscopic aquatic animals. In vitro cell infection assays revealed that while ABV can replicate in Aedes-derived cell lines, the virus does not replicate in vertebrate cells and displays only limited replication in Culex- and Anopheles-derived cells. A combination of SDS-PAGE and mass spectrometry analysis suggested that the ABV capsid precursor protein (pVP2) is larger than that of other birnaviruses and is partially resistant to trypsin digestion. Reactivity patterns of ABV-specific polyclonal and monoclonal antibodies indicate that the neutralizing epitopes of ABV are SDS sensitive. Our characterization shows that ABV displays a number of properties making it a unique member of the Birnaviridae and represents the first birnavirus to be isolated from Australian mosquitoes.

Cellular Synthesis and X-ray Crystal Structure of a Designed Protein Heterocatenane.

Herein, we report the biosynthesis of protein heterocatenanes using a programmed sequence of multiple post-translational processing events including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The approach is general, autonomous, and can obviate the need for any additional enzymes. The catenane topology was convincingly proven using a combination of SDS-PAGE, LC-MS, size exclusion chromatography, controlled proteolytic digestion, and protein crystallography. The X-ray crystal structure clearly shows two mechanically interlocked protein rings with intact folded domains. It opens new avenues in the nascent field of protein-topology engineering.

Yüksek dizi kapsama alanı ile fare beyin zarlarından VDAC (voltaj bağımlı anyon kanalı) kütle spektrometrik haritalama.

VDACs (voltage dependent anion channels) are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through mitochondrial membranes. The functional role of VDAC has been investigated in many studies, and different functions of VDAC have been shown. Posttranslational modifications of VDAC are significant for its regulation. The aim of our research was to develop approach for characterization of primary structure and posttranslational modifications of VDACs and other membrane proteins. Mouse brain membranes were isolated from mouse brains by differential centrifugation. Primary structure of mitochondrial isoform VDAC1 from mouse brain membranes has been identified almost completely (95 %, 258 of 283 amino acids) by combination of SDS-PAGE and LTQ-FTMS mapping of peptide mixtures after proteolytic degradation with trypsin. Sequence of each found peptide of VDAC has been analysed and confirmed according to accurate mass, isotopic distribution and MS/MS tandem analysis. Posttranslational modifications of VDAC’speptides have been shown. High sequence coverage of VDAC has been obtained, including 11 transmembranes domains. Extensive sequence coverage has been also detected for some other proteins at 30-34 kDa. A repressor of estrogen receptor activity has been identified with 76 % coverage, malate dehydrogenase with 55 % sequence coverage, syntaxin 1A and syntaxin 1B2 have been sequenced with 60 % and 65 % coverage, respectively. These results demonstrated that mass spectrometric mapping is reliable and sensitive approach for characterization of primary structure membrane proteins and identification of their posttranslational modifications.

Differentially expressed proteins are caused by increased chemokine ligand 2 in mice hippocampi after alcohol treatment

When alcohol enters the central nervous system (CNS) it disrupts the physiological balance, producing several negative effects on the nervous system, specifically learning and memory. It has been shown that the neuroimmune protein Chemokine (CC motif) Ligand 2 (CCL2), perturbs the negative effects alcohol has on the hippocampus, the region of the brain associated with learning and memory. CCL2 transgenic mice that overexpress this protein have been used to study the phenotypical and behavioral effects of alcohol use. Although the signaling cascade initiated by CCL2 is currently unknown, identification of proteins responsible for these effects is possible through the combination of SDS‐PAGE and high‐resolution mass spectrometry. Using these techniques, a molecular fingerprint was produced by identification of differentially expressed proteins located in hippocampus of CCL2‐transgenic mice when compared to wild type mice.Over a time course of ten days mice of varying ages consumed 1g/kg of 20% ethanol every day. After the alcohol treatment, the left‐hemisphere hippocampus was extracted for proteomic analysis and the right‐hemisphere hippocampus was used for quantification of CCL2 concentration. This ensured identical biological samples for cross‐comparison between CCL2 expression and the effects this had on the proteome of the hippocampus. ELISA was used to quantify CCL2 concentration. Proteins from the left hemisphere were extracted and subsequently resolved using large format SDS‐PAGE analysis. Differentially expressed proteins were identified by Quadropole‐Time of Flight tandem mass spectrometry. Identification of proteins was done using a combination of MassHunter Qualitative, ProFinder, and NCBI databases.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Brochosomins and other novel proteins from brochosomes of leafhoppers (Insecta, Hemiptera, Cicadellidae)

Brochosomes (BS) are secretory granules resembling buckyballs, produced intracellularly in specialized glandular segments of the Malpighian tubules and forming superhydrophobic coatings on the integuments of leafhoppers (Hemiptera, Cicadellidae). Their composition is poorly known. Using a combination of SDS-PAGE, LC-MS/MS, next-generation sequencing (RNAseq) and bioinformatics we demonstrate that the major structural component of BS of the leafhopper Graphocephala fennahi Young is a novel family of 21–40-kDa secretory proteins, referred to herein as brochosomins (BSM), apparently cross-linked by disulfide bonds. At least 28 paralogous BSM were identified in a transcriptome assembly of this species, most of which were detected in BS. Multiple additional BS-associated proteins (BSAP), possibly loosely attached to the outer and inner surfaces of BS, were also identified; some of these were glycine-, tyrosine- and proline-rich. BSM and BSAP together accounted for half of the 100 most expressed transcripts in the Malpighian tubules of G. fennahi. Except for several minor BSAP possibly related to cyclases, BSM and BSAP had no homologs among known proteins, thus representing taxonomically restricted gene families (orphans). Searching in 50 whole-body transcriptome assemblies of Hemiptera found homologs of BSM in representatives of all five families of the superfamily Membracoidea (Cicadellidae, Myerslopiidae, Aetalionidae, Membracidae, and Melizoderidae), but not in other lineages. Among the identified proteins only BSM were shared in common between all 17 surveyed leafhoppers known to produce BS. Combined CHN elemental and aminoacid analyses estimated the total protein content of BS from the integument of G. fennahi to be 60–70%.

English

Seed proteins have been evaluated for their nutritive value and biological activity. Proteases and proteinase inhibitors have been of immense benefit to both agriculture and therapeutic purposes. The seed proteins of Blighia sapida was evaluated for both structure and biological activity in this study. The matured dried seeds were pulverized and sequentially extracted using 10 mM Tris/HCl pH 7.4, 10 mM ammonium acetate and 10 mM sulphuric acid. Crude protein extracts were concentrated and the protein concentrations were estimated. Proteins were purified by 70% ammonium sulphate precipitation, Sephadex G50 reversed phase chromatography and finally by HPLC on a C18 column. Two bands were obtained from SDS-PAGE electrophoresis and they were identified by ESI/MS using in gel tryptic digestion. The seed protein from B. Sapida consists of two single polypeptide chains each with mass of about 24 to 27 KDa as established by a combination of SDS-PAGE and ESI/MS. Proteins exhibited protease activity, which was confirmed by zymography. Protease activity was characterized for effect of temperature, pH, divalent metal ions and chelating agents. The crude protein from the seed of B. sapida showed antimicrobial activity and the antifungal activity was comparable with the reference drug, Ticonazole. Keywords: Blighia sapida, chromatography, protease activity, in-gel trypsin digestion/mass spectrophotometry, antimicrobial activity. African Journal of Biotechnology , Vol 13(29) 2996-3007

Microbial population dynamics during spontaneous fermentation of Asparagus officinalis L. young sprouts

The aim of the present study was to assess the spontaneous fermentation of Asparagus officinalis L. young sprouts. For this purpose, asparagus fermentation was performed according to a traditional procedure originating from northern Greece. Young asparagus sprouts were cut, submerged in a brine solution (8 % NaCl) and placed in room temperature for fermentation to occur. Fermentation was monitored by measuring pH and total titratable acidity values as well as by qualitative and quantitative assessment of the microbiota dynamics. The latter was performed by classical microbiological techniques; clustering was performed by SDS-PAGE of whole cell proteins and rep-PCR and identification by sequencing of the 16S-rRNA gene. A culture-independent approach [PCR-denaturing gradient gel electrophoresis (DGGE)] was also applied in order to obtain a more integrated view of the microbiota dynamics. Lactic acid bacteria prevailed the fermentation forming a microbial consortium that was stable at species level; Lactobacillus sakei and Enterococcus faecium dominated until the 5th day while Weissella viridescens and W. cibaria dominated from the 7th day until the end of fermentation. Combination of SDS-PAGE with rep-PCR resulted in a very efficient clustering and differentiation also at sub-species level, revealing a succession at that level of all species throughout fermentation. On the other hand, application of PCR-DGGE was of limited usefulness.

Development and Validation of MRM Methods to Quantify Protein Isoforms of Polyphenol Oxidase in Loquat Fruits

Multiple reaction monitoring (MRM) is emerging as a promising technique for the detection and quantification of protein biomarkers in complex biological samples. Compared to Western blotting or enzyme assays, its high sensitivity, specificity, accuracy, assay speed, and sample throughput represent a clear advantage for being the approach of choice for the analysis of proteins. MRM assays are capable of detecting and quantifying proteolytic peptides differing in mass unique to particular proteins, that is, proteotypic peptides, through which different protein isoforms can be distinguished. We have focused on polyphenol oxidase (PPO), a plant conspicuous enzyme encoded by a multigenic family in loquat (Eriobotrya japonica Lindl.) and other related species. PPO is responsible for both the protection of plants from biotic stress as a feeding deterrent for herbivore insects and the enzymatic browning of fruits and vegetables. The latter makes fruit more attractive to seed dispersal agents but is also a major cause of important economic losses in agriculture and food industry. An adequate management of PPO at plant breeding level would maximize the benefits and minimize the disadvantages of this enzyme, but it would require a precise knowledge of the biological role played by each isoform in the plant. Thus, for the functional study of the PPOs, we have cloned and overexpressed fragments of three PPO isoforms from loquat to develop MRM-based methods for the quantification of each isoform. The method was developed using an ion trap instrument and validated in a QQQ instrument. It resulted in the selection of at least two peptides for each isoform that can be monitored by at least three transitions. A combination of SDS-PAGE and MRM lead to detect two out of three monitored isoforms in different gel bands corresponding to different processing stages of PPO. The method was applied to determine the amount of the PPO2 isoform in protein extracts from fruit samples using external calibrants.

Overview of Study on <i>Bacillus subtilis</i> Spores

This review documents my research for the past 29 years in the work of bacterial sporulation. The Gram-positive bacterium Bacillus subtilis forms spores when conditions are unsuitable for growth. The mature spores remain for long periods of starvation and are resistant to harsh environment. This property is attributed mainly to the unique figures of spore's outer layers, spore coat. The protein composition of the spores was comprehensively analyzed by a combination of SDS-PAGE and LC-MS/MS. The total of 154 proteins were identified and 69 of them were novel. The expression of the genes encoding them was dependent on sporulation-specific sigma factors, σF, σE, σG and σK. The expression of a coat protein gene, cotS, was dependent on σK and GerE. CotE is essential for the assembly of CotS in the coat layer. Many coat genes were identified by reverse genetics and the regulation of the gene expression was studied in detail. Some cot genes are functioned in the resistance to heat and lysozyme, and some of the coat proteins are involved in the specificity of germinants. The yrbA is essential in spore development, yrbA deficient cells revealed abnormal figures of spore coat structure and changed the response to germinants. The location of 16 coat proteins was determined by the observation of fluorescence microscopy using fluorescence-labelled proteins. One protein was assigned to the cortex, nine to the inner coat, and four to the outer coat. In addition, CotZ and CgeA appeared in the outermost layer of the spore coat.

Proteome profiling of peripheral mononuclear cells from human blood

Peripheral blood mononuclear cells (MNCs) are accessible through blood collection and represent a useful source for investigations on disease mechanisms and treatment response. Aiming to build a reference proteome database, we generated three proteome data sets from MNCs using a combination of SDS-PAGE and nanoflow LC-MS. Experiments were performed in triplicates and 514 unique proteins were identified by at least two non-redundant peptides with 95% confidence for all replicates. Identified proteins are associated with a range of dermatologic, inflammatory and neurological conditions as well as molecular processes, such as free radical scavenging and cellular growth and proliferation. Mapping the MNC proteome provides a valuable resource for studies on disease pathogenesis and the identification of therapeutic targets.

Disulfide proteomics for identification of extracellular or secreted proteins

The combination of SDS-PAGE and MS is one of the most powerful and perhaps most frequently used gel-based proteomics approaches in protein identification. However, one drawback of this method is that separation takes place under denaturing and reducing (R) conditions and as a consequence, all proteins with identical apparent molecular mass (Mr) will run together. Therefore, low-abundant proteins may not be easily identified. Another way of investigating proteins by proteomics is by analyzing subproteomes from a total proteome such as phosphoproteomics, glycoproteomics, or disulfide proteomics. Here, we took advantage of the property of secreted proteins to form disulfide bridges and investigated disulfide-linked proteins, using SDS-PAGE under nonreducing (NR) conditions. We separated sera from normal subjects and from patients with various diseases by SDS-PAGE (NR) and (R) conditions, followed by LC-MS/MS analysis. Although we did not see any detectable difference between the sera separated by SDS-PAGE(R), we could easily identify the disulfide-linked proteins separated by SDS-PAGE (NR). LC-MS/MS analysis of the disulfide-linked proteins correctly identified haptoglobin (Hp), a disulfide-linked protein usually found as a heterotetramer or as a disulfide-linked heteropolymer. Western blotting under NR and R conditions using anti-Hp antibodies confirmed the LC-MS/MS experiments and further confirmed that upon reduction, the disulfide-linked Hp heterotetramers and polymers were no longer disulfide-linked polymers. These data suggest that simply by separating samples on SDS-PAGEunder NR conditions, a different, new proteomics subset can be revealed and then identified.

Secretome Survey of Human Plexiform Neurofibroma Derived Schwann Cells Reveals a Secreted form of the RARRES1 Protein

To bring insights into neurofibroma biochemistry, a comprehensive secretome analysis was performed on cultured human primary Schwann cells isolated from surgically resected plexiform neurofibroma and from normal nerve tissue. Using a combination of SDS-PAGE and high precision LC-MS/MS, 907 proteins were confidently identified in the conditioned media of Schwann cell cultures combined. Label free proteome profiling revealed consistent release of high levels of 22 proteins by the four biological replicates of NF1 Schwann cell cultures relative to the two normal Schwann cell cultures. Inversely, 9 proteins displayed decreased levels in the conditioned media of NF1 relative to normal Schwann cells. The proteins with increased levels included proteins involved in cell growth, angiogenesis and complement pathway while proteins with decreased levels included those involved in cell adhesion, plasminogen pathway and extracellular matrix remodeling. Retinoic acid receptor responder protein-1 (RARRES1), previously described as an integral membrane tumor suppressor, was found exclusively secreted by NF1 Schwann cells but not by normal Schwann cells. All-trans retinoic acid modulated secretion of RARRES1 in a dose dependent manner. This study shows altered secretion of key proteins in NF1 derived Schwann cells. The potential implication of these proteins in neurofibroma biology is discussed.

Functional Interplay between Caspase Cleavage and Phosphorylation Sculpts the Apoptotic Proteome

Caspase proteases are principal mediators of apoptosis, where they cleave hundreds of proteins. Phosphorylation also plays an important role in apoptosis, although the extent to which proteolytic and phosphorylation pathways crosstalk during programmed cell death remains poorly understood. Using a quantitative proteomic platform that integrates phosphorylation sites into the topographical maps of proteins, we identify a cohort of over 500 apoptosis-specific phosphorylation events and show that they are enriched on cleaved proteins and clustered around sites of caspase proteolysis. We find that caspase cleavage can expose new sites for phosphorylation, and, conversely, that phosphorylation at the +3 position of cleavage sites can directly promote substrate proteolysis by caspase-8. This study provides a global portrait of the apoptotic phosphoproteome, revealing heretofore unrecognized forms of functional crosstalk between phosphorylation and caspase proteolytic pathways that lead to enhanced rates of protein cleavage and the unveiling of new sites for phosphorylation.

Dimerization of the Transmembrane Domain of Human Tetherin in Membrane Mimetic Environments

Tetherin/Bst-2 is a cell surface protein that can act as a restriction factor against a number of enveloped viruses, including HIV-1. It acts by tethering new virus particles to the host cell membrane, promoting their internalization and degradation. Tetherin is a type II membrane protein, with an N-terminal transmembrane domain, an extracellular coiled-coil domain, and a C-terminal GPI anchor. This double membrane anchor is important for anti-HIV activity, as is dimerization of the coiled-coil domain, but despite recent crystal structures of the coiled-coil ectodomains of human and mouse tetherin, the topology of tetherin with respect to host and viral membranes has yet to be determined. The tetherin transmembrane domain is also thought to mediate interactions with the HIV-1 encoded integral membrane protein Vpu, which is an antagonist of tetherin, through direct binding to the transmembrane region of Vpu. Using a combination of SDS-PAGE, size exclusion chromatography, and pyrene excimer fluorescence, we show that in the absence of the coiled-coil domain the transmembrane domain of human tetherin forms parallel homodimers in membrane mimetic environments. Transmembrane domain dimerization does not require disulfide bond formation and is favored in TFE, SDS micelles, and POPC liposomes. This observation has implications for functional models of tetherin, suggesting that both transmembrane domains in the dimeric molecule are inserted into the same lipid bilayer, rather than into opposing membranes.

Purification and biochemical characterization of a Ca 2+ - independent, thermostable and acidophilic α-amylase from Bacillus sp. RM16

Bacillus sp. RM16 was isolated from a hot spring in Karachi and screened for the production of α- amylase. The enzyme was obtained after 72 h cultivation of strain in Luria broth containing 1% starch (w/v). Enzyme Amy RM16 was purified to electrophoretic homogeneity by a series of sequential steps including precipitation with ammonium sulfate at 70% saturation, Q-Sepharose, Phenyl Sepharose and reversed phase chromatography. The purified enzyme is made up of a single polypeptide chain of 66 kDa as established by a combination of SDS-PAGE and zymographic analysis. In our experimental conditions, a total yield of 1.35% with specific activity of 6380U/mg was obtained providing 17 fold final purification of the enzyme. Biochemical characterization of the Amy RM16 such as optimum temperature and pH, substrate specificity and enzymatic susceptibilities towards different metal ions and inhibitors were also performed. Results of these studies revealed that, the enzyme is active at wide temperature range with optimum activity at 80°C and retained 85% of the activity for 3 h at 50°C and around 50% of remaining activity for 1 h at 80°C. The enzyme showed optimum activity at pH 5.0. On the other hand, Ca +2 and EDTA (1 to 5 mM) did not significantly affect the enzyme activity. The main substrate for the enzyme was found to be starch but it could also hydrolyze raw starch, dextrin, γ-cyclodextrin and pullulan. Key words: Ca 2+ -independent, Bacillus sp, thermostable α-amylases, low pH profile, enzyme, raw starch digestion, HPLC.

Renal and Urinary Proteomics

Renal and Urinary Proteomics

The Subunit Composition of Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) From Pichia pastoris

Respiratory complex I (NADH:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. It is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. Here we present a comprehensive analysis of the protein composition and post-translational modifications of complex I from Pichia pastoris, using a combination of proteomic and bioinformatic approaches. Forty-one subunits were identified in P. pastoris complex I, comprising the 14 core (conserved) subunits and 27 supernumerary subunits; seven of the core subunits are mitochondrial encoded. Three of the supernumerary subunits (named NUSM, NUTM, and NUUM) have not been observed previously in any species of complex I. However, homologues to all three of them are present in either Yarrowia lipolytica or Pichia angusta complex I. P. pastoris complex I has 39 subunits in common with Y. lipolytica complex I, 37 in common with N. crassa complex I, and 35 in common with the bovine enzyme. The mitochondrial encoded subunits (translated by the mold mitochondrial genetic code) retain their N-α-formyl methionine residues. At least eight subunits are N-α-acetylated, but the N-terminal modifications of the nuclear encoded subunits are not well-conserved. A combination of two methods of protein separation (SDS-PAGE and HPLC) and three different mass spectrometry techniques (peptide mass fingerprinting, tandem MS and molecular mass measurements) were required to define the protein complement of P. pastoris complex I. This requirement highlights the need for inclusive and comprehensive strategies for the characterization of challenging membrane-bound protein complexes containing both hydrophilic and hydrophobic components.

Proteomic analysis of proteins expressing in regions of rat brain by a combination of SDS-PAGE with nano-liquid chromatography-quadrupole-time of flight tandem mass spectrometry

BackgroundMost biological functions controlled by the brain and their related disorders are closely associated with activation in specific regions of the brain. Neuroproteomics has been applied to the analysis of whole brain, and the general pattern of protein expression in all regions has been elucidated. However, the comprehensive proteome of each brain region remains unclear.ResultsIn this study, we carried out comparative proteomics of six regions of the adult rat brain: thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala using semi-quantitative analysis by Mascot Score of the identified proteins. In order to identify efficiently the proteins that are present in the brain, the proteins were separated by a combination of SDS-PAGE on a C18 column-equipped nano-liquid chromatograph, and analyzed by quadrupole-time of flight-tandem-mass spectrometry. The proteomic data show 2,909 peptides in the rat brain, with more than 200 identified as region-abundant proteins by semi-quantitative analysis. The regions containing the identified proteins are membrane (20.0%), cytoplasm (19.5%), mitochondrion (17.1%), cytoskeleton (8.2%), nucleus (4.7%), extracellular region (3.3%), and other (18.0%). Of the identified proteins, the expressions of glial fibrillary acidic protein, GABA transporter 3, Septin 5, heat shock protein 90, synaptotagmin, heat shock protein 70, and pyruvate kinase were confirmed by immunoblotting. We examined the distributions in rat brain of GABA transporter 3, glial fibrillary acidic protein, and heat shock protein 70 by immunohistochemistry, and found that the proteins are localized around the regions observed by proteomic analysis and immunoblotting. IPA analysis indicates that pathways closely related to the biological functions of each region may be activated in rat brain.ConclusionsThese observations indicate that proteomics in each region of adult rat brain may provide a novel way to elucidate biological actions associated with the activation of regions of the brain.