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Description


The HUPO Proteomics Standards Initiative (PSI) defines standards for data representation in proteomics to facilitate data exchange and comparison between experiments, and further promote quality assessment. In particular, the MIAPE documents specify the minimum information to report when describing or publishing a proteomics experiment.
MIAPEGelDB is intended to help end-users to write such MIAPE documents, through a self-documenting web interface. The documents produced remain easily accessible by anybody through a stable URL unique for each documents.
This project has been published in Journal of Proteomics 2008 71(2).

Coverage


Currently, MIAPE Gel Electrophoresis document is implemented ( version 1.4, published in Nature Biotechnology (2008) 26: 863-864).
The database currently contains 114 published gels in 42 published experiments.

Published experiments

Test experiment


Description

This experiment is only an example

Gel documents

[as_xml] [as_html] [as_text] Test gel

SWISS-2DPAGE reference map (analytical gel)


Description

protocol (analytical gel) for SWISS-2DPAGE reference map

Gel documents

[as_xml] [as_html] [as_text] Human plasma (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human liver (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human cerebrospinal fluid (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human kidney (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human colorectal epithelium cells (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human erythroleukemia cell line (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human hepatoblastoma carcinoma derived cell line (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human hepatoblastoma carcinoma derived cell line secreted proteins (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human lymphoma (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human red blood cells (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human promyelocytic leukemia cells (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human macrophage like cell line (Analytical 2-DE)
[as_xml] [as_html] [as_text] Human platelet (Analytical 2-DE)
[as_xml] [as_html] [as_text] Soluble nuclear proteins and matrix from human liver tissue (Analytical 2-DE)
[as_xml] [as_html] [as_text] Saccharomyces cerevisiae (Analytical 2-DE)
[as_xml] [as_html] [as_text] Escherichia coli (Analytical 2-DE)
[as_xml] [as_html] [as_text] Escherichia coli, pH 4-5 (Analytical 2-DE)
[as_xml] [as_html] [as_text] Escherichia coli, pH 4.5-5.5 (Analytical 2-DE)
[as_xml] [as_html] [as_text] Escherichia coli, pH 5-6 (Analytical 2-DE)
[as_xml] [as_html] [as_text] Escherichia coli, pH 6-9 (Analytical 2-DE)
[as_xml] [as_html] [as_text] Escherichia coli, pH 6-11 (Analytical 2-DE)
[as_xml] [as_html] [as_text] Escherichia coli, pH 5.5-6.7 (Analytical 2-DE)
[as_xml] [as_html] [as_text] Dictyostelium discoideum (Analytical 2-DE)
[as_xml] [as_html] [as_text] Staphylococcus aureus (Analytical 2-DE)
[as_xml] [as_html] [as_text] Arabidopsis thaliana (Analytical 2-DE)
[as_xml] [as_html] [as_text] Mouse liver (Analytical 2-DE)
[as_xml] [as_html] [as_text] Mouse pancreatic islet cells (Analytical 2-DE)
[as_xml] [as_html] [as_text] Mouse brown adipose tissue (Analytical 2-DE)
[as_xml] [as_html] [as_text] Mouse white adipose tissue (Analytical 2-DE)
[as_xml] [as_html] [as_text] Mouse liver nuclei (Analytical 2-DE)
[as_xml] [as_html] [as_text] Mouse gastrocnemius muscle (Analytical 2-DE)

Cytoplasmic proteome reference map from cells of Corynebacterium glutamicum ATCC1406


Description

Cytoplasmic proteome reference map for a glutamic acid-producing Corynebacterium glutamicum ATCC14067

Gel documents

[as_xml] [as_html] [as_text] Reference map for Corynebacterium glutamicum ATCC14067, pH 4.5-5.5
[as_xml] [as_html] [as_text] Reference map for Corynebacterium glutamicum ATCC14067, pH 5-6

Proteomic analysis of rat olfactory bulb


Description

To study the biochemistry of the olfactory bulb by using proteomic methods

Gel documents

[as_xml] [as_html] [as_text] Proteomic analysis of rat olfactory bulb

Apis mellifera workerbee hemolymph proteins


Description

2DE reference map of the proteins that are present in the hemolymph af Apis mellifera workerbees

Gel documents

[as_xml] [as_html] [as_text] Apis mellifera (workerbee) hemolymph

Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome


Description

Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis

Gel documents

[as_xml] [as_html] [as_text] PHENOL_2
[as_xml] [as_html] [as_text] TCA-acetone_3
[as_xml] [as_html] [as_text] TCA-acetone-phenol_3

Reference map of multiple myeloma cells


Description

A two-dimensional gel electrophoresis (2DE) reference map of human multiple myeloma (MM) proteome is described here. Spots of 517 corresponding to 268 different proteins were identified on 2DE gels from plasma cells isolated from eight newly diagnosed MM patients. Proteins were classified into different categories based on their molecular functions and biological processes. This 2DE map of MM proteins will be an invaluable tool for further proteomics research that investigates proteomic changes associated with biomarker identification and carcinogenesis analysis of multiple myeloma.

Gel documents

[as_xml] [as_html] [as_text] Reference map of multiple myeloma cells

Pseudomonas putida UW4 proteome reference map


Description

Construction of the proteome reference map of a plant growth-promoting bacterium Pseudomonas putida UW4 using 2-D gel and mass spectrometry.

Gel documents

[as_xml] [as_html] [as_text] Pseudomonas putida UW4 proteome reference map

Differential proteome analysis of C. elegans after infection with Aeromonas hydrophila


Description

A search for C. elegans proteins of which the expression level is altered after infection with the gram-negative bacterium Aeromonas hydrophila. As an immune response is a dynamic process we looked at three different time-points after infection (1, 3 and 5 days). We identified a total of 65 different C. elegans proteins, some of which had already been linked to immunity. In addition, we isolated and identified a number of unknown proteins not yet associated with the immune response. Finally, 3 Aeromonas proteins were identified.

Gel documents

[as_xml] [as_html] [as_text] C. elegans proteome after infection with Aeromonas hydrophila
[as_xml] [as_html] [as_text] C. elegans proteome after 1 day of infection with Aeromonas hydrophila
[as_xml] [as_html] [as_text] C. elegans proteome after 3 days of infection with Aeromonas hydrophila
[as_xml] [as_html] [as_text] C. elegans proteome after 5 days of infection with Aeromonas hydrophila

Analysis of bovine placenta


Description

1 Placental sample

We analyzed protein expression level of 3 normal placentae obtained by afterbirth of AI-derived fetuses Korean Native Cattle (280 day post-insemination). The normal placentae were obtained from Hankyong National University in March to June, 2002. Placental samples were stored in liquid nitrogen just after collection until use.

2 Extraction of solubilized proteins from the bovine placenta.

For 2-D PAGE, soluble proteins of placenta were extracted as previously described [15] with some modifications by the authors. Briefly, lysis buffer A (containing 1% SDS, 1 mM PMSF, protease inhibitor cocktail [complete; Roche], 100 mM Tris-HCl, pH 7.0) for pH 4-7 was applied to placenta with equal volume to tissue weight (ex. 100 µL buffer / 100 mg wet weight), and samples were sonicated for 15sec and then chilled in ice. Lysis buffer B (7 M urea, 2 M thiourea, 4% CHAPS, 0.1 M DTT, 1 mM PMSF, protease inhibitor, 40 mM Tris-HCl, pH7.0) was applied to the sonicated samples. The samples were shaken gently for 1hr at room temperature with gentle shaking. In the case of pH 6-9 range, lysis buffer C (0.3% SDS, 3% DTT, 1 mM PMSF, protease inhibitor, 500 mM Tris-HCl, pH8.0) was added to placenta with equal volume to tissue weight, and samples were suspended with sonication for 15sec, and then chilled in ice. Each insoluble material was removed by centrifugation (15 000 X g, 20 min, 4C). To eliminate the nucleic acid in the supernatant, endonuclease (Benzonase; Sigma) was treated with 100 units/mL for 1hr at 30C. The solubilized protein extracts were quantified by Bradford protein assay (Bio-Rad) and then final protein samples were stored at –70C.

3 Separation of proteins by 2-D gel electrophoresis

3.1 First dimension

In the case of pH 4-7, 1 mg of preparative protein samples was mixed with rehydration buffer (6 M urea, 2 M thiourea, 4% CHAPS, 0.4% DTT, 2% v/v IPG buffer pH 4-7) to total volume of 450 µL. A mixture of samples was loaded onto an IPG strips (pH 4-7; 180 x 3 x 0.5 mm). The pre-cast immobilized dry strips were allowed to rehydrate overnight in swelling tray. After rehydration, first dimensional isoelectric focusing (IEF) was performed on the strip in a Multiphor II IEF system (Amersham Pharmacia Biotech). Sample proteins were resolved using the ISO-DALT 2-D system (Amersham Pharmacia Biotech). Automatic isoelectric focusing was carried out for with 1.5 × 105 Vh. Voltage started at 100 V and gradually increased to a final voltage of 8000 V. After the first dimensional IEF, IPG gel strip were placed in an equilibration solution (6 M urea, 2% SDS, 50% v/v glycerol, 2.5% acrylamide, 1.875 M Tris-HCl, pH 8.8) containing 5 mM TBP for 20min with gentle shaking.

2-DE on alkaline IPG gels (pH 6-9) was performed according to standard protocols [16, 28-29] with modifications. Alkaline IPG strips were rehydrated overnight in modified buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2.5% DTT, 10% v/v isopropanol, 5% v/v glycerol, 2% v/v IPG buffer pH 6-11). Rehydration of individual IPG strips was either performed in the swelling tray without sample. After rehydration, first dimensional isoelectric focusing (IEF) was performed on the strip and cup in a Multiphor II IEF system (Amersham Pharmacia Biotech). One mg of preparative protein samples was mixed with modified buffer to a total volume of 110 µL. A mixture of sample was loaded onto a cup at the anodic side. At the cathodic side a “CleanGel” paper wick, immersed in modified buffer containing 0.4% DTT, was added. Sample proteins were resolved using the ISO-DALT 2-D system (Amersham Pharmacia Biotech). Automatic isoelectric focusing was carried out for with 1.9 × 105 Vh with starting at 50 V and gradually increasing to a final voltage of 8000 V. After the first dimensional IEF, IPG gel strip were equilibrated twice for 15 min each with gentle shaking in 10 mL of an equilibration solution. DTT (1% w/v) was added to the first equilibration step and iodoacetamide (4% w/v) was added to the second step.

3.2 Second dimension

The second dimensional separation was performed on 8-16% linear gradient SDS polyacrylamide gels. The gels were placed into an ISO-DALT system (Hoefer Scientific Instruments, San Francisco, CA, USA). The gels (200 × 250 × 1.0 mm) were run overnight at 10-15 mA per gel until the bromophenol blue marker dye had disappeared at the bottom of the gel.

4 Staining and image analysis of the 2-DE gels
After 2-DE, gels for Mass Spectrometry were stained with method using colloidal Coomassie brilliant blue (CBB) G-250. The gels were fixed for 1 hr in fixation solution (30% v/v methanol, 10% v/v acetic acid) and stained the gel with colloidal CBB G250 for 24hr followed by being destained with 1% acetic acid. The stained gels were scanned at an optical resolution of 63.5 µm/pixel using a GS-710 calibrated densitometer (Bio-Rad). The gels were analyzed by Melanie III software (Swiss Institute for Bioinformatics, Geneva, Switzerland). Experimental variations such as stain intensities were analyzed with the scatter analysis and a set of gel normalization was done. A restriction in volume value was imposed for selecting spots whose volume (V=OD x area) was higher than 200. These calculations were applied to the percent volume (%vol) parameter representative of the protein expression. Intensity (optical density) was measured by summing pixels within each spots boundary (spot volume) and recorded as a percentage of the total spot intensity on the gel: %vol (spot volume / ∑ volumes of all spots resolved in the gel). Variations in abundance were calculated as the ratio of average values (%vol) for a group between the two classes. The process for validation of variant proteins was carried out by human operators.

5 MALDI-TOF mass spectrometry analysis

5.1 Protein digestion for mass spectrometry

In-gel digestion was performed mainly as previously described with some modifications. For coomassie-stained proteins, the gel slab was destained by using 120 L wash solution (50% v/v acetonitrile, 25 mM NH4HCO3, pH 7.8). Then the gel pieces were dehydrated with 50 µL of acetonitrile and dried of for 30 min with a vacuum centrifuge. The dried gel pieces were rehydrated with 5 µL of trypsin solution (trypsin at a concentration of 0.0012 µg/µL in 25 mM NH4HCO3, pH 7.8). If needed, additional ammonium bicarbonate buffer was added to completely cover the gel pieces. The digestion was performed at 37C overnight. After completion of the digestion, the supernatant was transferred to another Eppendorf tube. To extract residual peptides, the gel pieces were sonicated for 20 min at 30C in a solution of 50% acetonitrile/0.5% TFA. Extracted peptides were used for MALDI-TOF analysis.

5.2 MALDI-TOF-MS peptide mass fingerprinting

Mass spectrometric analysis of peptide mass fingerprinting (PMF) was performed using a Voyager-DE STR MALDI-TOF-MS (PerSeptive Biosystems, Framingham, MA, USA). Approximately 1 L of extracted peptide solution from each gel spot piece and the same volume of matrix solution (10 mg/mL -ciano-4-hydroxycinnamic acid, 0.1% v/v TFA, and 50% v/v acetonitrile) were loaded onto a MALDI sample plate (96 well) and crystallized. For each sample, the average of 500 spectra was obtained and scans were performed twice. Spectra were calibrated upon acquisition automatically using an external 3-point calibration. Peak assignment was performed manually using DataExplorerTM software that is part of the Voyager-DE STR MALDI-TOF-MS software package (PerSeptive Biosystems, Framingham, MA, USA) and spectra were saved as peak table files (*.pkt) to search against non-redundant protein sequence database on the internet (SWISS-PROT, IPI COW and/or NCBInr (2001/10/01) Data Bank).

6 Identification of proteins by LC-MS/MS

The resulting tryptic peptides were separated and analyzed using reversed phase capillary HPLC directly coupled to a Finnigan LCQ ion trap mass spectrometer (LC-MS/MS) (Zuo et al, 2001, with a slight modification). Both of a 0.1 ⅹ 20 mm trapping and a 0.075 ⅹ 130mm resolving column were packed with Vydac 218 MS low trifluoroactic acid C18 beads (5㎛ in size, 300Å in pore size; Vydac, Hesperia, CA, USA) and placed in-line. Following the peptides were bound to the trapping column for 10 min at with 5% (v/v) aqueous acetonitrile containing 0.1% (v/v) formic acid, then the bound peptides were eluted with a 50-min gradient of 5– 80% (v/v) acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.2㎕/min. For tandem mass spectrometry, a full mass scan range mode was m/z = 450– 2000Da. After determination of the charge states of an ion on zoom scans, product ion spectra were acquired in MS/MS mode with relative collision energy of 55%.
The individual spectra from MS/MS were processed using the TurboSEQUEST software (Thermo Quest, San Jose, CA). The generated peak list files were used to query either MSDB database or NCBI using the MASCOT program (http://www.matrixscience.com). Modifications of methionine and cysteine, peptide mass tolerance at 2 Da, MS/MS ion mass tolerance at 0.8 Da, allowance of missed cleavage at 2, and charge states (1, 2, and 3) were taken into account. Only significant hits as defined by MASCOT probability analysis were considered initially.

Gel documents

[as_xml] [as_html] [as_text] pH 4-7 map of bovine placenta protein
[as_xml] [as_html] [as_text] pH 6-9 map of bovine placenta protein

Phakopsora pachyrhizi germinating urediniospore 2-D gel


Description

Partial proteome map by 2D gel for Phakopsora pachyrhizi germinating urediniospores.

Gel documents

[as_xml] [as_html] [as_text] Phakopsora pachyrhizi germinating urediniospore 2-D gel

Proteomic analysis of rat cerebellum


Description

In this experiment we have made a two dimensional gel map of twenty days old rat cerebellum. Here we are providing the steps which we have followed for preparation of 2DE gels.

Gel documents

[as_xml] [as_html] [as_text] Proteomic analysis of rat cerebellum

A Comparative study on proteome of Brucella melitensisvaccine strain M 5 and a virulent strain,16M


Description

Brucella melitensis is a facultative intracellular bacterial pathogen causing causes brucellosis，a zoonotic disease primarily infecting sheep and goats，and characterized by undulant fever，arthritic pain and neurological disorders in humans．To understand the mechanism of virulence in B．melitensis，tWO dimension e1ectrophoresis was used tO separate the tota1 proteins of a vaccine strain M5 and a virulent strain 16M ，which were grown under identicallaboratory conditions．The proteome of vaccine strain M5 was computer—assisted analyzed and compared tO that of virulent strain 16M ．By means of matrix-assisted laser desorption／ionization time of flight mass spectrometry(MALDI—TOF-MS)，1 3 protein spots were identified．Peptide mass fingerprints were searched against the NCBInr database using the Program Mascot from Matrix Science．These proteins represent 8 discrete open reading frames(ORFs)．It was found that certain metabolic pathways may be altered in M5．involving alterations in expressions of 3-hydroxybutyrate dehydrogenase，4-hydroxybutyrate dehydrogenase，glycero1 trinitrate reductase,3-methyl一2一oxobutanoate dehydrogenase，sugar—binding protein，nickel—binding periplasmic protein precursor and modification of DNA protection during starvation protein，grpE protein was altered in M5．These findings provide new insight
for understanding M5 molecular attenuation mechanism．

Gel documents

[as_xml] [as_html] [as_text] Proteomes of laboratory-grown B．melitensis strains M5
[as_xml] [as_html] [as_text] Proteomes of laboratory-grown B．melitensis strains 16M

Identification of novel immunogenic proteins of Brucella melitensis M5 by immunoproteomic methodolog


Description

Brucella melitensis (B. melitensis) is the etiologic agent of sheep and goat brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. To date, no safe human vaccine is available against B. melitensis infection. Subunit vaccines of defined compositions based on purified protective antigens free of toxic components are likely to be favored. However, information regarding the profile of protective antigens of Brucella is not available yet. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. melitensis M5 which could be reacted with antisera produced by immunization of a whole bacterium. Eighty-eight protein spots (assigned to 65 proteins by MALDI-TOF-MS) from B. melitensis M5 were identified to be immunogenic by 2-DE immunoblotting. Of these 88 spots, 67 protein spots （53 proteins）were from the soluble proteome and 21 protein spots（12 proteins） were from the outer membrane. The elucidation of the immunome of B. melitensis identified a number of candidate proteins for developing vaccines against Brucella infection.

Gel documents

[as_xml] [as_html] [as_text] 2-DE and 2-DE Western blotting of soluble proteins of B. melitensis M5
[as_xml] [as_html] [as_text] 2-DE and 2-DE Western blotting of OMPs of B. melitensis M5

The proteome complement of Nicotiana tabacum BY2 cells


Description

To determine the repertoire of BY2 cell proteins, the soluble proteins from N. tabacum BY2 cells at the end of the exponential growth phase were isolated and analyzed by 2D gel electrophoresis followed by MALDI TOF/TOF identification.

Gel documents

[as_xml] [as_html] [as_text] gel448_BY2_ext_chloromethanol_1mg_A2

Differential proteome analysis of C. elegans after infection with Staphylococcus aureus


Description

The success of invertebrates throughout evolution is an excellent illustration of the efficiency of their defence strategies. An example of such a successful invertebrate is Caenorhabditis elegans. Over the years, this free-living ground nematode has proven to be a very useful model in the study of host-pathogen interactions. In this paper a proteomics approach was adopted to investigate the worm’s response at four time-points after infection with the gram-positive bacterium Staphylococcus aureus. This study resulted in the identification of 130 spots corresponding to 108 differentially expressed proteins.

Gel documents

[as_xml] [as_html] [as_text] C. elegans - S. aureus 1 hour
[as_xml] [as_html] [as_text] C. elegans - S. aureus 4 hours
[as_xml] [as_html] [as_text] C. elegans - S. aureus 8 hours
[as_xml] [as_html] [as_text] C. elegans - S. aureus 24 hours
[as_xml] [as_html] [as_text] C. elegans proteome after infection with Staphylococcus aureus

Aging Hippocampal Synaptoproteome


Description

In this study, hippocampal synaptosomes were isolated from Young-adult, Adult, and Aged rats, and alterations in the synaptoproteome were examined and identified by 2-DIGE and MS/MS.
This is a placeholder MIAPEGelDB file for this experiment. The MIAPEGelDB submission tools are inoperable. For the MIAPEGelDB data file please feel free to contact us at wfreeman@psu.edu.

Gel documents

[as_xml] [as_html] [as_text] Hippocampal synaptosome 2D gel

Basic proteins of the dlPFC region of the human brain


Description

To create a basic protein map (pH 6-11)of the dorso lateral prefrontal cortex (dlPFC) region of the human brain using 2-DE

Gel documents

[as_xml] [as_html] [as_text] Basic proteins of the dlPFC region of the human brain

Mining the vascular tissue proteome in AAAs


Description

Once thought to be a late consequence of atherosclerosis, it has become apparent that abdominal aortic aneurysms (AAAs) may be a local representation of a systemic disease of the vasculature. This experiment mined the tissue proteome of patients with AAAs to identify differential protein expression that may further elucidate the pathogeneisis of this disease. A comparative proteomic method was used on venous tissue (inferior mesenteric vein) harvested from AAA patients and matched controls (n=16 in each group).

Gel documents

[as_xml] [as_html] [as_text] Control Gel
[as_xml] [as_html] [as_text] AAA patients

HCR_LCR_Heart


Description

Difference in-gel electrophoresis of cardiac proteins from rats selected for either high- or low-running capacity.

Gel documents

[as_xml] [as_html] [as_text] HCR_LCR_Cardiac Map

Bee_Ovary_prot


Description

2D DIGE comparison of developed and rudimentary ovaries in honeybee workers. In addition a spot map.

Gel documents

[as_xml] [as_html] [as_text] Ovary workerbee proteome

Proteome changes in A549 cells following VOC exposure


Description

Styrene is a volatile organic compound that is widely used as an intermediate in many industrial settings. There are known adverse health effects at environmentally significant concentrations, but little is known about the molecular effect of exposure to styrene at sub-acute toxic concentrations. We exposed human lung epithelial cells, at a wide range of concentrations (1mg/m3–10 g/m3), to styrene and analyzed the effects on the proteome level by 2-DE, where 1380 proteins spots were detected and 266 were identified unambiguously by MS. A set of 16 protein spots were found to be significantly altered due to exposure to styrene at environmentally significant concentrations of 1–10mg/m3 (0.2–2.3 ppm).Treatment with styrene also caused the formation of styrene oxide–protein adducts, specifically for thioredoxin reductase 1. These results underline the relevance of oxidative stress as a primary molecular response mechanism of lung epithelial cells to styrene exposure at indoor-relevant concentrations.

Gel documents

[as_xml] [as_html] [as_text] Proteome of A549 cells

Kidney Glomeruli


Description

Differential proteome analysis of isolated kidney glomeruli of transgenic mice vs. wt controls.

Gel documents

[as_xml] [as_html] [as_text] GH vs wt
[as_xml] [as_html] [as_text] GIPRdn_4-7

Analysis of Pig oocyte


Description

1. Animals for oocyte
Ovaries from prepubertal gilts were collected at a local abattoir, and rinsed in PBS supplemented with 100 IU/ml penicillin and 50 µg/ml streptomycin. Ovaries were maintained at 37℃ during 1 to 2 h of transportation from the slaughterhouse to the laboratory.

2. Preparation of oocytes
Ovaries were obtained from prepubertal gilts at a local slaughter house and transported to laboratory in PBS solution supplemented with 100 IU/ml penicillin and 50 µg/ml streptomycin at 25-35℃. Cumulus-oocyte complexs (COCs) were collected from follicles with a diameter of 2-6 mm using 10-ml syringe fixed with an 18-gauge needle. COCs were washed three times in TL-Hepes containing 0.1% (w/v) polyvinyl alcohol (PVA).

3 Extraction of solubilized proteins from the bovine placenta.

For 2-D PAGE, soluble proteins of 12000 oocyte were extracted as previously described [15] with some modifications by the authors. Briefly, lysis buffer A (containing 1% SDS, 1 mM PMSF, protease inhibitor cocktail [complete; Roche], 100 mM Tris-HCl, pH 7.0) for pH 3-10 non-linear was applied to oocyte with equal volume to oocyte(ex. 100 µL buffer / 12000 oocyte), and samples were sonicated for 15sec and then chilled in ice. Lysis buffer B (7 M urea, 2 M thiourea, 4% CHAPS, 0.1 M DTT, 1 mM PMSF, protease inhibitor, 40 mM Tris-HCl, pH7.0) was applied to the sonicated samples. The samples were shaken gently for 1hr at room temperature with gentle shaking. Each insoluble material was removed by centrifugation (15 000 X g, 20 min, 4C). To eliminate the nucleic acid in the supernatant, endonuclease (Benzonase; Sigma) was treated with 100 units/mL for 1hr at 30C. The solubilized protein extracts were quantified by Bradford protein assay (Bio-Rad) and then final protein samples were stored at –70C.

4 Separation of proteins by 2-D gel electrophoresis

4.1 First dimension

In the case of pH 3-10, total protein (Approximately 1 mg) was mixed with rehydration buffer (6 M urea, 2 M thiourea, 4% CHAPS, 0.4% DTT, 2% v/v IPG buffer pH 3-10) to total volume of 450 µL. A mixture of samples was loaded onto an IPG strips (pH 3-10; 180 x 3 x 0.5 mm). The pre-cast immobilized dry strips were allowed to rehydrate overnight in swelling tray. After rehydration, first dimensional isoelectric focusing (IEF) was performed on the strip in a Multiphor II IEF system (Amersham Pharmacia Biotech). Sample proteins were resolved using the ISO-DALT 2-D system (Amersham Pharmacia Biotech). Automatic isoelectric focusing was carried out for with 1.5 × 105 Vh. Voltage started at 100 V and gradually increased to a final voltage of 8000 V. After the first dimensional IEF, IPG gel strip were placed in an equilibration solution (6 M urea, 2% SDS, 50% v/v glycerol, 2.5% acrylamide, 1.875 M Tris-HCl, pH 8.8) containing 5 mM TBP for 20min with gentle shaking.
4.2 Second dimension

The second dimensional separation was performed on 8-16% linear gradient SDS polyacrylamide gels. The gels were placed into an ISO-DALT system (Hoefer Scientific Instruments, San Francisco, CA, USA). The gels (200 × 250 × 1.0 mm) were run overnight at 10-15 mA per gel until the bromophenol blue marker dye had disappeared at the bottom of the gel.

5 Staining and image analysis of the 2-DE gels

After 2-DE, gels for Mass Spectrometry were stained with method using colloidal Coomassie brilliant blue (CBB) G-250. The gels were fixed for 1 hr in fixation solution (30% v/v methanol, 10% v/v acetic acid) and stained the gel with colloidal CBB G250 for 24hr followed by being destained with 1% acetic acid. The stained gels were scanned at an optical resolution. The gels were analyzed by Imagemaster 5.0 software (GE Healthcare Bio-Sciences, Uppsala, Sweden). Experimental variations such as stain intensities were analyzed with the scatter analysis and a set of gel normalization was done. A restriction in volume value was imposed for selecting spots whose volume (V=OD x area) was higher than 200. These calculations were applied to the percent volume (%vol) parameter representative of the protein expression. Intensity (optical density) was measured by summing pixels within each spots boundary (spot volume) and recorded as a percentage of the total spot intensity on the gel: %vol (spot volume / ∑ volumes of all spots resolved in the gel).

6 MALDI-TOF mass spectrometry analysis

6.1 Protein digestion for mass spectrometry

In-gel digestion was performed mainly as previously described with some modifications. For coomassie-stained proteins, the gel slab was destained by using 120 L wash solution (50% v/v acetonitrile, 25 mM NH4HCO3, pH 7.8). Then the gel pieces were dehydrated with 50 µL of acetonitrile and dried of for 30 min with a vacuum centrifuge. The dried gel pieces were rehydrated with 5 µL of trypsin solution (trypsin at a concentration of 0.0012 µg/µL in 25 mM NH4HCO3, pH 7.8). If needed, additional ammonium bicarbonate buffer was added to completely cover the gel pieces. The digestion was performed at 37C overnight. After completion of the digestion, the supernatant was transferred to another Eppendorf tube. To extract residual peptides, the gel pieces were sonicated for 20 min at 30C in a solution of 50% acetonitrile/0.5% TFA. Extracted peptides were used for MALDI-TOF analysis.

6.2 MALDI-TOF-MS peptide mass fingerprinting

Mass spectrometric analysis of peptide mass fingerprinting (PMF) was performed using a Voyager-DE STR MALDI-TOF-MS (PerSeptive Biosystems, Framingham, MA, USA). Approximately 1 L of extracted peptide solution from each gel spot piece and the same volume of matrix solution (10 mg/mL -ciano-4-hydroxycinnamic acid, 0.1% v/v TFA, and 50% v/v acetonitrile) were loaded onto a MALDI sample plate (96 well) and crystallized. For each sample, the average of 500 spectra was obtained and scans were performed twice. Spectra were calibrated upon acquisition automatically using an external 3-point calibration. Peak assignment was performed manually using DataExplorerTM software that is part of the Voyager-DE STR MALDI-TOF-MS software package (PerSeptive Biosystems, Framingham, MA, USA) and spectra were saved as peak table files (*.pkt) to search against non-redundant protein sequence database on the internet (SWISS-PROT, NCBInr (2010/05/01) Data Bank).

Gel documents

[as_xml] [as_html] [as_text] Analysis of Pig oocyte

Protochlamydia amoebophila UWE25 elementary body reference map


Description

The aim of this project was the construction of a 2-D proteome reference map of the infectious stage (the elementary bodies) of Protochlamydia amoebophila UWE25, an endosymbiont of Acanthamoeba spp. This reference map should facilitate further proteome analyses of P. amoebophila.

Gel documents

[as_xml] [as_html] [as_text] Protochlamydia amoebophila UWE25 elementary body reference proteome map

Analysis of Vigna mungo Cv T9 leaf proteome


Description

Analysis of Vigna mungo Cv T9 leaf proteome after Salicylic acid treatment

Gel documents

[as_xml] [as_html] [as_text] Proteomic analysis of salicylic acid treated Vigna mungo

Bee Hemolymph proteomics


Description

Hemolymph protein profiles of sterile and fertile honeybee workers in a queenless colony were compared by means of 2D-DIGE in order to reveal key molecular players in the repression of worker ovary activation

Gel documents

[as_xml] [as_html] [as_text] Bee Hemolymph proteomics

Basic proteins (pH 6-11) of human heart left ventricular tissue.


Description

Tissue lysate from the left ventricular region of the human heart was separated by 2-DE using pH 6-11 IPG strips. The basic proteins were visualised using an MS-compatible silver stain and were subsequently excised, digested and analysed by MS. Protein identifications were obtained by database searching. The aim of this experiment was to produce a 2-DE proteome map of the pH 6-11 region of the human heart to aid future 2-DE studies of human heart tissue and to complement previous 2-DE datasets.

Gel documents

[as_xml] [as_html] [as_text] 2-DE proteome map of the basic human heart proteins

Analysis of Caenorhabditis elegans antifungal response


Description

This set of experiments aims to characterise the protein changes that accompany infection of the nematode C. elegans by the fungus Drechmeria coniospora. This will help in the understanding of innate immune signalling.

Gel documents

[as_xml] [as_html] [as_text] f1INIp47g63261
[as_xml] [as_html] [as_text] f2INIp47g64149
[as_xml] [as_html] [as_text] f3inip47g64153
[as_xml] [as_html] [as_text] fnsinip47g63403
[as_xml] [as_html] [as_text] f1INIp310g63361
[as_xml] [as_html] [as_text] f2INIp310g58736
[as_xml] [as_html] [as_text] FNSinip310g63347
[as_xml] [as_html] [as_text] ftinip310g22125
[as_xml] [as_html] [as_text] FTinip47g63411

Proteome analysis of sperm from Mytilus edulis


Description

Characterisation of highly expressed proteins in sperm of Mytilus edulis

Gel documents

[as_xml] [as_html] [as_text] Mytilus edulis sperm

Lactobacillus acidophilus NCFM reference proteome


Description

Create a reference proteome of the whole cell extract of the probiotic Lactobacillus acidohilus NCFM

Gel documents

[as_xml] [as_html] [as_text] Lactobacillus acidophilus NCFM reference proteome (pI 6-11)

Analysis of leaf proteome of Vigna mungo


Description

Proteomic characterization of compatible and incompatible plant-virus interaction: A case study with Vigna mungo and Mungbean Yellow Mosaic India Virus

Gel documents

[as_xml] [as_html] [as_text] Analysis of leaf proteome of Vigna mungo (T9) control
[as_xml] [as_html] [as_text] Analysis of leaf proteome of Vigna mungo (T9) after MYMIV infection at 3 dpi
[as_xml] [as_html] [as_text] Analysis of leaf proteome of Vigna mungo (T9) after MYMIV infection at 7 dpi
[as_xml] [as_html] [as_text] Analysis of leaf proteome of Vigna mungo (T9) after MYMIV infection at 14 dpi
[as_xml] [as_html] [as_text] Analysis of leaf proteome of Vigna mungo (VM4) control
[as_xml] [as_html] [as_text] Analysis of leaf proteome of Vigna mungo (VM4) after MYMIV infection at 3 dpi
[as_xml] [as_html] [as_text] Analysis of leaf proteome of Vigna mungo (VM4) after MYMIV infection at 7 dpi
[as_xml] [as_html] [as_text] Analysis of leaf proteome of Vigna mungo (VM4) after MYMIV infection at 14 dpi

Flifactor alocis - total proteome


Description

Filifactor alocis, a Gram-positive anaerobic rod, is one of the most abundant bacteria identified in the periodontal pockets of periodontitis patients. There is a gap in our understanding of its pathogenicity and ability to interact with other periodontal pathogens. To evaluate the virulence potential of F. alocis and its ability to interact with Porphyromonas gingivalis W83, several clinical isolates of F. alocis were characterized. F. alocis showed nongingipain protease and sialidase activities. In silico analysis revealed the molecular relatedness of several virulence factors from F. alocis and P. gingivalis. In contrast to P. gingivalis, F. alocis was relatively resistant to oxidative stress and its growth was stimulated under those conditions. Biofilm formation was significantly increased in coculture. There was an increase in adherence and invasion of epithelial cells in coculture compared with P. gingivalis or F. alocis monocultures. In those epithelial cells, endocytic vesiclemediated internalization was observed only during coculture. The F. alocis clinical isolate had an increased invasive capacity in coculture with P. gingivalis compared to the ATCC 35896 strain. In addition, there was variation in the proteomes of the clinical isolates compared to the ATCC 35896 strain. Hypothetical proteins and those known to be important virulence factors in other bacteria were identified. These results indicate that F. alocis has virulence properties that may enhance its ability to survive and persist in the periodontal pocket and may play an important role in infection-induced periodontal disease.

Gel documents

[as_xml] [as_html] [as_text] Filifactor alocis-Total proteome
[as_xml] [as_html] [as_text]

Proteome reference maps for Xanthomonas oryzae pv. oryzae ZJ173


Description

Proteome reference maps of Xanthomonas oryzae pv. oryzae were constructed by two-dimensional gel electrophoresis, and 628 spots in the gels representing 469 different protein species were identified with MALDI-TOF-TOF MS.The identified proteins were assigned to different functional categories. Annotations of the 628 spots will greatly facilitate further comparative proteomic research of Xanthomonas oryzae pv. oryzae.

Gel documents

[as_xml] [as_html] [as_text] Proteome reference map for Xanthomonas oryzae pv. oryzae ZJ173 (pH 4-7)
[as_xml] [as_html] [as_text] Proteome reference map for Xanthomonas oryzae pv. oryzae ZJ173 (pH 3-10)

honeybee antenna


Description

Western Honeybee Drones and Workers (Apis mellifera ligustica) Have Stronger Olfactory Functions than Their Eastern Counterparts (Apis cerana cerana): A Proteome Study

Gel documents

[as_xml] [as_html] [as_text] honeybee antenna

Fusarium graminearum proteome reference map


Description

Construction of a proteome reference map of the fungi Fusarium graminearum strain 453. To create the map proteins were stained using cyanine dyes (2D-DiGE technique) and identifications were performed using a MALDI-Tof/Tof instrument.

Gel documents

[as_xml] [as_html] [as_text] Fusarium graminearum strain 453 reference map

Comparative 2-D gels of heat-stable proteins between Spartina pectinata and Spartina alterniflora


Description

Two-dimensional gels of heat-stable proteins from orthodox Spartina pectinata and recalcitrant Spartina alterniflora seeds were compared and computationally anazlyed to identify spots which are more abundant in orthodox S. pectinata than recalcitrant S. alterniflora. Those differentially expressed proteins in S. pectinata may be associated with seed desiccation tolerance.

Gel documents

[as_xml] [as_html] [as_text] Two-dimensional gel of Spartina pectinata
[as_xml] [as_html] [as_text] Two-dimensional gel of Spartina alterniflora

Analysis of low temperature stress modulted secretome of Hippophae rhamnoides


Description

Low temperture induced proteins were analyzed by 2-DE-nano-LC−MS/MS in shoot secretome of Hippophae rhamnoides (seabuckthorn). Seedlings were subjected to direct freezing stress (−5 °C), cold acclimation (CA), and subzero acclimation (SZA), and extracellular proteins (ECPs) were isolated using vacuum infiltration. Approximately 245 spots were reproducibly detected in 2-DE gels of LT treated secretome, out of which 61 were LT responsive. Functional categorization of 34 upregulated proteins showed 47% signaling, redox regulated, and defense associated proteins.

Gel documents

[as_xml] [as_html] [as_text] Analysis of freezing stress modulated secretome of Hippophae rhamnoides

Proteomic effects of membrane GR activation


Description

T-lymphoma cells (CCRF-CEM) were stimulated for 5, 15 or 90 minutes with BSA-conjugated Cortisol to specifically activate the membrane-bound isoform of the Glucocorticoid Receptor (mGR). Nuclear and cytosolic fractions were analyzed separately in order to evaluate eventual protein translocation. The aim of the study is to identify mGR downstream effectors.

Gel documents

[as_xml] [as_html] [as_text] CCB0_vs_CCB15_repl1
[as_xml] [as_html] [as_text] CCB0_vs_CCB15_repl2
[as_xml] [as_html] [as_text] CCB0_vs_CCB15_repl3
[as_xml] [as_html] [as_text] CCB5_vs_CCB90_repl1
[as_xml] [as_html] [as_text] CCB5_vs_CCB90_repl2
[as_xml] [as_html] [as_text] CCB5_vs_CCB90_repl3
[as_xml] [as_html] [as_text] NCB0_vs_NCB15_repl1
[as_xml] [as_html] [as_text] NCB0_vs_NCB15_repl2
[as_xml] [as_html] [as_text] NCB0_vs_NCB15_repl3
[as_xml] [as_html] [as_text] NCB5_vs_NCB90_repl1
[as_xml] [as_html] [as_text] NCB5_vs_NCB90_repl2
[as_xml] [as_html] [as_text] NCB5_vs_NCB90_repl3