Electrophoresis, Gel, Two-Dimensional ; methods ; Humans ; Proteomics ; methods ; Urinalysis ; methods

In this study,the protein in different Cordyceps samples,which include fresh sample( S1),22 ℃ drying sample( S2),37 ℃ drying sample( S3) and 60 ℃ drying sample( S4),were analyzed by sodium dodecylsupinate-polyacrylamide gel electrophoresis( SDS-PAGE) and two-dimensional electrophoresis( 2-DE). The total protein contents in Cordyceps samples were from 1. 655-4. 493 mg·g~(-1) and the protein contents in fresh sample was the highest. The results of SDS-PAGE showed that the mainly ranges of protein molecular weight of Cordyces samples were 10-100 kDa and the numbers of protein bands were 28 to 41,the fresh sample had the maximum number of protein bands. The 2-DE profiles were analyzed by PDQuest software. The resulted indicated that 488-876 protein spots were detected in different Cordyceps samples and the isoelectric point( pI) was distributed between 4. 5 and 6. 5,the protein molecular weight was distributed in 10-20 kDa and 25-100 kDa,the fresh sample had the maximum number of protein spots. Therefore,the drying process could decrease contents and species of protein in Cordyceps,and the different drying conditions had different effects on protein. These results provide a reference for improving the drying process of Cordyceps.
Cordyceps ; chemistry ; Desiccation ; methods ; Electrophoresis, Gel, Two-Dimensional ; Electrophoresis, Polyacrylamide Gel ; Fungal Proteins ; analysis ; Molecular Weight

OBJECTIVETo develop a method for extracting cytoskeletons and cytoskeleton-associated proteins for proteomic analysis.

METHODSA subcellular sequential proteome extraction method was exploited. The extraction procedure was optimized and controlled according to observed cell morphology changes and one- and two-dimensional electrophoresis images. The extraction efficiency and selectivity were evaluated by Western blotting and mass spectrometry.

RESULTSFour extracted fractions clearly displayed distinct patterns. Western blotting detected the fraction-marker proteins FAK, integrin-β1, histone H1 and cytokeratin 19 only in their expected fractions. About 90% of the protein spots in the cytoskeleton fraction were identified by mass spectrometry as cytoskeleton and/or its associated proteins.

CONCLUSIONThe subcellular proteome sequential fractionation method facilitates the detection of proteins of low abundance and shows a high reproducibility and selectivity, and thus can serve as an ideal pre-fractionation method prior to two-dimensional electrophoresis.

Cytoskeleton ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; methods ; Humans ; Proteome ; analysis ; Proteomics ; methods ; Subcellular Fractions

The aim of this study was to establish and optimize two-dimensional electrophoresis method for human bone marrow leukemia cells in order to obtain the profiles with high resolution and reproducibility. The total protein was extracted and separated by isoelectric focusing (IEF) and SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with silver nitrate or Coomassie brilliant blue, and then scanned and analyzed with PDQuest 7.4 analysis software. The effects of different protein preparation methods and electrophoresis conditions on the profiles were compared. The results indicated that by optimizing preparation of protein sample and electrophoresis protocols, clear profiles with 780 +/- 73 well separated protein spots on an average were obtained and the match rate was 82 +/- 5% between reproducible gels from leukemia cells of different sub-type. It is concluded that the two-dimensional electrophoresis method of proteome from human bone marrow leukemia cells is established successfully and is suitable for the further comparative proteomic research between leukemia of different types.
Bone Marrow Cells ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; methods ; Humans ; Leukemia ; metabolism ; Proteome ; analysis

OBJECTIVETo establish an effective separation system of 2-DE for the proteome of caudal gland, and provide foundation for revealing the mechanisms of histological development and pharmacological activities.

METHODThe total proteins of caudal gland were extracted by TCA/acetone precipitation, phenol extraction/methanol-ammonium acetate precipitation and trizol-base method respectively and separated by immobilized pH gradient (IPG) strips prior to SDS-PAGE. Loading protein sample size and isoelectric focusing conditions were optimized. The gels were stained with Coomassie brilliant blue, scanned and then analyzed using PDQuest 8.0 analysis software.

RESULTThe total proteins of caudal gland extracted by trizol-base method were the highest quality and could meet the needs of 2-DE. With 300 microg of proteins loaded on 7 cm pH 3-10 IPG strip followed by isoelectric focusing program II ,a satisfying 2-DE profiles were obtained. The total number of disticted protein spots was 209 with the optimized system.

CONCLUSIONA well-resolved 2-DE patterns of caudal gland were obtained by this optimized system. This method could be applied to prepare other similar tissue sample and 2-DE studies.

Animals ; Deer ; Electrophoresis, Gel, Two-Dimensional ; methods ; Proteins ; chemistry ; Scent Glands ; chemistry

OBJECTIVETo search the marker proteins of Guillain-Barré syndrome (GBS)-associated Campylobacter jejuni (C. jejuni) by comparing the protein maps of GBS-associated C. jejuni strains with that of non-GBS-associated C. jejuni strains.

METHODSThe whole-cell proteins of eight GBS-associated and eight non-GBS-associated C. jejuni strains were separated using the two-dimensional gel electrophoresis respectively. The differentially expressed proteins between the two sets of strains were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) after in-gel tryptic digestion.

RESULTSTwenty differentially expressed spots were found with seventeen identified ones using MSCOT database. These proteins were identified as wlaX protein and some other proteins involving in energy metabolism (malate dehydrogenase, triosephosphate isomerase, Ni/Fe-hydrogenase small chain, cysteine synthase, branched-chain amino acid aminotransferase), cell process (heat shock protein, iron-uptake ABC transport system periplasmic iron-binding protein, alkyl hydroperoxide reductase), cell envelope (flagellin, UDP-N-acetylenolpyruvoylglucosamine reductase) etc.

CONCLUSIONWlaX proteins were probably associated with LPS biosynthesis or virulence of C. jejuni. WlaX protein and flagellin protein were the possible marker-proteins of GBS-associated C. jejuni strains.

Campylobacter jejuni ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; methods ; Feces ; microbiology ; Guillain-Barre Syndrome ; microbiology ; Humans ; Proteomics

OBJECTIVETo optimize a pretreatment method of urine proteomics in children with primary nephrotic syndrome.

METHODSUrine from children with primary nephrotic syndrome was treated in different pH and isolated by cold acetone precipitation for different durations. Then the amounts and kinds of proteins were compared by quantify, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) in order to optimize a way to deal with urine protein.

RESULTSMost proteins were obtained at pH 2.7. The amounts of protein precipitated by acetone for 0.5 hr was obviously less than those precipitated for 1 and 2 hrs (P<0.05), while there was no significant difference between the amount of protein precipitated for 1 and for 2 hrs. Protein precipitated by cold acetone for 1 hr at pH 2.7 was selected as the best pretreatment method. Satisfactory 2-DE maps can be acquired.

CONCLUSIONSUrine protein can be best obtained at pH 2.7 and precipitated by cold acetone for 1 hr.

Child ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Hydrogen-Ion Concentration ; Nephrotic Syndrome ; urine ; Proteinuria ; urine ; Proteomics ; methods

OBJECTIVETo establish a urinary proteomic map on two-dimensional polyacrylamide gelelectrophoresis (2-DE) in children with idiopathic nephrotic syndrome (INS).

METHODSThe proteins from INS children were purified by four various means, separated by 2-DEand stained by silver.

RESULTSThe sequential preparation of urinary proteins by acetone precipitation and dislysis, when the sample was 300 microg, resulted in a clear background, well-resolved and reproducible 2-DE urinary protemic map in children with INS.

CONCLUSIONSA steady 2-DE technique for urinary protemic map in children with INS was established, which can be effectively applied in urinary proteomics of the disease.

Child ; Electrophoresis, Gel, Two-Dimensional ; Female ; Humans ; Male ; Nephrotic Syndrome ; urine ; Proteinuria ; urine ; Proteomics ; methods

OBJECTIVETo set up a rapid, efficient, reliable and accurate method for separation of Bacteriodes forsythus proteins.

METHODSBacteroides forsythus ATCC43037 cells were harvested by centrifugation, washed in Tris buffer to remove excess medium, and lysed by sonication. The sonicated lysis proteins were extracted step by step with ReadyPrep Sequential Extraction Kit (Bio-Rad). The supernatant of B. forsythus proteins were used for two-dimensional gel electrophoresis. The first dimension IEF (isoelectric focusing) was run with Immobiline DryStrip (pH 3-10) and the second dimension SDS-PAGE was run with Excelgel SDS, gradient 8-18 precast gel and buffer strips. The separated proteins were stained with Coomassie Brilliant Blue and silver staining kit (Plusone Silver Staining Kit, Protein, Pharmacia Biotech).

RESULTS1. The protein spots were clear when sample cups were used in the middle of IPG strip during IEF. 2. B. forsythus proteins were separated clearly by horizontal two-dimensional electrophoresis and most of B. forsythus whole-cell proteins were acidic proteins (P13-7).

CONCLUSIONHorizontal two-dimension electrophoresis is a useful method for separating B. forsythus proteins.

Bacterial Proteins ; isolation & purification ; Bacteroides ; isolation & purification ; Electrophoresis, Gel, Two-Dimensional ; methods

OBJECTIVETo optimize the method for preparing samples for amniotic fluid proteomics study.

METHODSPregnant rats were sacrificed with an overdose of Chloral hydrate at E17. The fetuses and amniotic fluid were harvested. The samples were processed by three different methods including trichloroacetic acid (TCA)-acetone precipitation (protocol 1), TCA-acetone precipitation combined with an Albumin and IgG Removal Kit (protocol 2), and a Centrifugal Filter concentrating combined with an Albumin and IgG Removal Kit (protocol 3). The samples were run through a two-dimensional electrophoresis gel, stained and analyzed with a Image Master 6.0 software. Protein spots were identified with a LCQ Deca XP mass spectrometer.

RESULTSThe total numbers of protein spots for samples processed by protocol 1, 2 and 3 were 253 ± 28, 749 ± 32 and 782 ± 27, respectively. And there was a significant difference between protocol 1 has and other two methods. Those with MW > 50 kDa were 57± 14, 45 ± 13 and 41 ± 14, respectively. Protocol 2 differed significantly from protocol 3. Protein number of samples with MW < 50 kDa was 196± 29, 702± 35 and 735 ± 29, respectively. Again, protocol 1 has differed significantly from other two methods.

CONCLUSIONBy removing albumin and IgG from the serum, low abundance proteins can be enriched with little loss of high abundance proteins. Centrifugal Filter concentrating combined with Albumin and IgG Removal Kit can be effectively applied for amniotic fluid proteomics study.

Amniotic Fluid ; metabolism ; Animals ; Electrophoresis, Gel, Two-Dimensional ; Female ; Pregnancy ; Proteome ; Proteomics ; methods ; Rats