Number Of Protein Spots Research Articles

Developing a reproducible method for the high-resolution separation of peritoneal dialysate proteins on 2-D gels

PurposeDespite recent progress in the proteomic analysis of peritoneal dialysate effluent (PDE), there remains unresolved problems in the development of an optimal sample preparation method. Experimental designWe examined five protocols for concentrating PDE proteins and the effects of immobilized pH gradient (IPG) strips with different pH ranges and sample loading techniques. In addition, we examined three kits for depleting high abundance proteins by SDS–PAGE and two-dimensional gel electrophoresis (2-DE). ResultsPDE proteins precipitated with 75% acetonitrile (ACN) showed the greatest number of protein spots by 2-DE, with over 800 distinct spots. Higher-resolution images were obtained using IPG strips with a pH range of 4–7. The ProteoPrep immunoaffinity albumin and IgG depletion kit removed high abundance proteins with higher efficiency and more compatibility with isoelectric focusing (IEF). Removing high abundance proteins also increased the resolution and improved the intensity of low abundance proteins. Conclusion and clinical relevanceHigh-resolution 2-DE images of PDE proteins were obtained by concentrating samples with 75% ACN, using pH 4–7 IPG strips, and depleting high abundance proteins. This optimized method will enable future studies to discover predictive biomarkers of disease in patients on dialysis.

The involvement of the mitochondrial peroxiredoxin PRXIIF in defining physiological differences between orthodox and recalcitrant seeds of two Acer species.

Norway maple (Acer platanoides L., orthodox) and sycamore (Acer pseudoplatanus L., recalcitrant) belong to the same genus and grow under similar climatic conditions, but their seeds differ in their tolerance to desiccation. The initial water content (WC) of the seeds used in this study was 50%, and they were dried to 40, 20 and 7%. The mitochondrial peroxiredoxin IIF (PRXIIF) was identified in seeds of both species by immunoblotting. Semiquantitative RT-PCR analyses indicated that the transcript level of PRXIIF in both types of seeds increased during different stages of desiccation and was higher in seeds of Norway maple than in sycamore. General proteome analyses showed important differences between orthodox and recalcitrant seeds. In sycamore seeds that had been desiccated to a 7% WC, the number of protein spots and the levels of those spots were lower than in desiccation-tolerant Norway maple seeds. Post-translational modifications of PRXIIF in seeds at a 50% WC were detected via 2D electrophoresis and subsequent western blot analysis. The detected shift in the pI values (± 0.3) in A. pseudoplatanus was possibly caused by phosphorylation because several potential phosphorylation sites were predicted in silico for that protein. The gene and amino acid sequences were obtained and aligned with known sequences of other plant PRXIIF genes and proteins. High values of sequence identity were noted between the PRXIIF protein sequences of Acer species, Populus trichocarpa Torr. & A. Gray and Arabidopsis thaliana (L.) Heynh. The involvement of PRXIIF in defining the physiological differences between desiccation-tolerant and desiccation-sensitive Acer seeds is discussed in the context of its role in mitochondrial redox homeostasis.

Comparison of different methods of rat amniotic fluid sample preparation for 2-d electrophoresis

To optimize the method for preparing samples for amniotic fluid proteomics study. Pregnant 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. The 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. By 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.

Expression and significance of 90K/Mac-2BP in prostate cancer

The aim of this study was to detect the differences in 90K/Mac-2BP expression in prostate cancer, benign prostatic hyperplasia and normal prostate tissues, as well as to study the significance of 90K/Mac-2BP in the early diagnosis and prognosis of prostate cancer. Comparative proteomic technologies were used in the present study. Total protein from 10 cases of prostate cancer, benign prostatic hyperplasia and normal prostate tissue was extracted and separated by two-dimensional electrophoresis (2-DE). Proteins expressed differentially by more than 2-fold were selected for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and biological information analysis. The 2-DE patterns of the proteins from the normal prostate, benign prostatic hyperplasia and prostate cancer tissues were successfully identified. The average numbers of protein spots were 3,066, 3,289 and 2,986, respectively. There were 31 spots with a difference of more than 2-fold. A total of 18 proteins were identified by MS and database searches. Of these 18 proteins, the most significant differential expression was that of 90K/Mac-2BP. Functional analysis demonstrated that 90K/Mac-2BP (Mac-2 binding protein) overexpression is correlated with the occurrence, proliferation, differentiation and metastasis of cancer cells. The proteomic approach used in the present study was effective and is feasible for identifying differentially expressed proteins in prostate cancer, benign prostatic hyperplasia and normal prostate tissues. 90K/Mac-2BP may be important for the early diagnosis and prognosis of prostate cancer and may also be associated with the molecular mechanisms of prostate cancer development.

Comparative proteomic analysis of casein and whey as prepared by chymosin-induced separation, isoelectric precipitation or ultracentrifugation

Fractionation of bovine milk was performed using chymosin-induced separation, isoelectric precipitation or ultracentrifugation as separation techniques prior to gel-based proteomic analysis. This approach allowed for comparative display and identification of proteins partitioned into casein and whey, respectively. Initially, three different staining methods (silver staining, colloidal Coomassie Blue G-250 or fluorescent Flamingo Pink staining) for two-dimensional gel electrophoresis (2-DGE) analysis were compared for their suitability as staining agent, especially in relation to their suitability to reveal differences in the casein fractions. Fluorescent staining proved to be the most appropriate for this purpose, giving a high sensitivity, and using this staining method, characteristic 2-DGE fingerprints were obtained for each casein and whey fraction from each separation method. A number of protein spots in both casein and whey fractions varied with separation method and these spots were subsequently identified using tandem mass spectrometry (MS). In rennet casein, proteolytic fragmentation of caseins (α(s1)-, α(s2),-, β- and κ-) was identified as a result of chymosin hydrolysis, whereas the 2-DGE profile of acid and ultracentrifuged casein was dominated by the presence of multiple isoforms of κ-caseins. Furthermore, casein remnants were identified in milk serum after ultracentrifugation. This study shows that gel-based proteomic analysis is suitable for characterisation of subtle variations in protein composition of milk fractions that occur as a consequence of different milk fractionation strategies.

Two dimensional SDS PAGE analysis of epididymal tissue proteins of normal and castrated bulls (Bos taurus) and identification of an androgen dependant protein

A study was undertaken with an objective of two dimensional SDS-PAGE analysis of protein profile in tissues of all caput, corpus and cauda of epididymis in both castrated and normal bulls and identification of androgen dependent protein/s in the epididymal tissues of bull (Bos taurus). Two dimensional SDS-PAGE analysis for protein spots of different molecular weight (MW) with pI 3.5 to 7.35 and pI > 7.35 to 9.3 between the three regions in the normal and castrated bull as well as between normal and castrated bull of a particular region revealed significant differences. Similarly, comparison between the three regions of the normal and castrated bull epididymis for the number of protein spots irrespective of the MW in pI range of 3.5 to 7.35 and pI range > 7.35 to 9.3 revealed significant differences. The number of protein spots found to be significantly higher in caput, corpus and cauda epididymis of the normal bull when compared to similar region of the castrated bull epididymis. Most of the proteins, which are secreted, are having MW between 20 and 85 kDa. Six proteins, which are known to be highly dependent on androgens, are also of acidic in nature except for one protein having basic pH. A protein spot (pI: 6.55–6.85, and MW: <20 kDa) which appeared at the same site in all the three regions of the epididymis in the normal bull but was absent at the same site in all the three regions of the castrated bull was subjected for identification of the protein by MALDI-MS. The results revealed that this protein is an interferon-stimulated protein and it could be ISG15/UCRP.

Proteome characterization of developing grains in bread wheat cultivars (Triticum aestivum L.).

BackgroundThe analyses of protein synthesis, accumulation and regulation during grain development in wheat are more complex because of its larger genome size compared to model plants such as Arabidopsis and rice. In this study, grains from two wheat cultivars Jimai 20 and Zhoumai 16 with different gluten quality properties were harvested at five development stages, and were used to displayed variable expression patterns of grain proteins.ResultsProteome characterization during grain development in Chinese bread wheat cultivars Jimai 20 and Zhoumai 16 with different quality properties was investigated by 2-DE and tandem MALDI-TOF/TOF-MS. Identification of 117 differentially accumulated protein spots representing 82 unique proteins and five main expression patterns enabled a chronological description of wheat grain formation. Significant proteome expression differences between the two cultivars were found; these included 14 protein spots that accumulated in both cultivars but with different patterns and 27 cultivar-different spots. Among the cultivar-different protein spots, 14 accumulated in higher abundance in Jimai 20 than in Zhoumai 16, and included NAD-dependent isocitrate dehydrogenase, triticin precursor, LMW-s glutenin subunit and replication factor C-like protein. These proteins are likely to be associated with superior gluten quality. In addition, some proteins such as class II chitinase and peroxidase 1 with isoforms in developing grains were shown to be phosphorylated by Pro-Q Diamond staining and phosphorprotein site prediction. Phosphorylation could have important roles in wheat grain development. qRT-PCR analysis demonstrated that transcriptional and translational expression patterns of many genes were significantly different.ConclusionsWheat grain proteins displayed variable expression patterns at different developmental stages and a considerable number of protein spots showed differential accumulation between two cultivars. Differences in seed storage proteins were considered to be related to different quality performance of the flour from these wheat cultivars. Some proteins with isoforms were phosphorylated, and this may reflect their importance in grain development. Our results provide new insights into proteome characterization during grain development in different wheat genotypes.

Establishment of two-dimensional gel electrophoresis profiles of the human acute promyelocytic leukemia cell line NB4

To explore optimum conditions for establishing a two‑dimensional gel electrophoresis (2-DE) map of the human acute promyelocytic leukemia (APL) cell line NB4 and to analyze its protein profiles, we extracted total proteins from NB4 cells using cell disruption, liquid nitrogen freeze-thawing and fracturing by ultrasound, and quantified the extracted protein samples using Bradford's method. 2-DE was applied to separate the proteins, which were silver-stained in the gel. Well‑separated protein spots were selected from the gel using the ImageMaster™ 2D Platinum analysis system. Moreover, the effects of various protein sample sizes (140, 160 and 180µg) on the 2-DE maps of the NB4 cells were determined and compared. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS), peptide mass fingerprinting (PMF) and database searching were used to identify the proteins. When the quantity of loading proteins was 160µg, clear, well-resolved, reproducible 2-DE proteomic profiles of the NB4 cells were obtained. The average number of protein spots in 3 gels was 1160±51 with an average matching rate of 81%. A total of 10 proteins were identified by mass spectrometry and database queries, certain proteins were products of oncogenes and others were involved in cell cycle regulation and signal transduction. In summary, 2-DE profiles of the proteome of NB4 cells were established and certain proteins were identified by MALDI-TOF-MS and PMF which lay the foundation of further proteomic research of NB4 cells. These data should be useful for establishing a human APL proteome database.

Proteomics of a toxic dinoflagellate Alexandrium catenella DH01: Detection and identification of cell surface proteins using fluorescent labeling

Alexandrium catenella DH01 is a toxic dinoflagellate species that is able to not only produce paralytic shellfish toxins, but also cause harmful algal blooms along the coast of China. In this study, we presented a new protocol for specific labeling and detection of the cell surface proteins (CSPs) of A. catenella DH01 cells using CyDye difference gel electrophoresis (DIGE) fluor minimal dyes. CSPs were identified using two-dimensional gel electrophoresis (2-DE) and MALDI TOF-TOF mass spectrometry (MS). The results showed that the fluorescent cyanine dye Cy3 could specifically label the CSPs of A. catenella DH01, with minimal labeling of intracellular proteins. Among three protein extraction methods evaluated, the Trizol method was the most efficient to extract CSPs with respect to protein spot number and resolution. Forty-one CSPs were separated and identified from A. catenella DH01 by 2-DE, in which 14 were identified in the protein database using MALDI TOF-TOF MS analysis. This work represents the first attempt to investigate the CSPs of A. catenella using the CyDye DIGE fluor dyeing method that provides a potentially important tool for future comprehensive characterization of CSPs and elucidation of the physiological functions of CSPs in dinoflagellates.

DMSO, an Organic Cleanup Solvent for TCA/Acetone-Precipitated Proteins, Improves 2-DE Protein Analysis of Rice Roots

Protein extraction is a critical step in a two-dimensional electrophoresis (2-DE)-based proteomic analysis. Dimethyl sulfoxide (DMSO), a small organic molecule, is widely used as a solvent in biological sciences. In this study, we modified the cleanup step of the commonly used trichloroacetic acid (TCA)/acetone method of protein extraction by using 20 % DMSO/acetone as a solvent to wash TCA–acetone-precipitated pellets. The improved protocol (TCA/acetone–DMSO) was compared with the TCA/acetone and phenol extraction method based on the protein yield, the number of spots, and resolution on 2-DE maps. TCA/acetone–DMSO washing increased protein yield, and improved the resolution in 2-DE with less background, streaking, and smearing. This method also produced the highest number of protein spots. To our knowledge, the present study is the first to use DMSO as a cleanup solvent for TCA/acetone-precipitated proteins to enhance their quality prior to 2-DE.

Comparison of urinary exosomal protein solubilization methods for two-dimensional gel electrophoresis

Background: Urinary exosomal proteins have recently emerged as important candidates for elucidating the mechanisms underlying physiological events and disease-related metabolism in the kidney. Here, we evaluated standard sample preparation methods for two-dimensional gel electrophoresis (2DE) to determine which one yielded the maximum protein recovery from urinary exosomes for protein identification.Materials and Methods: Urinary exosomes were purified from a healthy subject by using ultracentrifugation. The final pellets were dissolved with PBS or RIPA buffer. After being desalted, these exosomal protein solutions were each treated with 1 of 4 rehydration buffers (Rbs) containing detergents in the following formulations: CHAPS (Rb1), CHAPS and Triton X-100 (Rb2), dodecyl maltoside (Rb3), and ASB-14 (Rb4).Results: For all Rbs, a much greater number of protein spots was detected in the samples isolated with RIPA than with PBS. Only minor differences were observed in the number of protein spots for Rb1-3. The largest protein spots were detected using the combination of RIPA buffer and Rb4; however, the background on the 2DE gel was high in the region of >66 kD and at the lower pH values. For all combinations, the co-precipitation of the urinary Tamm-Horsfall protein masked the protein spots in the 66-100 kD region.Conclusion: For extracting a large number of proteins with a relatively clear background on silver-stained 2DE gels, the optimal exosomal protein-dissolving buffer is RIPA buffer. All of the evaluated Rbs, except for the one containing ASB-14 as a detergent, is suitable for solubilizing exosomal proteins on 2DE.

Fractional Precipitation of Plasma Proteome by Ammonium Sulphate:Case Studies in Leukemia and Thalassemia

Human plasma proteome is a comprehensive source of disease biomarkers. However, the >10 orders-wide dynamic concentration range of its constituent proteins necessitates depletion of abundant proteins from plasma prior to biomarker discovery. Our objective has been to develop a simple method that would deplete the most abundant proteins e.g. albumin and immunoglobulins, effectively facilitating identification of differentially regulated proteins in plasma samples. We employed ammonium sulphate based pre-fractionation of plasma followed by twodimensional gel electrophoresis (2DGE) for comparison of normal proteins with those from the plasma samples of the patients, after identification of proteins by MALDI-TOF/TOF tandem mass spectrometry. Fractional precipitation of the plasma samples by 20% ammonium sulphate from raw plasma doubled the number of protein spots after 2DGE and led to identification of 87 unique proteins, including several low-abundance proteins. Case studies done with fractional precipitation of the plasma samples of patients suffering from hematological diseases e.g. leukemia and thalassemia indicate the utility of such pre-fractionation in the detection of differentially regulated proteins.

Combined Use of a Solid-Phase Hexapeptide Ligand Library with Liquid Chromatography and Two-Dimensional Difference Gel Electrophoresis for Intact Plasma Proteomics

The intact plasma proteome is of great interest in biomarker studies because intact proteins reflect posttranslational protein processing such as phosphorylation that may correspond to disease status. We examined the utility of a solid-phase hexapeptide ligand library in combination with conventional plasma proteomics modalities for comprehensive profiling of intact plasma proteins. Plasma proteins were sequentially fractionated using depletion columns for albumin and immunoglobulin, and separated using an anion-exchange column. Proteins in each fraction were treated with a solid-phase hexapeptide ligand library and compared to those without treatment. Two-dimensional difference gel electrophoresis demonstrated an increased number of protein spots in the treated samples. Mass spectrometric studies of these protein spots with unique intensity in the treated samples resulted in the identification of high- and medium-abundance proteins. Our results demonstrated the possible utility of a solid-phase hexapeptide ligand library to reveal greater number of intact plasma proteins. The characteristics of proteins with unique affinity to the library remain to be clarified by more extensive mass spectrometric protein identification, and optimized protocols should be established for large-scale plasma biomarker studies.

Proteins That Bind to Misfolded Mutant Superoxide Dismutase-1 in Spinal Cords from Transgenic Amyotrophic Lateral Sclerosis (ALS) Model Mice

Mutant superoxide dismutase-1 (SOD1) has an unidentified toxic property that provokes ALS. Several ALS-linked SOD1 mutations cause long C-terminal truncations, which suggests that common cytotoxic SOD1 conformational species should be misfolded and that the C-terminal end cannot be involved. The cytotoxicity may arise from interaction of cellular proteins with misfolded SOD1 species. Here we specifically immunocaptured misfolded SOD1 by the C-terminal end, from extracts of spinal cords from transgenic ALS model mice. Associated proteins were identified with proteomic techniques. Two transgenic models expressing SOD1s with contrasting molecular properties were examined: the stable G93A mutant, which is abundant in the spinal cord with only a tiny subfraction misfolded, and the scarce disordered truncation mutant G127insTGGG. For comparison, proteins in spinal cord extracts with affinity for immobilized apo G93A mutant SOD1 were determined. Two-dimensional gel patterns with a limited number of bound proteins were found, which were similar for the two SOD1 mutants. Apart from neurofilament light, the proteins identified were all chaperones and by far most abundant was Hsc70. The immobilized apo G93A SOD1, which would populate a variety of conformations, was found to bind to a considerable number of additional proteins. A substantial proportion of the misfolded SOD1 in the spinal cord extracts appeared to be chaperone-associated. Still, only about 1% of the Hsc70 appeared to be associated with misfolded SOD1. The results argue against the notion that chaperone depletion is involved in ALS pathogenesis in the transgenic models and in humans carrying SOD1 mutations.

Optimizing protein recovery yield from serum samples treated with beads technology

Proteomics studies are often complicated by the wide dynamic range of the biological fluids, in which few highly abundant proteins obscure the signal of low abundant ones. To overcome this problem, several techniques have been developed on the basis of "depletion principles," namely immuno-subtraction with specific antibodies against the most-abundant proteins. Unfortunately, the probability of codepletion is a noteworthy drawback associated with these strategies. The ProteoMiner (PM) technology is a novel approach, consisting of a combinatorial library of hexapeptide ligands coupled to beads, that allows the capture of all species present in a proteome, but at much reduced protein concentration differences, simultaneously enhancing the concentration of the most dilute species. In this study, we evaluated the compatibility of the PM kit's elution reagent with 2-DE analysis, comparing five different purification methods on serum samples eluted from the beads: the "ReadyPrep 2-D Clean-up kit" and precipitation with organic solvents, as acetone/methanol, TCA/acetone, ACN, and chloroform/methanol. Considering protein recovery yield (quantity) and 2-DE spot pattern (quality), precipitation with ACN offered the most promising approach, showing the best spot resolution in all regions of the pH gradient and the greatest number of protein spots visualized on 2-D gels.

Leaf proteomic analysis in cassava (Manihot esculenta, Crantz) during plant development, from planting of stem cutting to storage root formation

Tuberization in cassava (Manihot esculenta Crantz) occurs simultaneously with plant development, suggesting competition of photoassimilate partitioning between the shoot and the root organs. In potato, which is the most widely studied tuber crop, there is ample evidence suggesting that metabolism and regulatory processes in leaf may have an impact on tuber formation. To search for leaf proteins putatively involved in regulating tuber generation and/or development in cassava, comparative proteomic approaches have been applied to monitor differentially expressed leaf proteins during root transition from fibrous to tuberous. Stringent cross comparison and statistical analysis between two groups with different plant ages using Student's t test with 95% significance level revealed a number of protein spots whose abundance were significantly altered (P<0.05) during week 4 to week 8 of growth. Of these, 39 spots were successfully identified by ion trap LC-MS/MS. The proteins span various functional categories from antioxidant and defense, carbohydrate metabolism, cyanogenesis, energy metabolism, miscellaneous and unknown proteins. Results suggested possible metabolic switches in the leaf that may trigger/regulate storage root initiation and growth. This study provides a basis for further functional characterization of differentially expressed leaf proteins, which can help understand how biochemical processes in cassava leaves may be involved in storage root development.

Evaluation of sample extraction methods for proteomic analysis of coniferous seeds

In this study, three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality (including the number of protein spots observed) in the two-dimensional gel electrophoresis (2-DE), obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis of the total released protein. Three protein extraction methods were: TCA-acetone precipitation, SDS extraction/acetone precipitation, and phenol extraction methanol/ammonium acetate precipitation. The results showed that TCA-acetone precipitation was the most effective method for protein extraction; it gave the highest yield of total protein (8.9 mg protein per g seed weight) and the greatest number of proteins spots (1,034 spots) on the 2-DE gel. Further, several proteins were identified by liquid chromatography mass spectrometry (LC MS/MS), which are legumin-like storage protein, similar to AMP binding/acetate-CoA ligase, similar to 40S ribosomal protein S20, actin, ascorbate peroxidase, Similar to cysteine synthase, and unknown protein. These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic analysis of other coniferous tree species.

Analysis of protein composition of rabbit aqueous humor following two different cataract surgery incision procedures using 2-DE and LC-MS/MS.

BackgroundThe aqueous humor (AH), a liquid of the anterior and posterior chamber of the eye, comprises many proteins with various roles and important biological functions. Many of these proteins have not been identified yet and their functions in AH are still unknown. Recently, our laboratory published the protein database of AH obtained from healthy rabbits which expanded known protein identifications by 65%. Our present study extends our previous work and analyses AH following two types of cataract surgery incision procedures (clear corneal and limbal incisions) by using two dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Although both incision protocols are commonly used during cataract surgeries, the difference in protein composition and their release into AH following each surgery has never been systematically compared and remains unclear. The first step, which is the focus of this work, is to assess the scale of the protein change, at which time does maximum release occurs and when possible, to identify protein changes.ResultsSamples of AH obtained prior to surgery and at different time points (0.5, 2, 12, 24 and 48 hours) following surgery (n = 3/protocol) underwent protein concentration determination, 2-DE and LC-MS/MS. There was a large (9.7 to 31.2 mg/mL) and rapid (~0.5 hour) influx of proteins into AH following either incision with a return to baseline quantities after 12 hours and 24 hours for clear corneal and limbal incision, respectively. We identified 80 non-redundant proteins, and compared to our previous study on healthy AH, 67.5% of proteins were found to be surgery-specific. In addition, 51% of those proteins have been found either in clear corneal (20%) or limbal incision (31%) samples.ConclusionsOur results imply that a mechanism of protein release into AH after surgery is a global response to the surgery rather than increase in amount of protective proteins found in healthy AH and a mechanism of protein release for each type of incision procedure could be different. Although the total protein concentration was increased (at 0.5 and 2 hour time points and between types of surgery) many of 2-DE protein spots were similar based on 2-DE and MS analyses, and only a small number of protein spots changed with either the time points or surgical conditions (0.4 -1.9%). This suggests that the high protein content is due to an increase in the concentration of the same proteins with only a few unique proteins being altered per time point and with the different surgery type. This is the first report on the comparison of AH protein composition following two different cataract surgery procedures and it establishes the basis for better understanding of protein release into AH during events such as cataract surgery or other possible intervention to the eyes.

The proteomic response of larvae of the Sydney rock oyster, Saccostrea glomerata to elevatedpCO2

The acidification of oceans is predicted to fundamentally alter marine ecosystems. Previous studies have found that elevated CO2 has an effect on adult calcification, fertilisation and larval development, perhaps because of the organisms' inability to regulate acid-base status, but little is known about the mechanisms that underlie such responses. This study investigated the growth response of larvae of a wild and selectively bred line of the Sydney rock oyster, Saccostrea glomerata, to elevated CO2 and measured the pattern of expression of proteins. Overall exposure to elevated CO2 caused a significant reduction in the shell length of D-veliger larvae of the wild, but not in the selectively bred line. Prior to this study, differences in growth between selectively bred and wild oysters have only been found following settlement. Proteome analysis of D-veliger larvae using two-dimensional gel electrophoresis detected a significantly greater number of protein spots in selectively bred compared to wild oyster...

Extraction of microbial proteome from soil: potential and limitations assessed through a model study

Proteomics is the study of functions and regulation of biological systems based on the analysis of the protein expression profile, and there is a general agreement that soil proteomics may be a tool for better soil management. Because of the ability of soils to stabilize extracellular proteins by various mechanisms, development of soil proteomics needs an assessment of the efficiency of protein extraction from various soil types. We evaluated the possibility of extraction of soil microbial proteome by inoculating Cupriavidus metallidurans CH34, which has a known proteome, into sterile sand, kaolinite, montmorillonite and a mixture of sand, kaolinite, montmorillonite, goethite and humic acids. One hour after inoculation, the viability of C. metallidurans was determined by the colony‐forming units method (CFU), the amount of extracted proteins was determined by the Bradford method and the bacterial proteome was analysed by the two‐dimensional gel electrophoresis technique (2D‐GE). The bacterial number was 2.5 × 106 CFU g−1 of soil in all microcosms, whereas the total extracted protein content varied from 98.1 to 1268 µg g−1 in the various microcosms, but was undetectable in the inoculated montmorillonite. The number of protein spots from the bacterial culture and the inoculated microcosms varied between 317 and 591, with 54 variable spots among the pure culture and the microcosms. No protein spots were detected in the 2D‐GE from the montmorillonite microcosm. The 2D‐GE of artificial soil microcosms showed a protein pattern that was different from those of pure culture and sand and kaolinite microcosms. The results confirm the importance of clay‐specific surface area and CEC in protein adsorption as montmorillonite alone had the largest sorptive capacity, and show that the artificial soil used also had a large sorptive capacity for microbial proteins. Globally, the results indicate that the extraction of proteins from soils is strongly influenced by the clay type and organic matter content, and that poor protein extraction efficiency may reduce the potential of soil proteomics.