Preprocessing of 2-Dimensional Gel Electrophoresis Images Applied to Proteomic Analysis: A Review / 基因组蛋白质组与生物信息学报·英文版

Various methods and specialized software programs are available for processing two-dimensional gel electrophoresis (2-DGE) images. However, due to the anomalies present in these images, a reliable, automated, and highly reproducible system for 2-DGE image analysis has still not been achieved. The most common anomalies found in 2-DGE images include vertical and horizontal streaking, fuzzy spots, and background noise, which greatly complicate computational analysis. In this paper, we review the preprocessing techniques applied to 2-DGE images for noise reduction, intensity normalization, and background correction. We also present a quantitative comparison of non-linear filtering techniques applied to synthetic gel images, through analyzing the performance of the filters under specific conditions. Synthetic proteins were modeled into a two-dimensional Gaussian distribution with adjustable parameters for changing the size, intensity, and degradation. Three types of noise were added to the images: Gaussian, Rayleigh, and exponential, with signal-to-noise ratios (SNRs) ranging 8-20 decibels (dB). We compared the performance of wavelet, contourlet, total variation (TV), and wavelet-total variation (WTTV) techniques using parameters SNR and spot efficiency. In terms of spot efficiency, contourlet and TV were more sensitive to noise than wavelet and WTTV. Wavelet worked the best for images with SNR ranging 10-20 dB, whereas WTTV performed better with high noise levels. Wavelet also presented the best performance with any level of Gaussian noise and low levels (20-14 dB) of Rayleigh and exponential noise in terms of SNR. Finally, the performance of the non-linear filtering techniques was evaluated using a real 2-DGE image with previously identified proteins marked. Wavelet achieved the best detection rate for the real image.

Comparative proteomic analysis of sequential isolates of Mycobacterium tuberculosis from a patient with pulmonary tuberculosis turning from drug sensitive to multidrug resistant.

Background & objectives: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). Methods: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. Interpretation & conclusions: This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings.

Hypobaric hypoxia-mediated protein expression in plasma of susceptible & tolerant rats.

Background & objectives: Low availability of oxygen at high altitudes has a great impact on the human life processes. There is a widespread interest and need to find out protein(s) that are possibly involved in mediating tolerance to hypobaric hypoxia. We undertook this study to identify and characterize protein expression in plasma of hypoxia susceptible and tolerant rats. Methods: Male albino Sprague Dawley rats were segregated into susceptible and tolerant groups on the basis of their gasping time when exposed to simulated hypobaric hypoxia of 32,000 ft (9,754 m) at 32ºC. Comparative proteome profiling of blood plasma of hypoxia susceptible and tolerant individuals was performed using 2-dimentional (2-D) gel electrophoresis. Results: Three proteins with higher expression levels were selected separately from tolerant and susceptible samples. Characterization of these proteins from tolerant sample using MALDI-TOF/TOF and MASCOT search indicated their homology with two different super-families viz. NADB-Rossmann superfamily (Rab GDP dissociation inhibitor β) and Transferrin superfamily (two Serotransferrins), having potential role in imparting tolerance against hypoxia. Three high level upregulated proteins were characterized from blood plasma of hypoxia susceptible animals showing similarity with threonine tRNA ligase (mitochondrial), carbohydrate sulphotransferase 7 and aspartate tRNA ligase (cytoplasmic) that play a role in ATP binding, carbohydrate metabolism and protein biosynthesis, respectively. Interpretation & conclusions: Our results indicated that rats segregated into hypoxia sensitive and tolerant based on their gasping time showed differential expression of proteins in blood plasma. Characterization of these differentially expressed proteins will lead to better understanding of molecular responses occurring during hypoxia and subsequently development of biomarkers for categorization of hypoxia susceptible and tolerant individuals.

Anti-drought differential proteome on interaction between Anoectochilus formosanus and mycorrhizal fungi / 中国药学杂志

OBJECTIVE: To study the proteomic difference between mycorrhizal fungi infected and uninfected plants and then to explore the possible anti-drought mechanism caused by mycorrhizal fungi. METHODS: The differential proteome of Anoectochilus formosanus infected by Rhizoctonia sp. growing in drought was investigated by means of 2D gel electrophoresis. RESULTS: Eight proteins were involved in photosynthesis, among them 7 proteins were correlated with CO2 fixation of darkreaction. Meanwhile, C4 pathway suited in drought condition was mobilized by the plants to utilize CO2 thoroughly. Five proteins were involved in glyoometabolism and li-pometabolism, and 3 in protein synthesis. CONCLUSION: Increased darkreaction of photosynthesis, especially the C4 pathway in infected fungi can enhance the capability of CO2 fixation and utilization when the stoma is closed. The glycometabolism, lipometabolism, protein synthesis, and resistance to pests of the infected plants are also enhanced in drought condition. Fungus of Rhizoctonia sp. can help plants improve their capability to resist drought.

Streptomycin induced protein expression analysis in Mycobacterium tuberculosis by two-dimensional gel electrophoresis & mass spectrometry.

Background & objectives: The resistance of Mycobacterium tuberculosis to streptomycin, a core drug for treatment of category II tuberculosis (TB) has posed a major challenge to the health providers as well as research workers worldwide and has severely compromised the therapeutic options. A significant proportion of streptomycin resistant M. tuberculosis isolates failed to show mutations in conventional targets like rpsL and rrs. Although efflux, permeability, etc. are also known to contribute, yet a substantial proportion of isolates remains resistant suggesting involvement of other unknown mechanism. A resistant isolate may show altered gene as well as protein expression under drug induced conditions and a whole cell proteome analysis under induced conditions might help in further understanding the mechanisms of drug resistance. The present study was therefore designed with the objective to identify proteins related to streptomycin resistance in M. tuberculosis isolate grown in presence and absence of streptomycin (SM). Methods: A clinical isolate of M. tuberculosis from Mycobacterial Repository Centre at the Institute (NJIL & OMD), Agra was grown in Sauton’s medium for 36 h with/without subinhibitory concentration of the drug (2 μg/ml) and the cell lysate of isolates was prepared by sonication and centrifugation. Two-dimensional (2D) gel electrophoresis was employed to study the protein profile. The selected proteins were finally identified by MALDI-TOF mass spectrometry. Results: Our study revealed eight inducible proteins (DnaK, fabG4, DNA-binding, hypothetical, two 14 kDa antigen and two 10 kDa chaperonin) that were upregulated in the presence of drug. Interpretation & conclusion: This preliminary study has thrown light on whether or not and how the resistant isolate responds to streptomycin at its non-toxic but sub-inhibitory concentration. An in-depth study of the upregulated proteins will give an insight into probable sites of drug action other than established primary sites.

Insights into the protein profiles of stress tolerant and mesophilic yeast by proteomics approach: a comparative study.

Proteins of the stress tolerant and mesophilic yeast were extracted using optimized protein extraction method and estimated by Bradford method. Immobilized pH gradient (IPG) strips were rehydrated with known concentrations of protein samples. Rehydrated IPG strips were run in isoelectric focusing (IEF) to separate the proteins on the basis of their pH gradient. 2DE gels were run, stained and image of the stress tolerant yeast was compared with the gel image of mesophilic yeast. The image analysis using the image master software resulted in the identification of differentially expressed spots in stress tolerant yeast. Among the differentially expressed spots, six were selected and characterized by MALDI-TOF as Enolase, Fructose bisphosphate aldolase, Alcohol dehydrogenase, 30KDa HSP, HSP70 and HSP90.

Study on Circulating Factor(s) in Patient with Recurrent Focal Segmental Glomerulosclerosis / 대한신장학회잡지

BACKGROUND: Although a significant number of studies were done on focal segmental glomerulosclerosis(FSGS), its pathogenesis has not been sufficiently established yet. Recent studies suggested certain types of circulating factor(s) played an important role in development and recurrence after renal transplantation of FSGS by modifying the glomerular permeability of albumin. The purpose of this study performed on animals and through molecular-biological experiments is to certify the role of circulating factor (s), which cause proteinuria, by manipulating plasma of a FSGS patient who showed massive of proteinuria and wide effacement of glomerular epithelial foot processes in histologic examination after renal transplantation. also, whose massive proteinuria decreased significantly after plasma exchange. METHODS: The patient's plasma prior to(plasma A) or post to(plasma B) plasma exchange were injected into tail veins of two groups of male Sprague-Dawley rats, six in each. The ratio of 24 hour urine protein and urine creatinine(Uprt/Ucr) was calculated for each case. The 2D gel electrophoresis was performed in plasma A and plasma B. The pattern of 2D gel electrophoresis of plasma A was compared to those of plasma B and healthy human serum. RESULTS: Compared to control group, there was no significant differences in 24-hour Uprt/Ucr afer injecting 1, 2, 3, 5 mL of plasma A(p>0.05). There was no significant difference in 24-hour Uprt/Ucr between the injecting groups of plasma A and plasma B(p>0.05). We were not able to observe any new protein which did not appear in plasma B or healthy human serum in 2D gel electrophoresis. CONCLUSION: These results suggest that the proteinuria developed in a few hours after renal transplantation and is related to wide effacement of glomerular epithelial foot processes, and that it may be induced by a certain factor which is eliminated by the plasma exchange or restrained by the immunosuppressive agents. However, we were not able to find certain circulating factor(s) which rapidly changes albumin permeability in the patient's plasma with FSGS.

Primary studies on retinal proteome of rds and C3B mice by two-dimensional gel electrophoresis / 眼科新进展

Objective　To apply two-dimensional (2-D) gel electrophoresis to resolve specially expressed proteins related to retinitis pigmentosa (RP) in the retina of rds mice.Methods　Proteins, prepared from the retinas of rds mice and normal C3B mice at different ages, were separated by using two-dimensional electrophoresis and then analyzed by 2-DE imaging analyzer.Results　2-D gel electrophoresis was established for the retinal proteome analysis. Retinal neuronal tissue was lysed by using chemical lysis solution and ultrasonic. Using carrier ampholyte to set up pH gradient as first dimension and casting vertical 12% SDS-acylamide-bis slab as second dimension, the major retinal proteins showed maps of proteome on 2-D gels clearly. Retinal proteome of rds and C3B mice at 37d has different expressive patterns. Some proteins only expressed in the retina of rds mice while another only in the retina of C3B mice and others had different level between the two kinds of mice.Conclusion　2-D gel electrophoresis is effective to separate specially expressed retinal proteins. The expressed proteins in the rds retina are different in quality and quantity from that in the normal C3B mice.