Two-dimensional polyacrylamide gel electrophoresis analysis of indomethacin-treated human colon cancer cells

AIM: To establish the two-dimensional gel electrophoresis (2-DE) profiles of indomethacin (IN)-treated human colon cancer cell line HCT116, and to provide a new way to study its anti-tumor molecular mechanism through analyzing a variety of protein maps.METHODS: Two-DE profiles of HCT116 were established in IN-treated and untreated groups. Total proteins were separated by immobilized pH gradient-based 2-DE. The gels were stained by silver, scanned by ImageScanner,and analyzed with Image Master software.RESULTS: Clear background, well-resolved and reproducible 2-DE patterns of HCT116 cells were acquired in IN-treated and untreated group. The average deviation of spot position was 0.896±0.177 mm in IEF direction and 1.106±0.289 mm in SDS-PAGE direction respectively. In IN-treated group,1 169±36 spots were detected and 1 061±32 spots were matched, the average matching rate was 90.6% in three gels. In untreated group, 1 256±50 spots were detected and 1 168±46 spots were matched, the average matching rate was 93.0% in three gels. Forty-five differential protein spots were displayed between IN-treated and untreated groups. Of which, 34 protein spots decreased and 9showed higher expression in IN-treated group, and only two protein spots showed an expression in untreated cells.CONCLUSION: Two-DE profiles of IN-treated and untreated HCT116 cells were established. Apparent 45 different protein spots were detected in IN-treated and untreated HCT116 cells. The analysis on differential protein spots may serve as a new way to study the molecule mechanism of IN-treated colon cancer.

Proteomic analysis of the serum in patients with idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension(IPAH) is a rare disease of unknown etiology.The exact pathogenesis of pulmonary arterial hypertension is still not well known.In the past decades,many protein molecules have been found to be in-volved in the development of IPAH.With proteomic techniques,profiling of human plasma proteome becomes more feasible in searching for disease-related markers.In present study,we showed the protein expression profiles of the serum of IPAH and healthy controls after depleting a few high-abundant proteins in serum.Thirteen spots had changed significantly in IPAH com-pared with healthy controls and were identified by LC-MS/MS.Alpha-1-antitrypsin and vitronectin were down-regulated in IPAH and may be valuable candidates for further explorations of their roles in the development of IPAH.

Extraction of Proteins from Apple Leaves for Two-Dimensional Electrophoresis Analysis

In order to develop an efficient method about protein extraction which is suitable for apple proteomic analysis, four protocols of total protein extraction from apple leaves, which are trichloroacetic acid/acetone precipitation method （TCA）, phenol extraction methanol/ammonium acetate precipitation method, Tris-HCl extraction method and modified Tris-HCl extraction method were compared. The results showed that the modified Tris-HCl extraction method was the most suitable method in protein extraction for two-dimensional electrophoresis （2-DE） from apple leaves based on the highest resolution and more informative spots of 2-DE gels with no apparent vertical or horizontal streaking.

基于蛋白质组学的假单胞菌耐溶剂机制研究

【目的】有机溶剂对微生物有强烈的毒害作用致使绝大多数微生物不能在较高的有机溶剂浓度下生长。为了探究微生物的耐溶剂性机制,由野生型假单胞菌Pseudomonas putida JUCS驯化获得一株能够在60%(V/V)的环己烷中生长的菌株P.putidaJUCT1。【方法】采用蛋白质二维电泳对P.putida JUCT1在不同溶剂条件下的蛋白组分表达量的差异进行分析比对。【结果】从总共22个表达量差异均超过50%的蛋白质中,选取了3个高丰度蛋白质,通过MALDI-TOF/TOF鉴定为:3-羟基异丁酸水解酶、蛋白质延伸因子EF-Ts、异分支酸水解酶超家族(编码基因分别为mmsB、tsf、PSEEN0851)。将这3个基因在大肠杆菌中重组表达,3个蛋白均能不同程度地提高E.coli JM109的耐溶剂性,其中3-羟基异丁酸脱氢酶(编码基因mmsB)对菌株的溶剂耐受性影响最为显著。【结论】证明了运用蛋白组学的方法研究微生物的耐溶剂性的可行性,并为构建适用于工业化应用的溶剂耐受性整体细胞生物催化剂提供理论依据。

Two-dimensional gel electrophoresis-based analysis provides global insights into the cotton ovule and fiber proteomes

Proteomic analysis of upland cotton was performed to profile the global detectable proteomes of ovules and fibers using two-dimensional electrophoresis(2DE).A total of 1,203 independent protein spots were collected from representative 2DE gels,which were digested with trypsin and identified by matrix-assisted laser desorption and ionization-time-offlight/time-of-flight(MALDI-TOF/TOF)mass spectrometry.The mass spectrometry or tandem mass spectrometry(MS or MS/MS)data were then searched against a local database constructed from Gossypium hirsutum genome sequences,resulting in successful identification of 975 protein spots(411 for ovules and 564 for fibers).Functional annotation analysis of the 975identified proteins revealed that ovule-specific proteins were mainly enriched in functions related to fatty acid elongation,sulfur amino acid metabolism and post-replication repair,while fiber-specific proteins were enriched in functions related to root hair elongation,galactose metabolism and D-xylose metabolic processes.Further annotation analysis of the most abundant protein spots showed that 28.96%of the total proteins in the ovule were mainly located in the Golgi apparatus,endoplasmic reticulum,mitochondrion and ribosome,whereas in fibers,27.02%of the total proteins were located in the cytoskeleton,nuclear envelope and cell wall.Quantitative real-time polymerase chain reaction(q RT-PCR)analyses of the ovule-specific protein spots P61,P93 and P198 and fiber-specific protein spots 230,477 and 511 were performed to validate the proteomics data.Protein-protein interaction network analyses revealed very different network cluster patterns between ovules and fibers.This work provides the largest protein identification dataset of 2DE-detectable proteins in cotton ovules and fibers and indicates potentially important roles of tissue-specific proteins,thus providing insights into the cotton ovule and fiber proteomes on a global scale.