基于iTraq技术的加拿大一枝黄花提取物作用下铜绿微囊藻细胞差异表达蛋白

为研究加拿大一枝黄花提取物对藻类生长抑制作用的分子机理,应用iTraq技术结合LC-MS-MS,分析了铜绿微囊藻细胞蛋白质的差异表达.共鉴定出261种差异蛋白(变化倍数≥1.5),其中表达上调的220种,表达下调的41种.GO功能分类显示,加拿大一枝黄花提取物通过影响细胞代谢过程、蛋白质催化和结合功能等方式抑制藻细胞生长.KEGG通路分析表明,通路大类中富集程度位于前3位的均是代谢通路,包括能量代谢、碳水化合物代谢和氨基酸代谢,通路中富集程度最高的是糖酵解/糖异生通路.本研究发现了加拿大一枝黄花提取物对藻细胞生长抑制作用的分子靶点,为从分子层面阐明其抑藻作用提供参考,也为研究植物化感物质的抑藻机理提供了新方法.

Chemometrics of differentially expressed proteins from colorectal cancer patients

AIM:To evaluate the usefulness of differentially expressed proteins from colorectal cancer (CRC) tissues for differentiating cancer and normal tissues.METHODS:A Proteomic approach was used to identify the differentially expressed proteins between CRC and normal tissues.The proteins were extracted using Tris buffer and thiourea lysis buffer (TLB) for extraction of aqueous soluble and membrane-associated proteins,respectively.Chemometrics,namely principal component analysis (PCA) and linear discriminant analysis (LDA),were used to assess the usefulness of these proteins for identifying the cancerous state of tissues.RESULTS:Differentially expressed proteins identified were 37 aqueous soluble proteins in Tris extracts and 24 membrane-associated proteins in TLB extracts.Based on the protein spots intensity on 2D-gel images,PCA by applying an eigenvalue > 1 was successfully used to reduce the number of principal components (PCs) into 12 and seven PCs for Tris and TLB extracts,respectively,and subsequently six PCs,respectively from both the extracts were used for LDA.The LDA classification for Tris extract showed 82.7% of original samples were correctly classified,whereas 82.7% were correctly classified for the cross-validated samples.The LDA for TLB extract showed that 78.8% of original samples and 71.2% of the cross-validated samples were correctly classified.CONCLUSION:The classification of CRC tissues by PCA and LDA provided a promising distinction between normal and cancer types.These methods can possibly be used for identification of potential biomarkers among the differentially expressed proteins identified.

Comparative proteomic study of cervical cancer with different radiotherapy sensitivity

Objective： To investigate the proteornic differences between the high-sensitivity （HS） group and low-sensitivity （LS） group of cervical cancer treated by radiotherapy and confirm the radiotherapy sensitivity associated proteins in early cervical cancer. Methods： The fresh carcinoma tissues were collected from 10 untreated cervical cancer patients and preserved in the -80 ℃ refrigeratory. The tissues were classified into two groups： high sensitivity group （HS） and low sensitivity group （LS）, according to their response to radiotherapy. In the first part of our experiment, protein separating was performed by using two-dimensional gel electrophoresis （2-DE） with Amersham 18 cm linear pH 3-10 immobilized pH gradient （IPG） strips. The images of the gels were acquired by the scanner and then analyzed by using PD-quest7.3 software to find the differentially expression protein-spots in each group. Then the differentially expressed protein-spots was incised from the gels and digested by trypsin. The peptide mass fingerprintings （PMF） was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and the proteins were identified by data searching in the Mascot-database. Part of differentially expression proteins were assayed by Western Blot. Results： Most of the gels were clear and successfully analyzed by PD-quest7.3 software. Most of the protein-spots concentrated on the area of 20-100Kda （Mw） and pH4-8. The average number of the protein-spots was 754 ± 64 in HS group and 777 ±48个 in LS group. The match rate was 87.6% between two groups. Five high expression proteins were found in HS group which were low expression in LS group, 3 high expression protein were found in LS group which were low expression in HS group. Reselts of Western Blot were in coincidence to proteomic result. Conclusion： The 2-DE gels image of HS group and LS group with early cervical cancer tissues treated by radiotherapy are successfully acquired. Some differentially expression proteins between the two groups are further confirmed by immunohistochemical assay.

Science Letters:Proteomic analysis of differentially expressed proteins in mice with concanavalin A-induced hepatitis

Objective： To find new protein biomarkers for the detection and evaluation of liver injury and to analyze the relationship between such proteins and disease progression in concanavalin A （Con A）-induced hepatitis. Methods： Twenty-five mice were randomly divided into five groups： an untreated group, a control group injected with phosphate buffered saline （PBS）, and groups with Con A-induced hepatitis evaluated at 1, 3 and 6 h. Two-dimensional gel electrophoresis （2-DE） and mass spectrometry （MS） were used to identify differences in protein expression among groups. Quantitative real-time polymerase chain reaction （qRT-PCR） was performed to verify the results. Results： In mice with Con A-induced hepatitis, expression levels of four proteins were increased： RIKEN, fructose bisphosphatase 1 （fbp1）, ketohexokinase （khk）, and Chain A of class pi glutathione S-transferase. Changes in fbpl and khk were confirmed by qRT-PCR. Conclusion： Levels of two proteins, fbp1 and khk, are clearly up-regulated in mice with Con A-induced hepatitis.

Label-free quantification of differentially expressed proteins in mouse liver cancer cells with high and low metastasis rates by a SWATH acquisition method

Label-free quantification is a valuable tool for the analysis of differentially expressed proteins identified by mass spectrometry methods.Herein,we used a new strategy:data-dependent acquisition mode identification combined with label-free quantification by SWATH acquisition mode,to study the differentially expressed proteins in mouse liver cancer metastasis cells.A total of 1528 protein groups were identified,among which 1159 protein groups were quantified and 249 protein groups were observed as differentially expressed proteins(86 proteins up-regulated and 163 down-regulated).This method provides a commendable solution for the identification and quantification of differentially expressed proteins in biological samples.