Separation and identification of differential protein in rat's bone with fluorosis and calcium supplementation intervention / 生物工程学报

In order to explore the mechanisms underlying the calcium alleviating fluorosis at protein level, we made an attempt to establish fluorosis and calcium supplementation rat models to isolate and identify bone differential proteins. The bone proteins of different groups were compared by two-dimensional electrophoresis (2-DE) and mass spectrometry (MALDI-TOF MS), and analyzed by gene ontology annotation, pathway enrichment and interaction networks. The 17 proteins were identified in the fluorosis group (F) and the fluorosis calcium supplement group (F+Ca), including type I collagen (Col1a1), actin (Actb), protein glutamine transferase 2 (Tgm2), compared with the control group (C). These differential proteins are enriched in 38 bone metabolic pathways such as focal adhesion, PI3K-Akt signaling pathway, and AMPK signaling pathway. And the functions of these proteins are mainly related to cytoskeleton, energy metabolism, substance transport, ion channel, and apoptosis. Therefore, it is speculated that calcium may alleviate the fluoride-induced bone damage by regulating the focal adhesion, PI3K-Akt, AMPK and other signaling pathway, but the specific mechanism needs further research.

Comparative Analysis of the Tear Protein Expression After Photorefractive Keratectomy Using Two-Dimensional Electrophoresis

PURPOSE:To investigate the change in the tear protein composition of patients who underwent refractive surgery. METHODS: Tear samples were collected before photorefrative keratectomy (PRK), on the first, the second, and the third postoperative day, and then a month after the operation from 40 eyes of 20 patients. These tear samples were analyzed using two-dimensional gel electrophoresis (2-DE). Matrix-associated laser desorption/ionization time of flight (MALDI-TOF) was employed for the identification of expressed proteins. Control tear samples were collected from 40 eyes of 20 healthy volunteers who had no history of ocular surgery or pathology. RESULTS: On the first postoperative day, lipocalin-1 precursor, lipocalin-1, and lysozyme were up-regulated. On the second postoperative day, serum albumin precursor and serum albumin were up-regulated. The tears collected on the third postoperative day and after 1 month had similar protein expression levels to the control group. Lipocalin 1 precursor and lysozyme were up-regulated and down-regulated after reftactive surgery, respectively. However, each protein had a different molecular weight and isopotential point. CONCLUSIONS: The tear protein composition changed uniquely in the early postoperative period, and proteins with different isopotential points were detected after PRK. We hypothesized that the healing process might influence the expression of the tear proteins.

Differential Analysis of Two-dimensional Gel Electrophoresis Profiles of Gastric Carcinoma and Paired Normal Gastric Mucosa / 中国医师杂志

Objective To establish two-dimensional electrophoresis profiles with high resolution and reproducibility from human gastric carcinoma and paired normal gastric mucosa tissues and to analyse the differential expression proteins between the two types of the tissues. Methods The total proteins of gastric carcinoma and paired normal gastric mucosa tissues were seperated by immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE).After silver staining, the differential expression proteins were analyzed by using Imaging Master 2D analysis software. Results ⑴Average protein spots were 1049?67, 1097?73 in gastric carcinoma and paired normal gastric mucosa respectively,the matched spots were 835?48, 953?56 in both the two types of tissues respectively, and the average matching rate was 79.6%, 86.7% respectively. ⑵A total of 769?45 protein spots were matched between the electrophoretic maps of 5 gastric carcinoma and paired normal gastric mucosa tissues. The 81 differential protein spots were identified between the two types of the tissues. 17 protein spots were expressed only in the gastric carcinoma and 24 protein spots were expressed only in the normal gastric mucosa. The expression levels of 26 protein spots in the gastric carcinoma were higher than those in the normal gastric mucosa, and the expression levels of 14 protein spots in the gastric carcinoma were lower than those in the normal gastric mucosa. Conclusion In this study, the well-resolved,reproducible 2-DE patterns of gastric carcinoma and paired normal gastric mucosa tissues were established and some differential proteins between the two types of tissues were found. These data will be helpful to further screen specific biomarkers of gastric carcinoma.

MALDI-TOF MS identification of protein of mitochondria from cultured hepatoma cells / 重庆医科大学学报

Objective:To explore the optimal conditions associated with proteolytic digestion in 2-DE gel,PMF analysis and database inquiry,and to construct a mass identification strategy in research for mitochondrial comparative proteomics.Methods:The BSA standard protein was incised from 2-DE gel by Coomassie Brilliant Blue dyeing and was mixed with matrix (CHCA),then analyzed by MALDI-TOF MS.The differentially expressed mitochondria protein L9,an unknown protein obtained from hepatoma cells QGY-7703,was identified by condition with BSA optimizing.The PMF was inquired by MS-Fit database.Results:The BSA was identified in Masses Matched number 10/11 and Sequence Overcast 19% of PMF,which proved to the reliability of experimental conditions.By database Inquiry about PMF from L9 protein,OXCT(3-oxoacid CoA transferase 1 precursor)was identified with Masses Matched number 9/13 and Sequence Overcast 24%.The OXCT was in accord with position on gel.Conclusion:This method is applicable to research for mitochondrial comparative proteomics.

Differential proteomic analysis of Pten~(L/L)MEFs and Pten~(△/△)MEFs cell lines / 西安交通大学学报(医学版)

Objective To study protein variation between PtenL/LMEFs and Pten△/△MEFs cells.Methods Two-dimensional electrophoresis(2-DE) was employed to compare the differential expression proteins between PtenL/LMEFs and Pten△/△MEFs cells.Six differential expression proteins were digested in gel by enzyme and the mass of generated peptides was measured by matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS).The data obtained from peptide mass fingerprinting(PMF) were searched using the Internet available database and five proteins were identified.Results Compared with that of PtenL/LMEFs,expression level of proteins including phosphoglycerate mutase 1(PGAM1) and peptidyl-prolyl cis-trans isomerase C(PPIC) was up-regulated,whereas expression level of Transgelin 2 was down-regulated in Pten△/△MEFs cells.B and F proteins were both identified to be peroxiredoxin-6.They had similar molecular weight but different PI which might be caused by post-translation modification.B protein was only expressed in Pten△/△MEFs cells.Conclusion The protein profile of Pten△/△MEFs cells displayed obvious difference compared to that of PtenL/LMEFs cells.The results implied that various distinct different proteins might lead to cancer.