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<p><b>OBJECTIVE</b>To establish a domestic database of Enterobacteria cloacae (E. cloacae), and improve the identification efficiency using peptide mass fingerprinting.</p><p><b>METHODS</b>Peptide mass fingerprinting was used for the identification and subtyping of E. cloacae. Eighty-seven strains, identified based on hsp60 genotyping, were used to construct and evaluate a new reference database.</p><p><b>RESULTS</b>Compared with the original reference database, the identification efficiency and accuracy of the new reference database was greatly improved at the species level. The first super reference database for E. cloacae identification was also constructed and evaluated. Based on the super reference database and the main spectra projection dendrogram, E. cloacae strains were divided into two clades.</p><p><b>CONCLUSION</b>Peptide mass fingerprinting is a powerful method to identify and subtype E. cloacae, and the use of this method will allow us to obtain more information to understand the heterogeneous organism E. cloacae.</p>
ABSTRACT
Ruminant placentas synthesize pregnancy-associated glycoproteins (PAGs) during pregnancy, which serve as biomarkers of pregnancy. The present study was conducted to verify, whether PAGs are expressed in buffalo placenta by using lectin-based affinity chromatography and peptide mass finger printing (PMF). Fetal cotyledonary tissues were collected from gravid uteri procured from slaughtered house. Proteins were extracted and subjected to wheat germ agglutinin (WGA) lectin affinity chromatography to isolate the PAGs. The isolated glycoproteins were separated by one-dimensional SDS-PAGE. PMF results of the 75 kDa protein revealed presence of two PAGs (PAG-7 and -11). The PAG-7 consisted of about 170 mass signals, of which 16 were assigned to corresponding/translated cDNA sequences of buffalo PAG-7, leading to sequence coverage of 40%. PMF result of PAG-11 showed 170 mass signals, of which 15 were assigned to buffalo PAG-11, leading to sequence coverage of 34%. In conclusion, the glycoprotein isolated from placental extract corresponding to 75 kDa band on SDS PAGE gel was a mixture of PAG-7 and -11, which may help in development of suitable diagnostics for pregnancy in buffalo.
Subject(s)
Amino Acid Sequence , Animals , Buffaloes , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Molecular Weight , Peptides/chemistry , Pregnancy Proteins/chemistryABSTRACT
With the rapid development of MS technologiesy, the demands for a more sophisticated MS interpretation algorithm haves grown as well. We have developed a new protein fingerprinting method using a binomial distribution, (fBIND). With the fBIND, we improved the performance accuracy of protein fingerprinting up to the maximum 49% (more than MOWSE) and 2% than(at a previous binomial distribution approach studied by of Wool et al.) as compared to the established algorithms. Moreover, we also suggest a the statistical approach to define the significance of transcription factors and motifs in the identified proteins based on the Gene Ontology (GO).
Subject(s)
Binomial Distribution , Fungal Proteins , Gene Ontology , Peptide Mapping , Transcription Factors , Wool , YeastsABSTRACT
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.
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Objective:Identification of some biochemical and physical properties for a new recombinant B7-2-PE40KDEL exotoxin fusion protein.Methods:12%SDS-PAGE separating and gel imaging analyzing,peptide mass fingerprinting,Western blot and MTT assasying were used respectively for identification of the protein.Results:Molecular weight of the recombinant B7-2-PE40KDEL was 72 628,5% of the difference to its theoretical value 69 561.The result of Western blot indicated that the purified recombinant B7-2-PE40KDEL could specifically bind with mAb anti-human B7-2 and the antibody against PEA,while the negative control did not.The recombinant B7-2-PE40KDEL digested with trypsin and then detected by MOLTI-TOF-MS.It was shown that the detected 15 peptides lied in the extracellular part of B7-2 and the truncated Pseudomonas extoxin PE40KDEL.Searching in the peptident data bank of Expasy website,we did not find any known proteins which was accordant with the above terms.The cytotoxic activity of the recombinant toxin with MTT method showed that the B7-2-PE40KDEL selectively killed Jurkat cell line which expressesed CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor.Conclusion:Recombinant B7-2-PE40KDEL exotoxin fusion protein we construct proves to be a new one with targeted killing bioactivity to B7:CD28 system.
ABSTRACT
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.