Rapid Identification and Subtyping of Enterobacter cloacae Clinical Isolates Using Peptide Mass Fingerprinting

Objective To establish a domestic database of Enterobacteria cloacae （E. cloacae）, and improve the identification efficiency using peptide mass fingerprinting. Methods 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. Results 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. Conclusion 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.

Evaluating Peptide Mass Fingerprinting-based Protein Identification

Identification of proteins by mass spectrometry （MS） is an essential step in proteomic studies and is typically accomplished by either peptide mass fingerprinting （PMF） or amino acid sequencing of the peptide. Although sequence information from MS/MS analysis can be used to validate PMF-based protein identification, it may not be practical when analyzing a large number of proteins and when highthroughput MS/MS instrumentation is not readily available. At present, a vast majority of proteomic studies employ PMF. However, there are huge disparities in criteria used to identify proteins using PMF. Therefore, to reduce incorrect protein identification using PMF, and also to increase confidence in PMF-based protein identification without accompanying MS/MS analysis, definitive guiding principles are essential. To this end, we propose a value-based scoring system that provides guidance on evaluating when PMF-based protein identification can be deemed sufficient without accompanying amino acid sequence data from MS/MS analysis.

Identification of proteins of human colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

AIM： To conduct bhe proteomic analysis of human colorectal carcinoma cell line, SW480 by using two-dimensional electrophoresis （2-DE） and matrix-assisted laser desorption /ionization-time of flight mass spectrometry （MALDITOFMS）. METHODS： The total proteins of human colorectal carcinoma cell line, SW480 were separated with 2-DE by using immobilized pH gradient strips and visualized by staining with silver nitrate. The gel images were acquired by scanner and 2-DE analysis software, Image Master 2D Elite. Nineteen distinct protein spots were excised from gel randomly and digested in gel by TPCK-trypsin. Mass analysis of the byptic digest peptides mixture was performed by using MALDI-TOF MS. Peptide mass fingerprints （PMFs） obtained by the MALDI-TOF analysis were used to search NCBI, SWISS-PROT and MSDB databases by using Mascot software. RESULTS： PMF maps of all spots were obtained by MALDI-TOF MS and thirteen proteins were preliminarily identified. CONCLUSION： The methods of analysis and identification of protein spots of tumor cells in 2-DE gel with silver staining by MALDI-TOF MS derived PMF have been established. Protein expression profile of SW480 has been obtained. It is demonstrated that a combination of proteomics and cell culture is a useful approach to comprehend the process of colon carcinogenesis.

Proteomic Study for Responses to Cadmium Stress in Rice Seedlings

A proteomic approach including two-dimensional electrophoresis and mass spectrometric （MALDI-TOF MS） analyses was used to investigate the responses to cadmium （Cd） stress in seedlings of rice （Oryza sativa L.） varieties Shanyou 63 and Aizaizhan. Cd stress significantly inhibited root and shoot growth, and affected the global proteome in rice roots and leaves, which induced or upregulated the expression of corresponding proteins in rice roots and leaves when rice seedlings were exposed to 0.1 or 1.0 mmol/L Cd. The Cd-induced proteins are involved in chelation and compartmentation of Cd, elimination of active oxygen free radicals, detoxification of toxic substances, degradation of denatured proteins or inactivated enzymes, regulation of physiologic metabolism and induction of pathogenesis-related proteins. Comparing the Cd-induced proteins between the two vadeties, the β-glucosidase and pathogenesis-related protein family 10 proteins were more drastically induced by Cd stress in roots and leaves of Aizaizhan, and the UDP-glucose protein transglucosylase and translational elongation factor Tu were induced by 0.1 mmol/L Cd stress in roots of Shanyou 63. This may be one of the important mechanisms for higher tolerance to Cd stress in Shanyou 63 than in Aizaizhan.

Two-dimensional Electrophoresis Analysis of Proteins in Response to Cold Stress in Extremely Cold-resistant Winter Wheat Dongnongdongmai 1 Tillering Nodes

The overwintering survival ratio of the cultivar Dongnongdongmai 1 with strong cold-resistance in paramos of Heilongjiang Province in China are over 85%. The tillering nodes are the most important organs for overwintering survival of winter wheat, because there are more substances associated with cold resistance in tillering nodes than those in leaves and roots. Proteins in the tillering nodes of the cold-resistant cultivar Dongnongdongmai 1 grown under field conditions with or without any lowtemperature stress were analyzed by 2-dimensional electrophoresis and identified by mass spectrometry. In the range of pH 4-7, the expression of 37 proteins showed obvious difference （±more than two fold） in the proteomic maps of cold-stressed and non-stressed tillering nodes, including a new protein spot. All proteins exhibiting the difference in expression were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, followed by a database search for protein identification and function prediction. Five groups of proteins were confirmed, namely stress-related proteins （22%）, metabolism-associated proteins （35%）, and signaling molecules （24%）, cell wall-binding proteins （5%）, unclear proteins （14%）. This indicated that tillering node cells supported the energy requirements of plant growth and stress resistance by signal transduction adapting to metabolism and structure.

Cloning, Expression and Characterization of Translationally Controlled Tumor Protein (TCTP) Gene from Flatfish Turbot (Scophthalmus maximus)

A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot （Scophthalmus maximus）, SmTCTP, was isolated with rapid amplification of cDNA Ends （RACE）. SmTCTP consisted of a 5＇ untranslated region （UTR） of 84 bp, a 3＇ UTR of 451 bp and an open reading frame （ORF） of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5＇UTR of SmTCTP started with a 5＇-terminal oligopyrimidine tract （5＇-TOP）, a typical feature for translationally controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known vertebrate TCTPs in a range of 58.8% to 64.1%. The length of fish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch （Lateolabraxjaponicus） is 170 aa in length, while that of zebrafish （Danio rerio） and rohu （Labeo rohita） is 171 aa in length. Northern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET3Oa-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further investigation of the biological function of TCTP in fish.

Synthetic protease inhibitor-induced inclusions in PC12 cells Potential proteomic characterization of six subunits in the 26S proteasome

Proteasome dysfunction during dopaminergic degeneration induces proteolytic stress, and is a contributing factor for the onset and formation of Lewy bodies. Results from our previous studies showed that synthetic proteasome inhibitor-induced inclusions in PC12 cells contained six subunits in the 26S proteasome. In the present study, mass spectrometry analysis of single protein spots resolved by two-dimensional gel electrophoresis and identified by bioinformatic analysis of peptide mass fingerprint （PMF） data were performed to comprehensively characterize the proteomic profile of the proteasome subunits. Results showed that six subunits in the 26S proteasome were characterized through accurate assignment by PMF data-specific protein identification in protein databases. Additionally, identification of one of the proteasome subunits was further confirmed using a subunit-specific antibody against non-adenosine triphosphatase subunit 11 of the 19S regulatory particle. Results suggest that the potential proteomic profile of six subunits in the 26S proteasome could be established from proteasome inhibitor-induced inclusions in PC12 cells.

A Novel Protein Found in Selenium-rich Silkworm Pupas

A novel protein was isolated and characterized in selenium-rich silkworm pupas. The peptide mass fingerprint of the protein was found to be new. Partial amino acid sequencing also confn-med to be a new protein. The novel protein had a molecular mass of about 80 kDa in the SDS-PAGE.