Optimization of Two-Dimensional Gel Electrophoresis for Kenaf Leaf Proteins

To establish a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis （2-DE） analysis in kenaf leaf tissues, three extraction methods （trichloroacetic acid/acetone, urea/thiourea, and phenol extraction methods） were applied to the extraction of kenaf leaf protein. The results were compared in regard to protein extraction efficiency, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and 2-DE gels. Furthermore, the 2-DE system was optimized for four aspects： the pH range of IPG （immobilized pH gradient） stripes, sampling methods, sample volumes, and concentration of polyacrylamide gels. The data presented showed that the phenol extraction method is the best method to perform 2-DE analysis of kenaf leaf protein. The protein extracted from phenol extraction method reached the purity of （26.40±0.859）%, showed （25.67±1.53） protein bands in one dimension SDS-PAGE gels, and （1 374±54.44） protein spots on 2-DE gels. The research also indicates that kenaf leaf protein spots were distributed mainly within the pH range of 4-8. More clear background with a better distribution effect and many protein spots could be obtained on 2-DE gels under the conditions of active rehydration loading, 24 cm IPG strips （linear pH gradient of 4-7）, 1.4 mg samples, and 12% SDS-PAGE gels.

Two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry for detection of protein expression profiles in the hippocampus following closed brain injury

Gene expression profile changes in brain regions following traumatic brain injury at the gene level cannot sufficiently elucidate gene expression time, expression amount, protein post-translational processing or modification. Therefore, it is necessary to quantitatively analyze the gene expression profile using proteomic techniques. In the present study, we established a rat model of closed brain injury using Marmarou＇s weight-drop device, and investigated hippocampal differential protein expression using two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry. A total of 364 protein peaks were detected on weak cation exchange-2 protein chips, including 37 differential protein peaks. 345 protein peaks were detected on immobilized metal affinity capture arrays-Cu, including 12 differential protein peaks Further examination of these differential proteins revealed that glucose-regulated protein and proteasome subunit alpha type 3 expression were significantly upregulated post-injury. These results indicate that brain injury can alter protein expression in the hippocampus, and that glucose-regulated protein and proteasome subunit alpha type 3 are closely associated with the occurrence and development of traumatic brain injury.

Comparative Proteomic Analysis of B. henselae Houston and B. henselae Marseille by Two-dimensional Gel Electrophoresis

To compare the protein difference between B. henselae Houston and B. henselae Marseille by two-dimensional gel electrophoresis. Method Protein samples were prepared by vorterx, ultrasonic treatment, and centrifugation. Protein concentrations were determined by Bradford method. Protein difference was compared by the first IEF and the second SDS-polyacrylamide gel electrophoresis. Results Protein concentrations in samples of Bartonella henselae Houston and Bartonella henselae Marseille were 2.117 μg/μL and 2.200 μg/μL respectively. Sample protein of 40 μg for IPG strips loading was perfect. The results of 2-DE in pH 4 to 7 IPG strips showed that the total protein spots of Bartonella henselae Houston and Bartonella henselae Marseille were 375 and 379 respectively, 95% of the spots were the same between the two strains of Bartonella henselae. Conclusion The procedure of 2-DE may prove successful for the proteomic analysis of Bartonella henselae. Bartonella henselae Houston and Bartonella henselae Marseille are different genotypes.

Establishment of two-dimensional gel electrophoresis for soybean protein isolate and its application

To optimize the conditions for the establishment of two-dimensional gel electrophoresis(2-DE)of soybean protein isolate(SPI),we investigated Ampholine mixture,anodic and cathodic electrolytes,loading amount of sample,acrylamide concentration,p H gradient and gel staining method in twodimensional gel electrophoresis to optimize the protein imaging conditions in two-dimensional gel.The results of mixed-level design experiments showed that Ampholine,loading amount and gel staining method had significant effect(P<0.05)on 2-DE of SPI.The optimal conditions were Ampholine mixture(pH 3–10+pH 5–7 or pH 4–6+pH 5–7),loading amount of 2 mg sample and silver staining.Although the acrylamide concentration of the gel,the p H gradient,the anodic and cathodic electrolyte solutions had significant statistical effects on the protein separation degree,the complexity of the protein composition and the visibility of the gel images were more inclined to the 12%gel,the 3–10 pH gradient and the H3PO4/NaOH electrolyte.According to the established conditions,the hydrolyzed products of SPI emulsion were determined by 2-DE,and the dynamic changes of protein in the process of enzymatic hydrolysis were described.

Protein expression of sensory and motor nerves Two-dimensional gel electrophoresis and mass spectrometry

The present study utilized samples from bilateral motor branches of the femoral nerve, as well as saphenous nerves, ventral roots, and dorsal roots of the spinal cord, to detect differential protein expression using two-dimensional gel electrophoresis and nano ultra-high performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry techniques. A mass spectrum was identified using the Mascot search. Results revealed differential expression of 11 proteins, including transgelin, Ig kappa chain precursor, plasma glutathione peroxidase precursor, an unnamed protein product （gil55628）, gfyceraldehyde-3-phosphate dehydrogenase-like protein, lactoylgfutathione lyase, adenyfate kinase isozyme 1, two unnamed proteins products （gil55628 and gi11334163）, and poly（rC）-binding protein 1 in motor and sensory nerves. Results suggested that these proteins played roles in specific nerve regeneration following peripheral nerve injury and served as specific markers for motor and sensory nerves.

Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

Objective To identify the sperm membrane proteins that are associated with antisperm antibody Methods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody. Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5～6.2, 4.6,5.1,5.5～5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher. Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two- dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.

In Vitro Protein Expression Profile of Campylobacter jejuni Strain NCTC11168 by Two-dimensional Gel Electrophoresis and Mass Spectrometry

Objective To investigate the protein expression profiles of the major food‐borne pathogen Campylobacter jejuni NCTC11168.Methods Membrane and soluble cellular proteins were extracted from the genome‐sequenced C.jejuni strain NCTC11168.Protein expression profiles were determined using two‐dimensional gel electrophoresis（2‐DE）.All the detected spots on the 2‐DE map were subjected to matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry（MALDI‐TOF/TOF） analysis.Results A total of 537 and 333 spots were detected from the whole cell and membrane‐associated proteins of C.jejuni NCTC11168 cultured on Columbia agar medium at 42 ℃ by 2‐DE and Coomassie Brilliant Blue staining,respectively.Analyses of whole cell and membrane‐associated proteins included 399 and 133 spots,respectively,which included 182 and 53 functional proteins identified by MALDI‐TOF/TOF analysis.Conclusion The comprehensive expression protein profiles of C.jeuni NCTC11168 obtained in this study will be useful for elucidating the roles of these proteins in further pathogenesis investigation.

Improvement of Two-Dimensional Gel Electrophoresis for Study of Corm Formation Related Proteins in vitro from Taro (Colocasia esculenta)

Efficient and reproducible sample preparation prior to 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) is a critical step in achieving accurate and reliable data. In this paper, we described a method to prepare protein samples of taro that was compatible with subsequent analysis using 2D-PAGE. We compared proteins from shoot basal region from 0 d and 2 d after the beginning of tuberization. By this method we got about (2 000) spots and high reproducibility. Additionally some changes of protein expression were found.

Differential analysis of two-dimensional gel electrophoresis profiles of spermatozoa protein in human normal motility sperm and idiopathic asthenospermia

Objective: To evaluate the application of two-dimensional electrophoresis in the research of differentially expressed proteins in the human asthenospermia. Methods: Two-dimensional gel electrophoresis was performed on 4 normal sperm samples from healthy men and 4 sperm samples from 4 asthenospermia patients. After silver staining, the differential expression proteins were analyzed by PDQuest 2D analysis software. Results: Six differential protein spots were identified. Four spots showed increased expression in the control gels compared with the patient gels. Conclusion: The protein profiles of differential expression between the normal spermatozoa and idiopathic asthenospermia were established and some differential proteins were found. The data of this study would establish the better fundament for further isolation and identification of differentially expressed proteins in human asthenospermia sperm.

Two-dimensional gel electrophoresis in bacterial proteomics

Two-dimensional gel electrophoresis(2-DE)is a gel-based technique widely used for analyzing the pro-tein composition of biological samples.It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal bio-physical separation techniques:isoelectric focusing(first dimension)and polyacrylamide gel electrophore-sis(second dimension).2-DE is ideally suited for ana-lyzing the entire expressed protein complement of a bacterial cell:its proteome.Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of envi-ronmental and medically-relevant bacteria.Here we give a broad overview of the basic principles and his-torical development of gel-based proteomics,and how this powerful approach can be applied for studying bacterial biology and physiology.We highlight specific 2-DE applications that can be used to analyze when,where and how much proteins are expressed.The links between proteomics,genomics and mass spectrometry are discussed.We explore how proteomics involving tandem mass spectrometry can be used to analyze(post-translational)protein modifications or to identify proteins of unknown origin by de novo peptide se-quencing.The use of proteome fractionation tech-niques and non-gel-based proteomic approaches are also discussed.We highlight how the analysis of pro-teins secreted by bacterial cells(secretomes or exo-proteomes)can be used to study infection processes or the immune response.This review is aimed at non-specialists who wish to gain a concise,compre-hensive and contemporary overview of the nature and applications of bacterial proteomics.

Comparative proteome analysis of Helicobacter pylori clinical strains by two-dimensional gel electrophoresis

Objective： To investigate the pathogenic properties of Helicobacterpylori by comparing the proteome map of H. pylori clinical strains. Methods： Two wild-type H. pylori strains, YN8 （isolated from biopsy tissue of a gastric cancer patient） and YN14 （isolated from biopsy tissue of a gastritis and duodenal ulcer patient）, were used. Proteomic analysis, using a pH range of 3-10 and 5-8, was performed. The individual proteins were identified by quadrupole time-of-flight （Q-TOF） mass spectrometer and protein database search. Results： Variation in spot patterns directed towards differential protein expression levels was observed between the strains. The gel revealed prominent proteins with several protein ＂families＂. The comparison of protein expressions of the two strains reveals a high variability. Differentially present or absent spots were observed. Nine differentially expressed protein spots identified by Q-TOF included adenosine triphosphate （ATP）-binding protein, disulfide oxidoreductase B （DsbB）-Iike protein, N utilization substance A （NusA）, ATP-dependent protease binding subunit/heat shock protein, hydantoin utilization protein A, seryl-tRNA synthetase, molybdenum ABC transporter ModD, and hypothetical proteins. Conclusions： This study suggests that H. pylori strains express/repress protein variation, not only in terms of the virulence proteins, but also in terms of physiological proteins, when they infect a human host. The difference of protein expression levels between H. pylori strains isolated from gastric cancer and gastritis may be the initiator of inflammation, and result in the different clinical presentation. In this preliminary study, we report seven differential proteins between strains, with molecule weights from approximately 10 kDa to approximately 40 kDa. Further studies are needed to investigate those proteins and their function associated with H. pylori colonization and adaptation to host environment stress.

PHProteomicDB:A Module for Two-dimensional Gel Electrophoresis Database Creation on Personal Web Sites

PHProteomicDB is a PHP-written module to help researchers in proteomics to share two-dimenslonal gel electrophoresis data using personal web sites. No technical or PHP knowledge is necessary except a few basics about web site management. PHProteomicDB has a user-friendly administration interface to enter and update data. It creates web pages on the fly displaying gel characteristics, gel pictures, and numbered gel spots with their related identifications pointing to their reference pages in protein databanks. The module is freely available at http：//www.huvec.com/index.php3？rub=Download.

Two-dimensional gel electrophoresis analysis of the proteomes expressed in the human hepatoma cell line BEL-7404 and normal liver cell line L-02

Proteome analysis technology has been used extensively in conducting discovery research of biology and has become one of the most essential technologies in functional genomics. The proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 have been separated by high resolution two-dimensional gel electrophoresis (2-DE) with immobilized pH gradient isoelectric focusing (IPG-IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension (IPG-DALT). The resulting images have been analyzed using 2-D analysis software. Quantitative analysis reveals that 7 protein spots are detected only in hepatoma BEL-7404 cells, 14 only in L-02 cells, and 78 protein spots show significant fluctuation in quantity in both cell lines (P【0.01). These protein spots have been displayed on a proteome differential expression map. Analysis for the reproducibility of 2-DE indicates that the positional variability in the IEF dimension

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.

Specific protein expression in a rat model of early focal cerebral ischemia:Fluorescent two-dimensional difference gel electrophoresis

BACKGROUND： The use of fluorescent two-dimensional difference gel electrophoresis （2D-DIGE） has been shown to compensate for the shortcomings of conventional two-dimensional gel electrophoresis, such as poor repeatability and large systematic errors. However, little information is presently available regarding the use of 2D-DIGE to investigate mechanisms of ischemic cerebrovascular diseases. Plasma and body fluids have been utilized in proteomic technology to study ischemic cerebrovascular diseases. OBJECTIVE： To perform proteomic analysis of fresh rat brain tissue in peripheral ischemic regions using 2D-DIGE 6 hours after middle cerebral artery occlusion （MCAO）, and to identify specific proteins closely associated with early ischemic cerebrovascular diseases. DESIGN, TIME AND SETTING： Proteomics-based, randomized, controlled, animal experiment was performed at the Laboratories of Neurology and Proteomics, Jilin University between January and April 2006. MATERIALS： 2, 3, 5-triphenyl tetrazolium chloride was purchased from Sigma, USA. Ettan DALTSix system, DeCyder DIA V5.0 differential analysis software, and Ettan matrix-assisted laser desorption/ionization time-of-flight mass spectrometer （MALDI-TOF-MS） were purchased from Amersham Bioscience, Sweden. METHODS： Eight healthy, male, Wistar rats were randomized to experimental and control groups, with four rats in each group. In the experimental group, rat models of focal cerebral ischemia were established by MCAO. In the control group, the internal and external carotid arteries were exposed and then immediately sutured, and the remaining procedures were identical to the experimental group. MAIN OUTCOME MEASURES： At 6 hours after cerebral ischemia, protein expression in the peripheral ischemia region of the experimental group was compared with the control group using 2D-DIGE. Protein spots that exhibited statistical differences between experimental and control groups with 〉 1.4 attributable risk were screened using DeCyder DIA V5.0 differential analysis software. Differential proteins were identified using MALDI-TOF-MS. RESULTS： Triphenyl tetrazolium chloride staining results revealed pink, normal brain tissue and white, ischemic brain tissue, suggesting successful MCAO establishment. The average matching rate of four 2D-DIGE gels was 92.4%. There were （1 758 ± 43） protein spots on each gel, with similar distribution modes. At 6 hours after focal cerebral ischemia, 13 protein spots exhibited marked expression changes, including significantly increased （n = 7） and decreased （n = 6） expression （P 〈 0.05）. MALDI-TOF-MS results revealed two differential protein spots： a-tubulin and heat shock protein 27, which were significantly decreased in the experimental group compared with the control group （P 〈 0.05）. CONCLUSION： Thirteen protein spots with expression changes were revealed by 2D-DIGE proteomics technology. Of them, a-tubulin and heat shock protein 27 expressions were markedly decreased during the early stage of cerebral ischemia. These two proteins were presumed to be proteins associated with early ischemic cerebrovascular diseases.

Analysis of Resistance-related Proteins in Rice Against Brown Planthopper by Two-dimensional Electrophoresis

A recombinant inbred population (RI) was constructed from a cross between B5, an introgression. line from the wild rice Oryza officinalis Wall. ex Watt, and susceptible cultivar Minghui 63 ( O. sativa L.). The brown planthopper ( BPH) resistances of RI lines were evaluated. Based on bulked segregant analysis (BSA), two protein bulks were made by extracting proteins from equally mixed seedlings of extremely resistant and susceptible plants selected from the RI population, respectively. Two-dimensional electrophoresis was used to detect the changes of polypeptide pattern. Results showed that a protein P40 ( pI 6.3, Mw 40 kD) was significantly reduced or vanished after BPH infestation for 48 h in the susceptible bulk, while it remained uninfluenced in the resistant bulk. In connection with the physiological changes of the resistant and susceptible lines subjected to BPH sucking, we suppose that the protein P40 is related to the interaction responses of lice plants to BPH infestation.

Study on the Optimization of Two-dimensional Electrophoresis Technology System for Rapeseed Proteome

[Objective] The research aimed to establish the two-dimensional electrophoresis（2-DE）technology which was suitable for the rapeseed proteome research.[Method] Xiangyou 17 was as the material.The sample preparation method,gel concentration and loading amount,etc.in 2-DE technology were optimized.[Result] The best extraction method of total protein of rapeseed was TCA-acetone method,and the protein spots on 2-DE map were the most.When IPG strip（pH 3-10）and 12% gel were used,and the loading amount was 250 μg,the two-dimensional electrophoresis map with the clear background,good repeatability and high protein spot resolution was obtained.[Conclusion] The research laid the foundation for carrying out the rapeseed proteomics research.

Cerebrospinal fluid diagnostic markers for two-dimensional electrophoresis-mass spectrometry in Parkinson’s disease patients

BACKGROUND： Previous studies have confirmed the existence of specific proteins in body fluid of Parkinson＇s disease （PD） patients. However, the existing research has contained several interference factors with poor reproducibility and has not focused on patients grouped according to disease duration. OBJECTIVE： To verify differential expression of proteins in cerebrospinal fluid of PD patients grouped in order of disease severity through the use of two-dimensional electrophoresis-mass spectrometry methods. DESIGN, TIME AND SETTING： The proteomic-based, case-control study was performed between September 2008 and June 2009 at the Key Laboratory of Neurology in the First Affiliated Hospital of Chongqing Medical University. PARTICIPANTS： A total of 52 outpatients and/or inpatients, who were admitted to the Department of Neurology in the First Affiliated Hospital of Chongqing Medical University between 2008 and 2009, were randomized into the present study. Among them, 27 PD patients served as the PD group and were assigned to three subgroups according to modified Webster, Hoehn, and Yahr rating scales： 14 = mild, 8 = moderate, and 5 = severe; non-PD group of 16 patients included 5 cases of viral meningitis, 3 cases of acute myelitis, 1 case of Guillain-Barre syndrome, 2 cases of tuberculous meningitis, 2 cases of restless legs syndrome, and 3 cases of essential tremor; control group （n = 9） consisted of muscular tension headache in 6 cases, as well as syncope, trigeminal neuralgia, idiopathic orthostatic hypotension in 1 case. METHODS： Cerebrospinal fluid was collected from the involved patients using the lumbar puncture method. Proteins in the cerebrospinal fluid were separated by two-dimensional electrophoresis. MAIN OUTCOME MEASURES： Characteristics of protein electrophoresis patterns were analyzed, differentially expressed proteins were detected using matrix-assisted laser desorption ionization time of flight mass spectrometry, and protein data were analyzed in the Mascot database. RESULTS： Five protein electropherograms were analyzed by PDQuest 8.0, and （789 ± 32） protein spots were observed. There were significant differences in four protein spots in each of the PD sub-groups compared with the non-disease and control groups. Expression was down-regulated in three protein spots and up-regulated in one protein spot; 100% repetition rate was observed in four protein spots. According to the Mascot database, protein spots with down-regulated expression were as follows： DNA-guided RNA polymerase III subunit RPC5 （score： 50 points）; double serine, threonine, and tyrosine protein kinase （score： 64 points, P 〈 0.05）; activity-regulated cytoskeleton-associated protein （score： 58 points, P 〈 0.05）. However, G2 mitotic-specific cyclin was up-regulated （score： 84 points, P 〈 0.05）. CONCLUSION： Differential protein expression in the cerebrospinal fluid of PD patients was detected by two-dimensional electrophoresis-mass spectrometry, revealing changes in DNA-guided RNA polymerase III subunit RPC5, double serine, threonine, and tyrosine protein kinase, activity-regulated cytoskeleton-associated protein, and G2 mitotic cell cyclin, with good reproducibility.

Two-dimensional Electrophoresis Analysis of Differential Protein Expression in Squamous Carcinoma of the Cervix

Objective： To establish and optimize the two-dimensional gel electrophoresis （2-DE） maps of squamous carcinoma of the cervix and to study the protein difference between squamous carcinoma of the cervix （SCC） and normal cervical tissue. Methods： Using Two-dimensional gel electrophoresis followed by computer-assisted image analysis, the differential proteins between squamous carcinoma of the cervical tissue and normal cervical tissue were compared. Then using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, the differential proteins were identified. Results： The well-resolved and reproducible two-dimensional gel electrophoresis patterns of squamous carcinoma of the cervix tissue and normal cervical tissue were obtained. After silver staining, the average matching ratio of squamous carcinoma of the cervix was 86.1%. There was a good reproducibility of spot position in 2-DE map, with average deviation in IEF direction of 0.95±0.13 mm, while in SDS-PAGE direction it was 1.20±0.18 mm. Ten protein spots were identified by mass spectrometry, some of which were involved in cell proliferation, cell apoptosis, intracellular enzymes, structural proteins, cycle regulation, and tumor occurrence. Conclusion： The differentially expressed proteins provide a fundamental basis for further study of human squamous carcinoma of the cervix and screening of its specific markers.

Differentially expressed phosphoproteins in diazoxide-pretreated ventricular myocytes by two-dimensional electrophoresis and mass spectrometry in vitro

Objectives To analyze and identify differentially expressed phosphoproteins associated with mitochondrial KATP channel opening. Methods： Adult rat ventricular myocytes were isolated, cultured, and identified, and pretreated without or with 100 μmol/L diazoxide for 10 min. Phosphoproteins prepared and enriched from the control and diazoxide-pretreated cells were separated by two-dimensional gel electrophoresis （2-DE） followed by sliver staining. The obtained interesting phosphoproteins were further identified by mass spectrometry. Results. Associated with diazoxide preconditioning, the proteins of chaperonin containing TCP-1 and hypothetical protein XP-346548 were phosphorylated significantly （P〈0. 01）, while the 94-kDa glucose-regulated protein, calpactin I heavy chain and ferritin were dephosphorylated markedly （P〈0. 01）. Conclusion： These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins in order to respond diazoxide preconditioning, and these phosphorylated protein may mediate the downstream signaling of cardioprotection by mitochondrial KATp channel opening induced by ischemic preconditioning.