Aims: Acinetobacter baumannii has been identified as one of the six most pathogenic bacteria that is the cause of most hospital bacterial infections according to Infectious Disease Society of America (IDSA). These nosocomial pathogens are notorious worldwide due to its ability in causing lethal infections among immunocompromised patients and its resistance to many strong antibiotics. This study aims to compare the expressed proteins of two A. baumannii strain, ATCC 19606 and a pathogenic clinically isolated strain known as AB-13. Methodology and results: AB-13 clinically strain was isolated from the lower respiratory tract of a patient with pneumonia. In this study, the proteomic profile of both ATCC 19606 and AB-13 are produced using 2-dimensional gel electrophoresis. The total protein contents were extracted, quantified and separated using 2-DE with a pH range of 4-7 to acquire the proteomic profile for comparison. The final analytical gel was analysed using Delta2D software and among the 324 protein spots successfully resolved, 10 spots exhibited signs of differential expression with 7 spots found to be downregulated and 3 spots upregulated (p< 0.01). These differences could signify the evolution AB-13 has undergone as it acquires traits ultimately aiding in its survivability, antimicrobial resistance and pathogenicity within varied environments especially during infections. Conclusion, significance and impact of study These findings support the presence of variation in AB-13 from a proteomic perspective, highlighting the pathogen’s evolution improving survivability and pathogenicity, warranting in-depth exploration towards understanding A. baumannii virulence and pathogenicity.
Acinetobacter baumannii--pathogenicity ; Two-Dimensional Difference Gel Electrophoresis ; Proteomics

Animals ; Electrophoresis, Gel, Two-Dimensional ; Male ; Muscles ; Physical Conditioning, Animal ; Proteome ; Rats ; Rats, Sprague-Dawley

two or more consecutive unexplained miscarriages and with no female factor abnormality as revealed by gynaecologic investigation including karyotyping and age matched fertile healthy volunteers (n=20). All samples were collected during 2013 to 2015 after getting institutional ethical approval and written consent from the participants. Seminal ejaculates were collected by masturbation after 2 to 3 days of sexual abstinence and analyzed according to World Health Organization 5th criteria 2010. Two-dimensional difference gel electrophoresis followed by mass spectrophotometric analysis was used to identify differentially expressed proteins (DEPs). Western blotting was used for validation of the key proteins.RESULTS: The data identified 36 protein spots to be differentially expressed by more than 2-fold change with p<0.05 considered as significant. Matrix-assisted laser desorption/ionization time of flight/mass spectrometry identified GPx4, JIP4, ZN248 to be overexpressed while HSPA2, GSTM5, TF3C1, CC74A was underexpressed in RPL group. Western blot analysis confirmed the differential expression of key redox associated proteins GPx4 and HSPA2 in the RPL group. Functional analysis revealed the involvement of key biological processes that includes spermatogenesis, response to oxidative stress, protein folding and metabolic process.CONCLUSIONS: The present study provides a snapshot of the altered protein expression levels consistent with the potential involvement of the sperm chromatin landscape in early embryonic development.]]>
Abortion, Spontaneous ; Biological Processes ; Blotting, Western ; Chromatin ; Embryo Loss ; Embryonic Development ; Female ; Healthy Volunteers ; Humans ; Karyotyping ; Male ; Masturbation ; Metabolism ; Oxidation-Reduction ; Oxidative Stress ; Pregnancy ; Prospective Studies ; Protein Folding ; Proteomics ; Sexual Abstinence ; Spectrum Analysis ; Spermatogenesis ; Spermatozoa ; Two-Dimensional Difference Gel Electrophoresis ; World Health Organization

To establish a two-dimensional gel electrophoresis (2-DE) map for comparative proteomic analysis of rat spinal cord with chronic morphine tolerance, and to detect differentially expression proteins that are associated with chronic morphine tolerance.
 Methods: Sixteen male SD rats received the intrathecal catheterization operation and they were randomly divided into a morphine tolerance group (MT group, n=8) and a saline group (NS group, n=8). The lumbar enlargement segments of the MT group and the NS group spinal cord were harvested and proteins were separated by 2-DE. Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The 2-DE maps were visualized after coomassie blue staining and analyzed using PDQuest analysis software. Identification of differential protein spots was conducted by MALDI-TOF-MS, and the Mascot query software was used to search Swiss-Prot database for bioinformatics analysis. Western blotting was used to verify the expression of some differentially expressed proteins.
 Results: A total of 1 000 spots were identified in 2-DE maps of rat spinal cord tissues from the MT group and the NS group, and 36 proteins were significantly differentially expressed in the MT group compared with the NS group. Identification was conducted by MALDI-TOF-MS and Swiss-Prot database through Mascot query software, and a total of 14 proteins were obtained. Among them, 2 protein spots were down-regulated in the MT group compared with that in the NS group, and 12 protein spots were up-regulated in the MT group compared with that in the NS group. Two kinds of proteins (NUDAA, ENOG) were verified by Western blotting and the results were consistent with proteomics data.
 Conclusion: The optimized 2-DE profiles for the proteome of spinal cord tissue in rats with chronic morphine tolerance is established preliminarily, which showed that morphine tolerance can cause changes in the expression of various proteins in the spinal cord.
Animals ; Electrophoresis, Gel, Two-Dimensional ; Male ; Morphine ; Proteome ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Spinal Cord

BACKGROUND: Irritable bowel syndrome (IBS) is one of the most common functional intestinal diseases, but its pathogenesis is still unknown. The present study aimed to screen the differentially expressed proteins in the mucosa of colon between IBS with diarrhea (IBS-D) patients and the healthy controls. METHODS: Forty-two IBS-D patients meeting the Rome III diagnostic criteria and 40 control subjects from July 2007 to June 2009 in Chinese PLA General Hospital were enrolled in the present study. We examined the protein expression profiles in mucosa of colon corresponding to IBS-D patients (n = 5) and controls (n = 5) using 2-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Secondly, Western blot and immunohistochemical analysis were carried out to validate the screened proteins in 27 IBS-D patients and 27 controls. Thirdly, high-performance liquid chromatography (HPLC) was further carried out to determine ATP concentration in the mucosa of colon between 10 IBS-D patients and 8 controls. Comparisons between 2 groups were performed by Student's t-test or Mann-Whitney U-test. RESULTS: Twelve differentially expressed proteins were screened out. The α-enolase (ENOA) in the sigmoid colon (0.917 ± 0.007 vs. 1.310 ± 0.100, t = 2.643, P = 0.017) and caecum (0.765 ± 0.060 vs. 1.212 ± 0.122, t = 2.225, P = 0.023), Isobutyryl-CoA dehydrogenase (ACAD8) in the sigmoid colon (1.127 ± 0.201 vs. 1.497 ± 0.392, t = 7.093, P = 0.008) of the IBS-D group were significantly lower while acetyl-CoA acetyltransferase (CT) in the caecum (2.453 ± 0.422 vs. 0.931 ± 0.652, t = 8.363, P = 0.015) and ATP synthase subunit d (ATP5H) in the sigmoid (0.843 ± 0.042 vs. 0.631 ± 0.042, t = 8.613,P = 0.007) of the IBS-D group was significantly higher, compared with the controls. The ATP concentration in the mucosa of the sigmoid colon in IBS-D group was significantly lower than that of control group (0.470 [0.180, 1.360] vs. 5.350 [2.230, 7.900], U = 55, P < 0.001). CONCLUSIONS Many proteins related to energy metabolism presented differential expression patterns in the mucosa of colon of the IBS-D patients. The abnormalities in energy metabolism may be involved in the pathogenesis of IBS which deserves more studies to elucidate.
Adenosine Triphosphate ; metabolism ; Adult ; Blotting, Western ; Colon ; metabolism ; pathology ; Diarrhea ; enzymology ; metabolism ; pathology ; Electrophoresis, Gel, Two-Dimensional ; Energy Metabolism ; genetics ; physiology ; Female ; Humans ; Immunohistochemistry ; Intestinal Mucosa ; enzymology ; metabolism ; pathology ; Irritable Bowel Syndrome ; enzymology ; metabolism ; pathology ; Male ; Mass Spectrometry ; Middle Aged ; Proteome ; metabolism

In this study,the protein in different Cordyceps samples,which include fresh sample( S1),22 ℃ drying sample( S2),37 ℃ drying sample( S3) and 60 ℃ drying sample( S4),were analyzed by sodium dodecylsupinate-polyacrylamide gel electrophoresis( SDS-PAGE) and two-dimensional electrophoresis( 2-DE). The total protein contents in Cordyceps samples were from 1. 655-4. 493 mg·g~(-1) and the protein contents in fresh sample was the highest. The results of SDS-PAGE showed that the mainly ranges of protein molecular weight of Cordyces samples were 10-100 kDa and the numbers of protein bands were 28 to 41,the fresh sample had the maximum number of protein bands. The 2-DE profiles were analyzed by PDQuest software. The resulted indicated that 488-876 protein spots were detected in different Cordyceps samples and the isoelectric point( pI) was distributed between 4. 5 and 6. 5,the protein molecular weight was distributed in 10-20 kDa and 25-100 kDa,the fresh sample had the maximum number of protein spots. Therefore,the drying process could decrease contents and species of protein in Cordyceps,and the different drying conditions had different effects on protein. These results provide a reference for improving the drying process of Cordyceps.
Cordyceps ; chemistry ; Desiccation ; methods ; Electrophoresis, Gel, Two-Dimensional ; Electrophoresis, Polyacrylamide Gel ; Fungal Proteins ; analysis ; Molecular Weight

Glutamate leads to neuronal cell damage by generating neurotoxicity during brain development. The objective of this study is to identify proteins that differently expressed by glutamate treatment in neonatal cerebral cortex. Sprague-Dawley rat pups (post-natal day 7) were intraperitoneally injected with vehicle or glutamate (10 mg/kg). Brain tissues were isolated 4 h after drug treatment and fixed for morphological study. Moreover, cerebral cortices were collected for protein study. Two-dimensional gel electrophoresis and mass spectrometry were carried out to identify specific proteins. We observed severe histopathological changes in glutamate-exposed cerebral cortex. We identified various proteins that differentially expressed by glutamate exposure. Identified proteins were thioredoxin, peroxiredoxin 5, ubiquitin carboxy-terminal hydrolase L1, proteasome subunit alpha proteins, isocitrate dehydrogenase, and heat shock protein 60. Heat shock protein 60 was increased in glutamate exposed condition. However, other proteins were decreased in glutamate-treated animals. These proteins are related to anti-oxidant, protein degradation, metabolism, signal transduction, and anti-apoptotic function. Thus, our findings can suggest that glutamate leads to neonatal cerebral cortex damage by regulation of specific proteins that mediated with various functions.
Animals ; Brain ; Cerebral Cortex ; Chaperonin 60 ; Electrophoresis, Gel, Two-Dimensional ; Glutamic Acid ; Humans ; Infant, Newborn ; Isocitrate Dehydrogenase ; Mass Spectrometry ; Metabolism ; Neurons ; Peroxiredoxins ; Proteasome Endopeptidase Complex ; Proteolysis ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Thioredoxins ; Ubiquitin Thiolesterase

Various methods and specialized software programs are available for processing two-dimensional gel electrophoresis (2-DGE) images. However, due to the anomalies present in these images, a reliable, automated, and highly reproducible system for 2-DGE image analysis has still not been achieved. The most common anomalies found in 2-DGE images include vertical and horizontal streaking, fuzzy spots, and background noise, which greatly complicate computational analysis. In this paper, we review the preprocessing techniques applied to 2-DGE images for noise reduction, intensity normalization, and background correction. We also present a quantitative comparison of non-linear filtering techniques applied to synthetic gel images, through analyzing the performance of the filters under specific conditions. Synthetic proteins were modeled into a two-dimensional Gaussian distribution with adjustable parameters for changing the size, intensity, and degradation. Three types of noise were added to the images: Gaussian, Rayleigh, and exponential, with signal-to-noise ratios (SNRs) ranging 8-20 decibels (dB). We compared the performance of wavelet, contourlet, total variation (TV), and wavelet-total variation (WTTV) techniques using parameters SNR and spot efficiency. In terms of spot efficiency, contourlet and TV were more sensitive to noise than wavelet and WTTV. Wavelet worked the best for images with SNR ranging 10-20 dB, whereas WTTV performed better with high noise levels. Wavelet also presented the best performance with any level of Gaussian noise and low levels (20-14 dB) of Rayleigh and exponential noise in terms of SNR. Finally, the performance of the non-linear filtering techniques was evaluated using a real 2-DGE image with previously identified proteins marked. Wavelet achieved the best detection rate for the real image.
Algorithms ; Animals ; Electrophoresis, Gel, Two-Dimensional ; methods ; Humans ; Image Processing, Computer-Assisted ; methods ; Proteins ; analysis ; Proteomics ; methods ; Software

OBJECTIVE: To investigate the effect of moderate-intensity aerobic exercise on the differential expression of rat atrial muscle Proteomics and genes, which provide research basis for the rehabilitation of chronic cardiovascular diseases and exercise -induced cardiac remodeling research. METHODS: Twenty male SD rats were randomly divided into control group and experimental group (=10) according to body weight. Rats in the experimental group were trained (6 days per week),which lasted for 4 weeks of moderate-intensity aerobic exercise at a rate of 24 m·min for 40 min (load intensity equivalent to 60%~70% VO). The proteins were separated by two dimensional gel electrophoresis, and the tandem time-of- flight mass spectrometer technique was used to identify 13 candidate target protein spots. The expression levels of these 13 protein spots were up-regulated more than 5 times or down -regulated to below 1/5. The mRNA of six target proteins were detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: By software analysis, the experimental group compared with the control group, there were 8 protein points which their expression reduced more than 4/5 and 5 protein points up-regulated more than 5 times, 13 proteins were identified by mass spectrometry protein spots, the final identification results acquired 8 proteins and a unknown protein of molecular mass 54 KDa, such as:pyruvate dehydrogenase E1α1, mitochondrial aconitate hydratase, protein disulfide isomerase A3, methylmalonic acid semialdehyde dehydrogenase, mitochondrial dihydrolipoic acid dehydrogenase, isovaleryl coenzyme A dehydrogenase, glutathione synthetase, mitogen-activated protein kinase 3 and so on. Compared with the control group, the mRNA expression of methylmalonic acid semialdehyde dehydrogenase in the atrial muscle of rats was decreased after 4 weeks of moderate aerobic exercise (<0.05), the mRNA expression levels of mitochondria Ⅱ lipoic acid dehydrogenase, protein disulfide isomerase A3, mitochondrial aconitate hydratase and glutathione synthetase were decreased (>0.05); The mRNA expression level of isopentenyl-CoA dehydrogenase was increased (>0.05). The results indicated that the mRNA expression level was not completely consistent with the changes in mass spectrometry identification results. CONCLUSIONS The 4 weeks moderate-intensity aerobic exercise induced ignificant changes of rats atrial muscle protemics. The majority of the 13 identified target proteins in this experiment are energy metabolism enzymes. The majority of the expression of the target protein and the mRNA expression in the atrial muscle is inconsistent and different. Exercise may affect the regulation of gene transcription or downstream translation and modification of these target proteins, resulting in the change of differential expression.
Animals ; Electrophoresis, Gel, Two-Dimensional ; Male ; Muscles ; Physical Conditioning, Animal ; Proteomics ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To investigate the innate characters of 3 endometriosis (EMT) syndromes, blood stasis (BS), qi stagnation and blood stasis (QSBS) as well as Shen (Kidney) deficiency and blood stasis (KDBS) in terms of proteomics, lay a molecular biological basis for the differentiation of various blood stasis syndromes of EMT, establish a EMT microscopic syndrome differentiation and diagnosis system in terms of proteomics, discover the evolution principles and therapeutic targets of these EMT syndromes, and search their signifificant molecular markers and genetic intervention targets. METHODS: Six specimens from the ectopic and entopic endometrium tissues of patients with EMT in each syndrome, BS, QSBS as well as KDBS, in the early proliferative phase of the menstrual cycle, and 6 specimens from normal endometrium tissues in the early proliferative phase of the menstrual cycle were obtained. Three groups were formed in each syndrome by mixing two random specimens in equal amount, and then their respective two-dimensional electrophoresis graphs were obtained after total protein extraction. Finally, the detected differences in protein expression were identifified through matrix-assisted laser desorption Ionization-time of flflight mass spectrometry (MALDI-TOF/MS) and protein database. RESULTS: The results of differential proteins expressed in each syndrome were shown as follows: BS syndrome had 2 differential proteins in entopic endometrium and 1 differential protein in ectopic endometrium; KDBS syndrome had 3 in entopic endometrium and 3 in ectopic endometrium; and QSBS syndrome had 3 in entopic endometrium and 4 in ectopic endometrium. It was found out that annexin was highly expressed in both entopic and ectopic endometrium of KDBS syndrome; and myosin light chain 3 was highly expressed in both entopic and ectopic endometrium of QSBS syndrome. CONCLUSION There are differential protein expressions among the 3 EMT syndromes, which might be the inner origin of syndrome characters, and these differential proteins might be the candidate biomarkers for the pathogenesis of various EMT syndromes.
Adult ; Electrophoresis, Gel, Two-Dimensional ; Endometriosis ; blood ; metabolism ; Female ; Humans ; Mass Spectrometry ; Proteome ; metabolism ; Proteomics ; methods ; Syndrome