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.

Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry

Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round- headed and normal spermatozoa. Two-dimensional （2-D） fluorescence difference gel electrophoresis （DIGE） coupled with mass spectrometry （MS） was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry （MS/MS） exhibited significant changes （paired t-test, P 〈 0.05） in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.

Identification of differentially expressed proteins in SH-SY5Y cells treated with resveratrol

To gain insight into the molecular mechanisms of resveratrol-mediated neuroprotection, two-dimensional difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to identify proteins differentially-expressed in SH-SY5Y cells treated with resveratrol. Compared with the control group, resveratrol treatment significantly affected the expression of four proteins： endoplasmic reticulum oxidoreductin 1-like protein alpha, p21-activated kinase 1, Archain 1, and T cell receptor beta chain. The former three were downregulated and the latter was upregulated. These proteins are primarily associated with endoplasmic reticulum stress, intracellular trafficking, and immune function.

Valproic acid alters differential protein expression in SH-SY5Y cells

This study sought to identify differentially expressed proteins in SH-SY5Y cells treated with valproic acid, using two-dimensional difference gel electrophoresis analysis. Three proteins were unambi-guously identified： the eukaryotic translation initiation factor 4A isoform 1 and ATP6V1B2 protein were downregulated, while the heterogeneous nuclear ribonucleoprotein K was upregulated. Moreover, all three proteins are associated with altered expression due to oxidative stress. Ma-trix-assisted laser desorption/ionization-time of flight mass spectrometry and protein immunoblotting assay confirmed the differential expression of eukaryotic translation initiation factor 4A isoform 1. The results indicate that valproic acid exerts an antioxidation effect by regulating the expression of eukaryotic translation initiation factor 4A isoform 1.

Characterization of Acute Renal Allograft Rejection by Human Serum Proteomic Analysis

To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis （2-D DIGE） and reversed phase high-performance liquid chromatography （RP-HPLC） followed by electrospray ionization mass spectrometry （ESI-MS） were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups： acute rejec- tion （AR）, stable renal function （SRF） and normal volunteer （N）. Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differentially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were excised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor, apolipoprotein A-IV precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor, etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into understanding the mechanisms and potential treatment strategy of acute rejection.

Systemic Nuclear Proteomics Researches on Change of Jurkat T Lymphocyte Cells Under Radiation

Radiation causes severe constraint on numerous pathological functions of cells, such as cell growth, nuclear genetic material expression and cell functions. In this study, we performed proteomic profiling of a nuclear fraction of Jurkat T lymphocyte cells under radiation along different time course by means of 2DE and MALDI TOF-MS. We found 24 protein spots whose expression had changed after radiation, including relevant proteins, genetic material proteins, metabolism proteins, molecular chaperon and nuclear membrane proteins. Based on the above it is concluded that the combination of fluorescence labeled 2D-PAGE and MALDI-TOF MS is more precisely and effectively to elucidate the protein changes in Jurkat T lymphocyte cells after irradiation.

Original article Preliminary analysis of cerebrospinal fluid proteome in patients with neurocysticercosis

Background Neurocysticercosis is the infection of the nervous system by the larvae of Taenia solium （T. solium）. Despite continuous effort, the experimental diagnosis of neurocysticercosis remains unresolved. Since the cerebrospinal fluid （CSF） contacts with the brain, dynamic information about pathological processes of the brain is likely to be reflected in CSF. Therefore, CSF may serve as a rich source of putative biomarkers related to neurocysticercosis. Comparative proteomic analysis of CSF of neurocysticercosis patients and control subjects may find differentially expressed proteins.Methods Two-dimensional difference in gel electrophoresis （2D-DIGE） was used to investigate differentially expressed proteins in CSF of patients with neurocysticercosis by comparing the protein profile of CSF from neurocysticercosis patients with that from control subjects. The differentially expressed spots/proteins were recognized with matrix-assisted laser desorption/ionization-time of flight-time of flight （MALDI-TOF-TOF） mass spectrometry.Results Forty-four enzyme digested peptides were obtained from 4 neurocysticercotic patients. Twenty-three were identified through search of the NCBI protein database with Mascot software, showing 19 up-expressed and 4 down-expressed. Of these proteins, 26S proteosome related to ATP- and ubiquitin-dependent degradation of proteins and lipocalin type prostaglandin D synthase involved in PGD2-synthesis and extracellular transporter activities were up-expressed, while transferrin related to iron metabolism within the brain was down-expressed.Conclusions This study established the proteomic profile of pooled CSF from 4 patients with neurocysticercosis, suggesting the potential value of proteomic analysis for the study of candidate biomarkers involved in the diagnosis or pathogenesis of neurocysticercosis.

Quantitative proteomics analysis of parthenogenetically induced pluripotent stem cells

Parthenogenetic embryonic stem(pES)cells isolated from parthenogenetic activation of oocytes and embryos,also called parthenogenetically induced pluripotent stem cells,exhibit pluripotency evidenced by both in vitro and in vivo differentiation potential.Differential proteomic analysis was performed using differential in-gel electrophoresis and isotope-coded affinity tag-based quantitative proteomics to investigate the molecular mechanisms underlying the developmental pluripotency of pES cells and to compare the protein expression of pES cells generated from either the in vivo-matured ovulated(IVO)oocytes or from the in vitro-matured(IVM)oocytes with that of fertilized embryonic stem(fES)cells derived from fertilized embryos.A total of 76 proteins were upregulated and 16 proteins were downregulated in the IVM pES cells,whereas 91 proteins were upregulated and 9 were downregulated in the IVO pES cells based on a minimal 1.5-fold change as the cutoff value.No distinct pathways were found in the differentially expressed proteins except for those involved in metabolism and physiological processes.Notably,no differences were found in the protein expression of imprinted genes between the pES and fES cells,suggesting that genomic imprinting can be corrected in the pES cells at least at the early passages.The germline competent IVM pES cells may be applicable for germ cell renewal in aging ovaries if oocytes are retrieved at a younger age.

Comparative proteomic analysis of rat juvenile and adult dura

Background It has been widely observed that infants and young children can reossify large calvarial defects when they are younger than 2 years of age; afterwards, they lose this regenerative potential. Previous studies have implicated that the dura mater serves as a key regulator of calvarial regeneration. However, the molecular mechanism of calvarial reossification remains elusive. Methods In order to identify the proteins that may participate in this process, we performed a proteome-wide comparison of the protein expression levels of immature and mature dura using 2D electrophoresis and MALDI-TOF mass spectrometry. The Western blotting was used to verify the results of the 2D electrophoresis/MALDI-TOF mass spectrometry. Results Eleven proteins were found to express with significant differences in the immature and the mature dura. Among them, the emergence of vimentin, tropomyosin, 13-actin and y-actin were further confirmed by the Western blotting analysis. Conclusion The proteins and proteomic profiles provide a better understanding of the molecular mechanism of calvarial regeneration.

Proteomic analysis of the alteration of protein expression in the placenta of Down syndrome

Background Down syndrome （DS） is the most common form of human aneuploidy, and there is no effective therapy for the chromosomal abnormalities. We aimed to unravel the molecular mechanisms underlying DS and to provide clues to prenatal screening. Methods A series of proteomics-based experiments was conducted using 19 patients with DS fetuses and 17 normal pregnancies. The proteome of placenta was investigated as displayed by two-dimensional difference gel electrophoresis （2D-DIGE）, and comparisons were made between placentas that developed under DS and normal pregnancy conditions. Multivariate analysis of the resulting protein patterns revealed DS-specific protein expression. Matrix-assisted laser desorption/ionization （MALDI） time-of-flight/time-of-flight （TOF/TOF） high-resolution tandem mass spectrometer （MS）-based identification was successful for 12 out of 17 selected protein spots. Results Among those, three proteins involved in the resist of reactive oxygen species （ROS） and neurogenesis were more abundant in the DS placenta （superoxide dismutase 1, endoplasmic reticulum protein 29 and heat shock protein beta-l）, while peroxiredoxin-6 involved in cell defense mechanism against ROS was expressed at a higher level in the normal pregnancies. Conclusion Knowledge of the DS placenta proteome emphasizes the role of proteins involved in anti-oxidation during DS, and may form the basis of a potential approach to minimize the incidence of DS in the clinical settina.