Yongqian Zhang

Study of rat hypothalamic proteome by HPLC/ESI ion trap and HPLC/ESI-Q-TOF MS.

The proteomic profile of hypothalamus, a key organ of CNS, is explored here by employing two widely used MS techniques, i.e. HPLC/ESI‐ion trap and HPLC/ESI‐quadrupole‐TOF MS. Strong cation exchange is used for the fractionation of peptides and protein search engine MASCOT is employed for data query. One hundred and thirty six proteins with 10 973 peptides were identified by HPLC/ESI‐ion trap MS, while 140 proteins with 32 183 peptides were characterized by HPLC/ESI‐quadrupole‐TOF MS. Among the total 198 proteins identified in both experiments, 78 proteins were common in both sets of conditions. The rest of the 120 proteins were identified distinctly in both MS strategies, i.e. 58 unique proteins were found using the quadrupole‐TOF while 62 were found with the HPLC/ESI‐ion trap. Moreover, these proteins were classified into groups based on their functions performed in the body. Results presented here identified some important signal and cellular defense proteins inevitable for survival in stressed conditions. Additionally, it is also shown that any single MS strategy is not reliable for good results due to loss of data depending on sensitivity of the instrument used.

Combination of HPLC chromatogram and hypoglycemic effect identifies isoflavones as the principal active fraction of Belamcanda chinensis leaf extract in diabetes treatment.

In previous study, we demonstrated the hypoglycemic effect of aqueous extract of Belamcanda chinensis leaves in rats. Here, we separated the aqueous extract of B. chinensis leaves and investigated the spectrum-effect relationships between HPLC chromatograms and hypoglycemic activities of different isolates from B. chinensis leaf extract. Sequential solvent extraction with petroleum ether, chloroform, acetic ester and n-butanol provided several isolates showing similar hypoglycemic activities, making it difficult to discriminate the active fractions. Stepwise elution through HP20 macroporous resin by water, 40% and 95% ethanol provided isolates with distinct hypoglycemic activities, representing a simple, rapid and efficient preparative separation method. Combination of HPLC chromatogram and pharmacological effect targeted a hypoglycemic activity-related region in HPLC chromatogram. Each peak in this region was analyzed by UV spectrum scan. Most of them were flavonoids in which tectoridin and swertisin were known flavonoids with anti-diabetic activities. In together, this work provides a general model of combination of HPLC chromatography and pharmacological effect to study the spectrum-effect relationships of aqueous extract from B. chinensis leaves, which can be used to find principle components of B. chinensis on pharmacological activity.

Differential expression of synaptic proteins in unilateral 6-OHDA lesioned rat model-A comparative proteomics approach.

Parkinsons disease (PD) is characterized as a movement disorder due to lesions in the basal ganglia. As the major input region of the basal ganglia, striatum plays a vital role in coordinating movements. It receives afferents from the cerebral cortex and projects afferents to the internal segment of the globus pallidus and substantia nigra pars reticulate. Additionally, accumulating evidences support a role for synaptic dysfunction in PD. Therefore, the present study explores the changes in protein abundance involved in synaptic disorders in unilateral lesioned 6‐OHDA rat model. Based on 18O/16O‐labeling technique, striatal proteins were separated using online 2D‐LC, and identified by nano‐ESI‐quadrupole‐TOF. A total of 370 proteins were identified, including 76 significantly differentially expressed proteins. Twenty‐two downregulated proteins were found in composition of vesicle, ten of which were involved in neuronal transmission and recycling across synapses. These include N‐ethylmaleimide‐sensitive fusion protein attachment receptor proteins (SNAP‐25, syntaxin‐1A, syntaxin‐1B, VAMP2), synapsin‐1, septin‐5, clathrin heavy chain 1, AP‐2 complex subunit beta, dynamin‐1, and endophilin‐A1. Moreover, MS result for syntaxin‐1A was confirmed by Western blot analysis. Overall, these synaptic changes induced by neurotoxin may serve as a reference for understanding the functional mechanism of striatum in PD.

Differential expression of specific cellular defense proteins in rat hypothalamus under simulated microgravity induced conditions: Comparative proteomics

Microgravity severely halts the structural and functional cerebral capacity of astronauts especially affecting their brains due to the stress produced by cephalic fluid shift. We employed a rat tail suspension model to substantiate simulated microgravity (SM) in brain. In this study, comparative mass spectrometry was applied in order to demonstrate the differential expression of 17 specific cellular defense proteins. Gamma‐enolase, peptidyl‐prolyl cis‐trans isomerase A, glial fibrillary acidic protein, heat shock protein HSP 90‐alpha, 10 kDa heat shock protein, mitochondrial, heat shock cognate 71 kDa protein, superoxide dismutase 1 and dihydropyrimidinase‐related protein 2 were found to be upregulated by HPLC/ESI‐TOF. Furthermore, five differentially expressed proteins including 60 kDa heat shock protein, mitochondrial, heat shock protein HSP 90‐beta, peroxiredoxin‐2, stress‐induced‐phosphoprotein, and UCHL‐1 were found to be upregulated by HPLC/ESI‐Q‐TOF MS. In addition, downregulated proteins include cytochrome C, superoxide dismutase 2, somatic, and excitatory amino acid transporter 1 and protein DJ‐1. Validity of MS results was successfully performed by Western blot analysis of DJ‐1 protein. This study will not only help to understand the neurochemical responses produced under microgravity but also will give future direction to cure the proteomic losses and their after effects in astronauts.

Comparative proteomic analysis of human SH-SY5Y neuroblastoma cells under simulated microgravity.

Microgravity is one of the most important features in spaceflight. Previous evidence has shown that neurophysiological impairment signs occurred under microgravity. The present study was undertaken to explore the change in protein abundance in human SH-SY5Y neuroblastoma cells that were grown in a microgravity environment. The comparative proteomic method based on the (18)O labeling technique was applied to investigate the up-regulated proteins and down-regulated proteins in SH-SY5Y under simulated microgravity. Twenty-two differentially abundant proteins were quantified in human SH-SY5Y neuroblastoma cells. The cell microfilament network was disrupted under simulated microgravity, which was determined by the immunocytochemistry. The concentration of reactive oxygen species, malondialdehyde, and free Ca2+ ion significantly increased, and the level of ATP significantly decreased under simulated microgravity. However, there was no obvious cell apoptosis observed under simulated microgravity. These results provide new molecular evidence for the change in protein abundance in SH-SY5Y cells under simulated microgravity, which might unfold biological mechanisms and the development of effective countermeasures to deal with microgravity-related neurological problems. We believe that the state-of-the-art proteomic assay may be a means by which aerospace scientists will begin to understand the underlying mechanisms of space life activities at the protein level.

Effect of Prolonged Simulated Microgravity on Metabolic Proteins in Rat Hippocampus: Steps toward Safe Space Travel.

Mitochondria are not only the main source of energy in cells but also produce reactive oxygen species (ROS), which result in oxidative stress when in space. This oxidative stress is responsible for energy imbalances and cellular damage. In this study, a rat tail suspension model was used in individual experiments for 7 and 21 days to explore the effect of simulated microgravity (SM) on metabolic proteins in the hippocampus, a vital brain region involved in learning, memory, and navigation. A comparative (18)O-labeled quantitative proteomic strategy was used to observe the differential expression of metabolic proteins. Forty-two and sixty-seven mitochondrial metabolic proteins were differentially expressed after 21 and 7 days of SM, respectively. Mitochondrial Complex I, III, and IV, isocitrate dehydrogenase and malate dehydrogenase were down-regulated. Moreover, DJ-1 and peroxiredoxin 6, which defend against oxidative damage, were up-regulated in the hippocampus. Western blot analysis of proteins DJ-1 and COX 5A confirmed the mass spectrometry results. Despite these changes in mitochondrial protein expression, no obvious cell apoptosis was observed after 21 days of SM. The results of this study indicate that the oxidative stress induced by SM has profound effects on metabolic proteins.

Effects of simulated microgravity on the expression of presynaptic proteins distorting the GABA/glutamate equilibrium ‐ a proteomics approach

Microgravity may cause cognition‐related changes in the animal nervous system due to the resulting uneven flow of fluids in the body. These changes may restrict the long‐term stay of humans in space for various purposes. In this study, a rat tail suspension model (30o) was used to explore the effects of 21 days of prolonged simulated microgravity (SM) on the expression of proteins involved in cognitive functions in the rat hippocampus. SM decreased the content of γ‐aminobutyric acid (GABA) and increased the content of glutamate (Glu) in the rat hippocampus. A comparative 18O‐labeled quantitative proteomics strategy was applied to detect the differential expression of synaptic proteins under SM. Fifty‐three proteins were found to be differentially expressed under SM. Microgravity induces difficulty in the formation of the SNARE complex due to the down‐regulation of vesicle‐associated membrane protein 3(VAMP3) and syntaxin‐1A. Synaptic vesicle recycling may also be affected due to the dysregulation of syntaxin‐binding protein 5 (tomosyn), rab3A and its effector rim2. Both processes are disturbed, indicating that presynaptic proteins mediate a GABA/Glu imbalance under SM. These findings provide clues for understanding the mechanism of the GABA/Glu equilibrium in the hippocampus induced by microgravity in space and represent steps toward safe space travel.

Distribution and accumulation of caffeine in rat tissues and its inhibition on semicarbazide-sensitive amine oxidase.

Wistar rats were treated with caffeine or 2-bromoethylamine, the effect of caffeine on the activity of semicarbazide-sensitive amine oxidase (SSAO) in rat serum and tissues was studied using various LC-MS methods. Caffeine was found to present in all tissues after administration for 10 days and accumulated for 25 days. The level of caffeine was high in brain and liver, and the SSAO activity in all tissues was found to be inhibited by caffeine. As the concentration of caffeine increased, the SSAO activity decreased. The inhibition ratio was correlated to the levels of caffeine present. We presume that caffeine may treat with SSAO activity associated diseases.

Optimization and quality assessment of the post-digestion 18O labeling based on urea for protein denaturation by HPLC/ESI-TOF mass spectrometry.

The post-digestion (18)O labeling method decouples protein digestion and peptide labeling. This method allows labeling conditions to be optimized separately and increases labeling efficiency. A common method for protein denaturation in proteomics is the use of urea. Though some previous studies have used urea-based protein denaturation before post-digestion (18)O labeling, the optimal (18)O labeling conditions in this case have not been yet reported. Present study investigated the effects of urea concentration and pH on the labeling efficiency and obtained an optimized protocol. It was demonstrated that urea inhibited (18)O incorporation depending on concentration. However, a urea concentration between 1 and 2M had minimal effects on labeling. It was also demonstrated that the use of FA to quench the digestion reaction severely affected the labeling efficiency. This study revealed the reason why previous studies gave different optimal pH for labeling. They neglect the effects of different digestion conditions on the labeling conditions. Excellent labeling quality was obtained at the optimized conditions using urea 1-2 M and pH 4.5, 98.4+/-1.9% for a standard protein mixture and 97.2+/-6.2% for a complex biological sample. For a 1:1 mixture analysis of the (16)O- and (18)O-labeled peptides from the same protein sample, the average abundance ratios reached 1.05+/-0.31, demonstrating a good quantitation quality at the optimized conditions. This work will benefit other researchers who pair urea-based protein denaturation with a post-digestion (18)O labeling method.

Differential proteomic analysis of white adipose tissues from T2D KKAy mice by LC-ESI-QTOF

Type 2 diabetes (T2D) has become a worldwide increasingly social health burden for its high morbidity and heightened prevalence. As one of the main tissues involved in uptake of glucose under the stimulation of insulin, WAT plays very important role in metabolic and homeostasis regulation. We performed a differential proteomics study to investigate alterations in epididymis fat pad of high fat diet fed T2D KKAy mice compared to normal fed C57BL/6J mice, by 18O‐labeling relative quantitative technique. Among 329 confidently identified proteins, 121 proteins showed significant changes with CV ≤ 20% (fold changes of >2 or <0.5 as threshold). According to GO classification, we found that altered proteins contained members of biological processes of metabolic process, oxidative stress, ion homeostasis, apoptosis and cell division. In metabolic, proteins assigned to fatty acid biosynthesis (FAS etc.) were decreased, the key enzyme (ACOX3) in β‐oxidation process was increased. Increased glycolysis enzymes (ENOB etc.) and decreased TCA cycle related enzymes (SCOT1 etc.) suggested that glucose metabolism in mitochondria of T2D mice might be impaired. Elevated oxidative stress was observed with alterations of a series of oxidordeuctase (QSOX1 etc.). Besides, alterations of ion homeostasis (AT2C1 etc.) proteins were also observed. The enhancement of cell proliferation associated proteins (ELYS etc.) and inhibition of apoptosis associated proteins (RASF6 etc.) in WAT might contributed to the fat pad and body weight gain. Overall, these changes in WAT may serve as a reference for understanding the functional mechanism of T2D.