Publications (30)87.95 Total impact
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Article: Purification and Partial Characterization of a Novel Neurotoxic Protein from Eggs of Black Widow Spiders (Latrodectus tredecimguttatus).
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ABSTRACT: Up to now, there have been a few reports on the toxic components purified from black widow spider (Latrodectus tredecimguttatus) eggs. In the present study, a novel neurotoxic protein was purified from the eggs by gel filtration combined with ion-exchange chromatography. Its molecular weight was 23.752 kDa determined by electrospray mass spectrometry. The protein could block the neuromuscular transmission in mouse-isolated phrenic nerve-hemidiaphragm preparations completely in a reversible manner and activate tetrodotoxin-sensitive sodium current in rat dorsal root ganglion cells. The N-terminal sequence of the protein was identified by the Edman degradation to be N-S-I-A-D-D-R-Y-R-W-P-G-Y-P-G-A-G-L-I-P-Y-I-I-D-S-. When the sequence was used to search against protein database with a sequence query in Mascot engine there was no matched sequence or protein whereas the Basic Local Alignment Search Tool (BLAST) analysis indicated that no significant similarity was found. These results demonstrated that the protein (named Latroeggtoxin-I) is a novel neurotoxic protein purified from the eggs of black widow spiders.Journal of Biochemical and Molecular Toxicology 05/2013; · 1.38 Impact Factor -
Article: Sodium laurate, a novel protease- and mass spectrometry-compatible detergent for mass spectrometry-based membrane proteomics.
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ABSTRACT: The hydrophobic nature of most membrane proteins severely complicates their extraction, proteolysis and identification. Although detergents can be used to enhance the solubility of the membrane proteins, it is often difficult for a detergent not only to have a strong ability to extract membrane proteins, but also to be compatible with the subsequent proteolysis and mass spectrometric analysis. In this study, we made evaluation on a novel application of sodium laurate (SL) to the shotgun analysis of membrane proteomes. SL was found not only to lyse the membranes and solubilize membrane proteins as efficiently as SDS, but also to be well compatible with trypsin and chymotrypsin. Furthermore, SL could be efficiently removed by phase transfer method from samples after acidification, thus ensuring not to interfere with the subsequent CapLC-MS/MS analysis of the proteolytic peptides of proteins. When SL was applied to assist the digestion and identification of a standard protein mixture containing bacteriorhodoposin and the proteins in rat liver plasma membrane-enriched fractions, it was found that, compared with other two representative enzyme- and MS-compatible detergents RapiGest SF (RGS) and sodium deoxycholate (SDC), SL exhibited obvious superiority in the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.PLoS ONE 01/2013; 8(3):e59779. · 4.09 Impact Factor -
Article: Protein Compositional Analysis of the Eggs of Black Widow Spider (Latrodectus tredecimguttatus): Implications for the Understanding of Egg Toxicity.
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ABSTRACT: Previous work found that high-molecular-weight fractions in the egg extract of Latrodectus tredecimguttatus exhibited strong toxicities. For investigating the possible relationship of proteins in the eggs with the toxic effect, the protein composition of the eggs was analyzed using proteomic strategies and compared with that of the spider's venom. SDS-PAGE showed that the proteins of eggs were primarily distributed in the molecular weight range of higher than 55 kDa as well as around 34 kDa, having high abundance proteins with molecular weights of about 60 kDa and 130 kDa. A total of 157 proteins were identified from the egg extract, which were involved in important cellular functions and processes including catalysis, transport, and metabolism regulation. Comparison indicated that the protein composition of eggs is more complex than that of venom, and there are few similarities between the protein composition of the two materials, demonstrating that the eggs have their own distinct toxic mechanism.Journal of Biochemical and Molecular Toxicology 12/2012; · 1.38 Impact Factor -
Article: Development and evaluation of an entirely solution-based combinative sample preparation method for membrane proteomics.
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ABSTRACT: The hydrophobic nature of many membrane proteins, especially integral membrane proteins, brings great difficulties to their analysis. To improve the analysis of membrane proteins, an entirely solution-based combinative sample preparation (CSP) method was developed and its application to the shotgun analysis of rat liver membrane proteomes was evaluated in this study. This CSP method comprehensively uses the strong ability of sodium dodecyl sulfate (SDS) to lyse the membranes and solubilize hydrophobic membrane proteins, the high efficiency of the optimized acetone precipitation method in sample cleanup and protein recovery, and the advantages of sodium deoxycholate (SDC) in improving protein solubilization/digestion as well as being compatible with trypsin activity. Compared with two other representative sample preparation methods, the SDC-assisted membrane-lysing method and the tube gel method, the newly established CSP method exhibited superiority in the recovery and identification of hydrophobic peptides, larger peptides, and highly hydrophobic membrane proteins with multiple transmembrane domains. The CSP method has characteristics of easy operation, low cost, and suitability for treating protein samples in various volumes, particularly large volumes, thereby having potential in the analysis of membrane proteomes with mass spectrometry.Analytical Biochemistry 09/2012; · 3.00 Impact Factor -
Article: Sample preparation for the analysis of membrane proteomes by mass spectrometry.
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ABSTRACT: The low abundance and highly hydrophobic nature of most membrane proteins make their analysis more difficult than that for common soluble proteins. Successful membrane protein identification is largely dependent on the sample preparation including the enrichment and dissolution of the membrane proteins. A series of conventional and newly developed methods has been applied to the enrichment of low-abundance membrane proteins at membrane and/or protein levels and to the dissolution of hydrophobic membrane proteins. However, all the existing methods have inherent advantages and limitations. Up to now, there has been no unique method that can universally be employed to solve all the problems and more efforts are needed in improving sample preparation for the analysis of membrane proteomes.Protein & Cell 09/2012; 3(9):661-8. -
Article: Shotgun analysis of membrane proteomes using a novel combinative strategy of solution-based sample preparation coupled with liquid chromatography-tandem mass spectrometry.
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ABSTRACT: Although much effort has been made in the field of membrane proteomics, the analysis of membrane proteins particularly integral membrane proteins with poor water solubility still presents a great challenge. In this paper, 2% SDS was used to extract membrane proteins and experimental conditions for the application of acetone precipitation method to the cleanup of SDS-solubilized membrane protein sample were optimized. For improving the re-dissolution and trypsinolysis of acetone-precipitated proteins, several commonly used additives, urea, methanol and sodium deoxycholate (SDC), were employed and compared. The results showed that, when the pre-cooled acetone-to-sample ratio was 6:1 (v/v) with one additional washing step, residual SDS in the protein sample could be lowered to below 0.01% and more than 90% of the proteins were precipitated and therefore recovered. 1% SDC-containing buffer could improve the re-dissolution and digestion of the acetone precipitated proteins more efficiently than the others. Using the combinative sample preparation strategy developed, 398 proteins were identified from the rat liver membrane-enriched fraction, including 188 membrane proteins. Compared with other three representative solution-based sample preparation methods commonly used in membrane proteomics, the newly developed combinative strategy increased the number of identified total proteins and membrane proteins on average by 29.2% and 28.5%, respectively. This combinative strategy was demonstrated to be easily operated at low cost and suitable for the analysis of membrane proteins varying in type and sample volume, etc.Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 06/2012; 901:18-24. · 2.78 Impact Factor -
Article: Gel-absorption-based sample preparation method for shotgun analysis of membrane proteome.
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ABSTRACT: Membrane proteins solubilized in a starting buffer containing high concentration of sodium dodecyl sulfate (SDS) are directly entrapped and immobilized into gel matrix when the membrane protein solution is absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts are removed by washing, the proteins are subjected to in-gel digestion and the tryptic peptides are extracted and analyzed by CapLC-MS/MS. The newly developed method not only avoids protein loss and the adverse protein modifications during gel-embedment but also improves the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides. Thus, this method facilitates the identification of membrane proteins especially the integral membrane proteins.Methods in molecular biology (Clifton, N.J.) 01/2012; 869:385-92. -
Article: Electrophoretically driven SDS removal and protein fractionation in the shotgun analysis of membrane proteomes.
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ABSTRACT: SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins.Electrophoresis 01/2012; 33(2):316-24. · 3.30 Impact Factor -
Article: Quantitative proteomic analysis of membrane proteins involved in astroglial differentiation of neural stem cells by SILAC labeling coupled with LC-MS/MS.
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ABSTRACT: Membrane proteins play a critical role in the process of neural stem cell self-renewal and differentiation. Here, we apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the self-renewing and the astroglial differentiating cells. High-resolution analysis on a linear ion trap-Orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 700 distinct membrane proteins during the astroglial differentiation. Of the 735 quantified proteins, seven cell surface proteins display significantly higher expression levels in the undifferentiated state membrane compared to astroglial differentiating membrane. One cell surface protein transferrin receptor protein 1 may serve as a new candidate for NSCs surface markers. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that most of overexpressed membrane proteins in the astroglial differentiation neural stem cells are involved in cellular growth, nervous system development, and energy metabolic pathway. Taken together, this study increases our understanding of the underlying mechanisms that modulate complex biological processes of neural stem cell proliferation and differentiation.Journal of Proteome Research 12/2011; 11(2):829-38. · 5.11 Impact Factor -
Article: Shotgun proteomics and network analysis of ubiquitin-related proteins from human breast carcinoma epithelial cells.
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ABSTRACT: Protein ubiquitination via the covalent attachment of ubiquitin (Ub) plays an important role in the regulation of the stability, function or localization of multiple proteins in eukaryotic cells. Comprehensive investigation of the proteomics related to ubiquitination will gain the insight into the Ub-mediated regulatory mechanism. In the present study, the combination of polyUb affinity purification, SDS-PAGE separation, and liquid chromatography-tandem mass spectrometry analysis (GeLC-MS/MS) was employed to analyze the Ub-related proteins in human MDA-MB-231 breast carcinoma epithelial cells after treatment with the proteasome inhibitor MG132. A total of 260 non-redundant Ub-related proteins were identified from the cells. These proteins were shown to be involved in a host of critical cellular functions and processes, including transcription, translation, Ub-proteasome pathway, cell cycle, heat shock response, transport, etc. The interaction network analysis by STRING indicated that the identified Ub-related proteins formed eleven clusters, the three most highly ranked network clusters were mainly involved in protein translation, RNA transcription and processing, and Ub-proteasome pathway, suggesting that there were obvious ubiquitination-mediated alternations in gene expression of human MDA-MB-231 cells. The proteomic profiling and their interaction network analysis in this study would help to our systematic understanding of the Ub-related cellular protein functions and the related biological processes in human disease tissue cells.Molecular and Cellular Biochemistry 08/2011; 359(1-2):375-84. · 2.06 Impact Factor -
Article: Blue native/SDS-PAGE combined with iTRAQ analysis reveals advanced glycation end-product-induced changes of synaptosome proteins in C57 BL/6 mice.
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ABSTRACT: Evidence shows that administration of high-level D-galactose induces the production of advanced glycation end-products (AGEs) that have been implicated in the development of diabetic complications such as neuropathy. The deterioration of learning and memory during neuropathy might be associated with the altered expression of proteins in synapse. To evaluate AGE-induced protein network alterations in synapse, blue native/SDS-PAGE and iTRAQ proteomic methods were used to screen for differentially expressed synaptic proteins of cerebral cortex in D-galactose-induced C57 BL/6 mice. In total, the expression level of 84 proteins is changed during AGE accumulation. The significantly differentially expressed proteins mainly participate in neurotransmission, energy metabolism and signal transduction pathway, suggesting that energy metabolism is damaged and neurotransmission is attenuated in synapse. The results of in vivo activities of malondialdehyde and superoxide dismutase suggested that AGE accumulation in the brain leads to the generation of reactive oxygen species. Therefore, elucidating the differentially expressed proteins underlying the AGE accumulation will open a new window to the mechanism of learning and memory impairments in neuropathy.Electrophoresis 07/2011; · 3.30 Impact Factor -
Article: Improvement of hydrophobic integral membrane protein identification by mild performic acid oxidation-assisted digestion.
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ABSTRACT: Integral membrane proteins (IMPs) are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of IMPs make them difficult to analyze. In proteomic analyses, hydrophobic peptides including transmembrane domains are often underrepresented, and this reduces the sequence coverage and reliability of the identified IMPs. Here we report a new strategy, mild performic acid oxidation treatment (mPAOT), for improvement of IMP identification. In the mPAOT strategy, the hydrophobicity of IMPs is significantly decreased by oxidizing their methionine and cysteine residues with performic acid, thereby improving the solubility and enzymolysis of these proteins. The application of the mPAOT strategy to the analysis of IMPs from human nasopharyngeal carcinoma CNE1 cell line demonstrated that many IMPs, including those with high hydrophobicity, could be reliably identified.Analytical Biochemistry 12/2010; 407(2):196-204. · 3.00 Impact Factor -
Article: Dried polyacrylamide gel absorption: a method for efficient elimination of the interferences from SDS-solubilized protein samples in mass spectrometry-based proteome analysis.
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ABSTRACT: Sample preparation holds an important place in MS-based proteome analysis. For effective proteolysis and MS analysis, it is essential to eliminate the interferences while extracting the analytes of interest from complex mixtures. To address this, herein we describe a new dried polyacrylamide gel absorption method. In this method, the protein sample prepared using high concentration of SDS was directly and completely absorbed by vacuum-dried polyacrylamide gel, and then the interfering substances including SDS and some other salts were efficiently removed by in-gel washing steps while retaining the denatured proteins in the gel, thus offering a clean environment amenable to downstream buffer exchange, proteolytic digestion and digest recovery, etc. In combination with in-gel digestion and LC-MS/MS, the newly developed method was applied to the proteome analyses of membrane-enriched fraction and whole tissue homogenate. It was demonstrated that the method is suitable for the analysis of a complex biological sample and can be widely used for sample cleanup in shotgun proteome analyses.Electrophoresis 11/2010; 31(23-24):3816-22. · 3.30 Impact Factor -
Article: Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry.
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ABSTRACT: A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.Analytical Biochemistry 09/2010; 404(2):204-10. · 3.00 Impact Factor -
Article: Evaluation and optimization of removal of an acid-insoluble surfactant for shotgun analysis of membrane proteome.
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ABSTRACT: Due to its compatibility with protease activity at high concentration, sodium deoxycholate (SDC) can be used to effectively improve the solubilization and enzymolysis of membrane proteins and has received increasing attention in the field of membrane proteome analysis in recent years. SDC can be removed from digests by means of acidification followed by centrifugation (i.e. acid precipitation, AP) or extraction with ethyl acetate (i.e. phase transfer, PT) so as not to interfere with the downstream analyses like LC-MS/MS. In this study, the two strategies were systematically evaluated, compared and optimized. The results of the study demonstrated that both of the AP and PT strategies led to a certain amount of tryptic peptides being lost, and in PT strategy even more peptides were lost during SDC removal process. However, the lost peptides could be mostly recovered by washing the pellet and solid content produced during AP and PT, respectively. By recovering the lost peptides, the identification efficiency of proteins, especially transmembrane and low abundance ones, was significantly improved. Comparatively, after optimization by recovering the lost peptides, AP strategy was superior to PT strategy because the former not only could achieve the comparable identification efficiency with the latter but also was more economical, safer and easier to operate than the latter.Electrophoresis 08/2010; 31(16):2705-13. · 3.30 Impact Factor -
Article: Analysis of integral membrane proteins by heat gel-embedment combined with improved in-gel digestions.
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ABSTRACT: Analysis of integral membrane proteins (IMPs) presents a special challenge because of their hydrophobic nature and low abundance. Here, a new method was developed, which involved heat gel-embedment and improved in-gel digestion of the proteins. Membrane protein lysate containing detergents was mixed with acrylamide solution and the proteins were embedded when the gel polymerized. For comparison, the protein embedment was made at different temperatures (25, 35 or 45 degrees C), and the in-gel digestions were performed in the presence of 0.1% RapiGest reagent (ALS), 0.1% sodium deoxycholate and 10% ACN, respectively. The resultant peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry. Compared with that at 25 degrees C, gel-embedment at 45 degrees C improved the protein embedment and thus protein identification, with the identified IMPs increased by 27%. 0.1% sodium deoxycholate was more efficient than 0.1% ALS and 10% ACN in terms of improving the digestion and tryptic digest recovery of the gel-embedded proteins particularly the hydrophobic IMPs. Out of the 326 IMPs identified by heat gel-embedment combined with improved in-gel digestion strategies, 149 (46%) proteins had at least two mapped transmembrane domains. These results indicate that our newly developed protocol could facilitate the high throughput analysis of integral membrane proteome.Electrophoresis 12/2009; 30(23):4109-17. · 3.30 Impact Factor -
Article: Enrichment and proteomic analysis of plasma membrane from rat dorsal root ganglions.
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ABSTRACT: Dorsal root ganglion (DRG) neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. Plasma membrane (PM) of DRG cells plays important roles in their functions. PM proteins are main performers of the functions. However, mainly due to the very low amount of DRG that leads to the difficulties in PM sample collection, few proteomic analyses on the PM have been reported and it is a subject that demands further investigation. By using aqueous polymer two-phase partition in combination with high salt and high pH washing, PMs were efficiently enriched, demonstrated by western blot analysis. A total of 954 non-redundant proteins were identified from the plasma membrane-enriched preparation with CapLC-MS/MS analysis subsequent to protein separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or shotgun digestion. 205 (21.5%) of the identified proteins were unambiguously assigned as PM proteins, including a large number of signal proteins, receptors, ion channel and transporters. The aqueous polymer two-phase partition is a simple, rapid and relatively inexpensive method. It is well suitable for the purification of PMs from small amount of tissues. Therefore, it is reasonable for the DRG PM to be enriched by using aqueous two-phase partition as a preferred method. Proteomic analysis showed that DRG PM was rich in proteins involved in the fundamental biological processes including material exchange, energy transformation and information transmission, etc. These data would help to our further understanding of the fundamental DRG functions.Proteome Science 11/2009; 7:41. · 2.33 Impact Factor -
Article: Development of cationic colloidal silica-coated magnetic nanospheres for highly selective and rapid enrichment of plasma membrane fractions for proteomics analysis.
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ABSTRACT: PM (plasma membrane) proteins play critical roles in many biological processes and are often used as molecular targets for drug discovery. In PM proteome research, fast and highly selective methods for PM preparation are highly desirable for efficient PM protein identification. In the present study, an improved PM isolation technique involving coating intact cells with synthesized cationic silica-coated magnetic nanospheres was developed and applied to the proteomic analysis of the PM from human erythroleukaemia K562 cells. Western blotting characterization and protein identification of the prepared PM indicated that the PM enrichment method using the prepared magnetic nanospheres is a fast and inexpensive strategy with high specificity. Our results demonstrate the potential of these cationic silica-coated magnetic nanospheres for high-throughput identification of PM proteins from cells.Biotechnology and Applied Biochemistry 10/2009; 54(4):213-20. · 1.53 Impact Factor -
Article: Improvement of gel-separated protein identification by DMF-assisted digestion and peptide recovery after electroblotting.
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ABSTRACT: In-gel digestion of gel-separated proteins is a major route to assist in proteomics-based biological discovery, which, however, is often embarrassed by its inherent limitations such as the low digestion efficiency and the low recovery of proteolytic peptides. For overcoming these limitations, many efforts have been directed at developing alternative methods to avoid the in-digestion. Here, we present a new method for efficient protein digestion and tryptic peptide recovery, which involved electroblotting gel-separated proteins onto a PVDF membrane, excising the PVDF bands containing protein of interest, and dissolving the bands with pure DMF (> or =99.8%). Before tryptic digestion, NH(4)HCO(3) buffer was added to moderately adjust the DMF concentration (to 40%) in order for trypsin to exert its activity. Experimental results using protein standards showed that, due to actions of DMF in dissolving PVDF membrane and the membrane-bound substances, the proteins were virtually in-solution digested in DMF-containing buffer. This protocol allowed more efficient digestion and peptide recovery, thereby increasing the sequence coverage and the confidence of protein identification. The comparative study using rat hippocampal membrane-enriched sample showed that the method was superior to the reported on-membrane tryptic digestion for further protein identification, including low abundant and/or highly hydrophobic membrane proteins.Electrophoresis 09/2009; 30(20):3626-35. · 3.30 Impact Factor -
Article: An in vivo membrane density perturbation strategy for identification of liver sinusoidal surface proteome accessible from the vasculature.
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ABSTRACT: Liver sinusoidal endothelial cells (LSEC), the predominant nonparenchyma cells in liver, play critical roles in many important physiological and pathological processes by virtue of their unique location at the blood-tissue interface. To uncover the protein composition of LSEC plasma membrane (PM) comprehensively and give implications for the tissue microenvironment heterogeneity, we have developed an in vivo modified membrane density perturbation method for purification of the PM fraction. The proteins were separated and identified by SDS-PAGE combined with LC-MS/MS (GeLC-MS/MS). A total of 837 nonredundant proteins were identified, including a number of proteins previously reported to be localized to the PM of LSEC, as well as others not described. A diversity of membrane proteins involved in signaling, traffic, transporting and adhesion functions were identified. Our results demonstrated that the in vivo membrane density perturbation was an effective strategy to purify LSEC PM.Journal of Proteome Research 12/2008; 8(1):123-32. · 5.11 Impact Factor
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2013
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Hunan Agricultural University
Changsha, Jiangsu Sheng, China
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2006–2012
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Hunan University
Changsha, Hunan, China
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