Caiyun Fang

Highly Efficient Enrichment Method for Glycopeptide Analyses: Using Specific and Nonspecific Nanoparticles Synergistically

We invented a new method for highly efficient and specific enrichment of glycopeptides using two different nanomaterials synergistically. One is boronic-acid-functionalized Fe3O4 nanoparticles, enriching glycopeptides through formation of cyclic boronate esters between the boronic acid groups and the cis-diol groups on glycopeptides. The other nanomaterial is conventional poly(methyl methacrylate) nanobeads, which have strong adsorption toward nonglycopeptides. By optimizing the proportion of these two materials, extremely high sensitivity and selectivity are achieved in analyzing the standard glycopeptides/nonglycopeptides mixture solutions. Since the washing step is not necessary for these conditions, the enrichment process is simplified and the recovery efficiency of target glycopeptides reaches 90%. Finally, this approach is successfully applied to analyze human serum with the sample volume as little as 1 μL, in which 147 different N-glycosylation peptides within 66 unique glycoproteins are identified. All these performances by the synergistic enrichment are much better than employing one specific enrichment agent alone.

Proteomic characterization of larval and adult developmental stages in Echinococcus granulosus reveals novel insight into host–parasite interactions

UNLABELLED Cystic hydatid disease is an important zoonosis caused by Echinococcus granulosus infection. The expression profiles of its parasitic life stages and host-Echinococcus interactions remain to be elucidated. Here, we identified 157 adult and 1588 protoscolex proteins (1610 in all), including 1290 novel identifications. Paramyosins and an antigen B (AgB) were the dominant adult proteins. Dog proteins (30) identified in adults indicated diminished local inflammation caused by adult infection. The protoscolex expresses proteins that have been reported to be antigens in other parasites, such as 6-phosphofructokinase and calcineurin B. Pathway analyses suggested that E. granulosus uses both aerobic and anaerobic carbohydrate metabolisms to generate ATP. E. granulosus expresses proteins involved in synthesis and metabolism of lipids or steroids. At least 339 of 390 sheep proteins identified in protoscolex were novel identifications not seen in previous analyses. IgGs and lambda light chains were the most abundant antibody species. Sheep proteins were enriched for detoxification pathways, implying that host detoxification effects play a central role during host-parasite interactions. Our study provides valuable data on E. granulosus expression characteristics, allowing novel insights into the molecular mechanisms involved in host-parasite interactions. BIOLOGICAL SIGNIFICANCE In this study, the Echinococcus granulosus adult worm proteome was analyzed for the first time. The protein identification of E. granulosus protoscoleces was extended dramatically. We also identified the most abundant host proteins co-purified with Echinococcus. The results provide useful information pertaining to the molecular mechanisms behind host-Echinococcus interaction and Echinococcus biology. This data also increases the potential for identifying vaccine candidates and new therapeutic targets, and may aid in the development of protein probes for selective and sensitive diagnosis of echinococcosis infection. In addition, the data collected here represents a valuable proteomic resource for subsequent genome annotation.

Integration of Cardiac Proteome Biology and Medicine by a Specialized Knowledgebase

Rationale: Omics sciences enable a systems-level perspective in characterizing cardiovascular biology. Integration of diverse proteomics data via a computational strategy will catalyze the assembly of contextualized knowledge, foster discoveries through multidisciplinary investigations, and minimize unnecessary redundancy in research efforts. Objective: The goal of this project is to develop a consolidated cardiac proteome knowledgebase with novel bioinformatics pipeline and Web portals, thereby serving as a new resource to advance cardiovascular biology and medicine. Methods and Results: We created Cardiac Organellar Protein Atlas Knowledgebase (COPaKB; www.HeartProteome.org), a centralized platform of high-quality cardiac proteomic data, bioinformatics tools, and relevant cardiovascular phenotypes. Currently, COPaKB features 8 organellar modules, comprising 4203 LC-MS/MS experiments from human, mouse, drosophila, and Caenorhabditis elegans, as well as expression images of 10 924 proteins in human myocardium. In addition, the Java-coded bioinformatics tools provided by COPaKB enable cardiovascular investigators in all disciplines to retrieve and analyze pertinent organellar protein properties of interest. Conclusions: COPaKB provides an innovative and interactive resource that connects research interests with the new biological discoveries in protein sciences. With an array of intuitive tools in this unified Web server, nonproteomics investigators can conveniently collaborate with proteomics specialists to dissect the molecular signatures of cardiovascular phenotypes.

Regulation of Acetylation Restores Proteolytic Function of Diseased Myocardium in Mouse and Human

Proteasome complexes play essential roles in maintaining cellular protein homeostasis and serve fundamental roles in cardiac function under normal and pathological conditions. A functional detriment in proteasomal activities has been recognized as a major contributor to the progression of cardiovascular diseases. Therefore, approaches to restore proteolytic function within the setting of the diseased myocardium would be of great clinical significance. In this study, we discovered that the cardiac proteasomal activity could be regulated by acetylation. Histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid and sodium valproate) enhanced the acetylation of 20S proteasome subunits in the myocardium and led to an elevation of proteolytic capacity. This regulatory paradigm was present in both healthy and acutely ischemia/reperfusion (I/R) injured murine hearts, and HDAC inhibition in vitro restored proteolytic capacities to baseline sham levels in injured hearts. This mechanism of regulation was also viable in failing human myocardium. With 20S proteasomal complexes purified from murine myocardium treated with HDAC inhibitors in vivo, we confirmed that acetylation of 20S subunits directly, at least in part, presents a molecular explanation for the improvement in function. Furthermore, using high-resolution LC-MS/MS, we unraveled the first cardiac 20S acetylome, which identified the acetylation of nine N-termini and seven internal lysine residues. Acetylation on four lysine residues and four N-termini on cardiac proteasomes were novel discoveries of this study. In addition, the acetylation of five lysine residues was inducible via HDAC inhibition, which correlated with the enhancement of 20S proteasomal activity. Taken as a whole, our investigation unveiled a novel mechanism of proteasomal function regulation in vivo and established a new strategy for the potential rescue of compromised proteolytic function in the failing heart using HDAC inhibitors.

Coating cells with cationic silica-magnetite nanocomposites for rapid purification of integral plasma membrane proteins.

This study developed a simple and rapid purification method for plasma membrane with high yields from adherent cells. The plasma membrane (PM) sheets could be absorbed specifically by the cationic silica–magnetite nanocomposites (CSMN) under acidic conditions, and recovered directly in cell‐lysis‐buffer with no need for precipitation. The binding between CSMN and PM sheets was confirmed by electron microscopy. Western blot analysis demonstrated a >10‐fold relative enrichment factor. Up to 422 integral membrane proteins were identified from 107 Huh7 cells. Notably, we found 29 Ras family proteins by classification according to their biological functions. The whole enrichment procedure took <30 min. The CSMN‐based procedure demonstrates a simple, economical and efficient enrichment of integral PM proteins in proteomic study.

Isoflurane protects the myocardium against ischemic injury via the preservation of mitochondrial respiration and its supramolecular organization.

BACKGROUND:Isoflurane has been demonstrated to limit myocardial ischemic injury. This effect is hypothesized to be mediated in part via effects on mitochondria. We investigated the hypothesis that isoflurane maintains mitochondrial respiratory chain functionality, in turn limiting mitochondrial damage and mitochondrial membrane disintegration during myocardial ischemic injury. METHODS:Mice (9–12 weeks of age) received isoflurane (1.0 minimum alveolar concentration) 36 hours before a 30-minute coronary artery occlusion that was followed by 24 hours of reperfusion. Cardiac mitochondria were isolated at a time point corresponding to 4 hours of reperfusion. 2,3,5-Triphenyltetrazoliumchloride staining was used to determine myocardial infarct size. Mitochondrial respiratory chain functionality was investigated using blue native polyacrylamide gel electrophoresis, as well as specific biochemical assays. Mitochondrial lipid peroxidation was quantified via the formation of malondialdehyde; mitochondrial membrane integrity was assessed by Ca2+-induced swelling. Protein identification was achieved via liquid chromatography mass spectrometry/mass spectrometry. RESULTS:Thirty-one mice were studied. Mice receiving isoflurane displayed a reduced myocardial infarct size (P = 0.0011 versus ischemia/reperfusion [I/R]), accompanied by a preserved activity of respiratory complex III (P = 0.0008 versus I/R). Isoflurane stabilized mitochondrial supercomplexes consisting of oligomers from complex III/IV (P = 0.0086 versus I/R). Alleviation of mitochondrial damage after isoflurane treatment was further demonstrated as decreased malondialdehyde formation (P = 0.0019 versus I/R) as well as a diminished susceptibility to Ca2+-induced swelling (P = 0.0010 versus I/R). CONCLUSIONS:Our findings support the hypothesis that isoflurane protects the heart from ischemic injury by maintaining the in vivo functionality of the mitochondrial respiratory chain. These effects may result in part from the preservation of mitochondrial supramolecular organization and minimized oxidative damage, circumventing the loss of mitochondrial membrane integrity.

Proteome Dynamics and Proteome Function of Cardiac 19S Proteasomes

Myocardial proteasomes are comprised of 20S core particles and 19S regulatory particles, which together carry out targeted degradation of cardiac proteins. The 19S complex is unique among the regulators of proteasomes in that it affects both the capacity and specificity of protein degradation. However, a comprehensive molecular characterization of cardiac 19S complexes is lacking. In this investigation, we tailored a multidimensional chromatography-based purification strategy to isolate structurally intact and functionally viable 19S complexes from murine hearts. Two distinct subpopulations of 19S complexes were isolated based upon (1) potency of activating 20S proteolytic activity, and (2) molecular composition using a combination of immuno-detection, two-dimensional-differential gel electrophoresis, and MS-based approaches. Heat shock protein 90 (Hsp90) was identified to be characteristic to 19S subpopulation I. The physical interaction of Hsp90 with 19S complexes was demonstrated via multiple approaches. Inhibition of Hsp90 activity using geldanamycin or BIIB021 potentiated the ability of subpopulation I to activate 20S proteasomes in the murine heart, thus demonstrating functional specificity of Hsp90 in subpopulation I. This investigation has advanced our understanding of the molecular heterogeneity of cardiac proteasomes by identifying molecularly and functionally distinct cardiac 19S complexes. The preferential association of Hsp90 with 19S subpopulation I unveils novel targets for designing proteasome-based therapeutic interventions for combating cardiac disease.

Identification of Palmitoylated Transitional Endoplasmic Reticulum ATPase by Proteomic Technique and Pan Antipalmitoylation Antibody

Protein palmitoylation plays a significant role in a wide range of biological processes such as cell signal transduction, metabolism, apoptosis, and carcinogenesis. For high-throughput analysis of protein palmitoylation, approaches based on the acyl-biotin exchange or metabolic labeling of azide/alkynyl-palmitate analogs are commonly used. No palmitoylation antibody has been reported. Here, the palmitoylated proteome of human colon cancer cell lines SW480 was analyzed via a TS-6B-based method. In total, 151 putative palmitoylated sites on 92 proteins, including 100 novel sites, were identified. Except for 3 known palmitoylated transmembrane proteins, ATP1A1, ZDHHC5, and PLP2, some important proteins including kinases, ion channels, receptors, and cytoskeletal proteins were also identified, such as CLIC1, PGK1, PPIA, FKBP4, exportin-2, etc. More importantly, the pan antipalmitoylation antibody was developed and verified for the first time. Our homemade pan antipalmitoylation antiserum could differentiate well protein palmitoylation from mouse brain membrane fraction and SW480 cells, which affords a new technique for analyzing protein palmitoylation by detecting the palmitic acid moiety directly. Furthermore, the candidate protein transitional endoplasmic reticulum ATPase (VCP) identified in SW480 cells was validated to be palmitoylated by Western blotting with anti-VCP antibody and the homemade pan antipalmitoylation antibody.

Stroma derived COL6A3 is a potential prognosis marker of colorectal carcinoma revealed by quantitative proteomics

Colorectal cancer (CRC) represents the third most common cancer in males and second in females worldwide. Here, we performed a quantitative 8-plex iTRAQ proteomics analysis of the secreted proteins from five colonic fibroblast cultures and three colon cancer epithelial cell lines. We identified 1114 proteins at 0% FDR, including 587 potential secreted proteins. We further recognized 116 fibroblast-enriched proteins which were significantly associated with cell movement, angiogenesis, proliferation and wound healing, and 44 epithelial cell-enriched proteins. By interrogation of Oncomine database, we found that 20 and 8 fibroblast-enriched proteins were up- and downregulated in CRC, respectively. Western blots confirmed the fibroblast-specific secretion of filamin C, COL6A3, COL4A1 and spondin-2. Upregulated mRNA and stroma expression of COL6A3 in CRC, which were revealed by Oncomine analyses and tissue-microarray-immunohistochemistry, indicated poor prognosis. COL6A3 expression was significantly associated with Dukes stage, T stage, stage, recurrence and smoking status. Circulating plasma COL6A3 in CRC patients was upregulated significantly comparing with healthy peoples. Receiver operating characteristic curve analysis revealed that COL6A3 has better predictive performance for CRC with an area under the curve of 0.885 and the best sensitivity/specificity of 92.9%/81.3%. Thus we demonstrated that COL6A3 was a potential diagnosis and prognosis marker of CRC.

Proteomic analysis of cell lines expressing small hepatitis B surface antigen revealed decreased glucose-regulated protein 78 kDa expression in association with higher susceptibility to apoptosis.

Accumulating evidence suggests a key role of hepatocyte apoptosis in the pathogenesis of viral hepatitis B. It was found in this study that stable expression of small hepatitis B surface antigen (SHBs) in HepG2 and Huh7 cells increased susceptibility to apoptosis. Proteomic analysis of SHBs expressing HepG2 cells revealed 43 down‐regulated and 38 up‐regulated proteins. Some have been implicated in apoptosis, including glucose‐regulated protein 78 kDa (GRP78), heterogeneous nuclear ribonucleoprotein H3 (hnRNP H), Rho GDP dissociation inhibitor (GDI), cystatin B, far upstream element‐binding protein (FUSEbp), and TNF receptor‐associated protein 1 (TRAP1). Differential expression of GRP78 and several other proteins was confirmed by Western blot analysis. Replenishing GRP78 improved cellular resistance to apoptosis, whereas reduction of GRP78 by siRNA increased susceptibility even in the absence of SHBs. Taken together, these results suggest that HBsAg plays a pro‐apoptotic role through down‐regulation of GRP78. J. Med. Virol. 82:14–22, 2010.