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1.  Stable Isotope Labeling with Amino Acids in Drosophila for Quantifying Proteins and Modifications 
Journal of proteome research  2012;11(9):4403-4412.
Drosophila melanogaster is a common animal model for genetics studies, and quantitative proteomics studies of the fly are emerging. Here we present in detail the development of a procedure to incorporate stable isotope labeled amino acids into the fly proteome. In the method of Stable Isotope Labeling with Amino acids in Drosophila melanogaster (SILAC fly), flies were fed with SILAC labeled yeast grown with modified media, enabling near complete labeling in a single generation. Biological variation in proteome among individual flies was evaluated in a series of null experiments. We further applied the SILAC fly method to profile proteins from a model of fragile X syndrome, the most common cause of inherited mental retardation in human. The analysis identified a number of altered proteins in the disease model, including actin-binding protein profilin and microtubulin-associated protein futsch. The change of both proteins was validated by immunoblotting analysis. Moreover, we extended the SILAC fly strategy to study the dynamics of protein ubiquitination during the fly life span (from day 1 to day 30), by measuring the level of ubiquitin along with two major polyubiquitin chains (K48 and K63 linkages). The results show that the abundance of protein ubiquitination and the two major linkages do not change significantly within the measured age range. Together, the data demonstrate the application of the SILAC principle in Drosophila melanogaster, facilitating the integration of powerful fly genomics with emerging proteomics.
PMCID: PMC3443408  PMID: 22830426
SILAC; Drosophila melanogaster; proteomics; mass spectrometry; fragile X syndrome; ubiquitin
2.  Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis 
Journal of proteome research  2012;11(9):4722-4732.
Protein ubiquitination is an essential posttranslational modification regulating neurodevelopment, synaptic plasticity, learning and memory, and its dysregulation contributes to the pathogenesis of neurological diseases. Here we report a systematic analysis of ubiquitinated proteome (ubiquitome) in rat brain using a newly developed monoclonal antibody that recognizes the diglycine tag on lysine residues in trypsinized peptides (K-GG peptides). Initial antibody specificity analysis showed that the antibody can distinguish K-GG peptides from linear GG peptides or pseudo K-GG peptides derived from iodoacetamide. To evaluate the false discovery rate of K-GG peptide matches during database search, we introduced a null experiment using bacterial lysate that contains no such peptides. The brain ubiquitome was then analyzed by this antibody enrichment with or without strong cation exchange (SCX) prefractionation. During SCX chromatography, although the vast majority of K-GG peptides were detected in the fractions containing at least three positive charged peptides, specific K-GG peptides with two positive charges (e.g. protein N-terminal acetylated and C-terminal non-K/R peptides) were also identified in early fractions. The reliability of C-terminal K-GG peptides was also extensively investigated. Finally, we collected a dataset of 1786 K-GG sites on 2064 peptides in 921 proteins and estimated their abundance by spectral counting. The study reveals a wide range of ubiquitination events on key components in presynaptic region (e.g. Bassoon, NSF, SNAP25, synapsin, synaptotagmin, and syntaxin) and postsynaptic density (e.g. PSD-95, GKAP, CaMKII, as well as receptors for NMDA, AMPA, GABA, serotonin, and acetylcholine). We also determined ubiquitination sites on amyloid precursor protein and alpha synuclein that are thought to be causative agents in Alzhermer’s and Parkinson’s disorders, respectively. As K-GG peptides can also be produced from Nedd8 or ISG15 modified proteins, we quantified these proteins in the brain and found that their levels are less than 2% of ubiquitin. Together, this study demonstrates that a large number of neuronal proteins are modified by ubiquitination, and provides a feasible method for profiling the ubiquitome in the brain.
PMCID: PMC3443409  PMID: 22871113
ubiquitin; synapse; antibody; proteomics; mass spectrometry
3.  Wild-Type Mitochondrial DNA Copy Number in Urinary Cells as a Useful Marker for Diagnosing Severity of the Mitochondrial Diseases 
PLoS ONE  2013;8(6):e67146.
The genotype-phenotype relationship in diseases with mtDNA point mutations is still elusive. The maintenance of wild-type mtDNA copy number is essential to the normal mitochondrial oxidative function. This study examined the relationship between mtDNA copy number in blood and urine and disease severity of the patients harboring A3243G mutation. We recruited 115 A3243G patients, in which 28 were asymptomatic, 42 were oligo-symptomatic, and 45 were poly-symptomatic. Increase of total mtDNA copy number without correlation to the proportion of mutant mtDNA was found in the A3243G patients. Correlation analyses revealed that wild-type mtDNA copy number in urine was the most important factor correlated to disease severity, followed by proportion of mutant mtDNA in urine and proportion of mutant mtDNA in blood. Wild-type copy number in urine negatively correlated to the frequencies of several major symptoms including seizures, myopathy, learning disability, headache and stroke, but positively correlated to the frequencies of hearing loss and diabetes. Besides proportion of mutant mtDNA in urine, wild-type copy number in urine is also an important marker for disease severity of A3243G patients.
PMCID: PMC3695014  PMID: 23826218
4.  Thrombolytic effects of Douchi Fibrinolytic enzyme from Bacillus subtilis LD-8547 in vitro and in vivo 
BMC Biotechnology  2012;12:36.
Today, thrombosis is one of the most widely occurring diseases in modern life. Drugs with thrombolytic functions are the most effective methods in the treatment of thrombosis. Among them, Douchi fibrinolytic enzyme (DFE) is a promising agent. DFE was isolated from Douchi, a typical and popular soybean-fermented food in China, and it can dissolve fibrin directly and efficiently. A strain, Bacillus subtilis LD-8547 produced DFE with high fibrinolytic activity has been isolated in our lab previously.
In the study, thrombolytic effect of DFE from Bacillus subtilis LD-8547 was studied in vitro and in vivo systematically. The results showed that DFE played a significant role in thrombolysis and anticoagulation in vitro. And the thrombolytic effects correlated with DFE in a dose-dependent manner. In vivo, the acute toxicity assay showed that DFE had no obvious acute toxicity to mice. Test of carrageenan-induced thrombosis in mice indicated that the DFE significantly prevented tail thrombosis, and arterial thrombosis model test indicated that Douchi fibrinolytic enzyme DFE had thrombolytic effect on carotid thrombosis of rabbits in vivo. Other results in vivo indicated that DFE could increase bleeding and clotting time obviously.
The DFE isolated from Bacillus subtilis LD-8547 has obvious thrombolytic effects in vitro and in vivo. This function demonstrates that this enzyme can be a useful tool for preventing and treating clinical thrombus.
PMCID: PMC3434014  PMID: 22748219
Thrombolytic effects; Douchi Fibrinolytic enzyme; in vitro; in vivo
5.  Improved radiosensitizing effect of the combination of etanidazole and paclitaxel for hepatocellular carcinoma in vivo 
Hepatocellular carcinoma (HCC) is one of the most critical global health issues. Potential curative therapies, including surgical resection, are offered to only a limited number of patients. Therefore, new and effective treatment strategies are required. Recently, radiotherapy with hypoxic radiosensitizers has shown promise in cancer therapy. Our previous study demonstrated that radiosensitization produced by etanidazole and paclitaxel was additive in vitro. This study was carried out to determine the synergistic effect of the two drugs in murine HCC H22 cell xenograft-bearing BALB/c mice in vivo. The morphology of the transplanted tumors was observed. The drug content in the blood and tumors of mice was measured by high-performance liquid chromatography. The radiosensitizing effect on H22 cell xenograft-bearing mice was evaluated in terms of tumor growth inhibition and survival. Expression of hypoxia inducible factor-1α (HIF-1α) was studied using immunohistochemistry. The morphological consequences on the H22 xenografts were consistent with the pathological characteristics of HCC. There was no significant difference in drug content in the blood and tumors between single drug and combination administration. The combination of the two drugs improved the radiosensitizing effect in vivo compared to single drug administration in an animal model. The changes in HIF-1α expression indirectly verified the above-mentioned results. This study may provide a new combination of radiosensitizers for HCC radiotherapy.
PMCID: PMC3438662  PMID: 22969885
hepatocellular carcinoma; paclitaxel; etanidazole; combination radiosensitization
6.  Submicroscopic subtelomeric aberrations in Chinese patients with unexplained developmental delay/mental retardation 
BMC Medical Genetics  2010;11:72.
Subtelomeric imbalance is widely accepted as related to developmental delay/mental retardation (DD/MR). Fine mapping of aberrations in gene-enriched subtelomeric regions provides essential clues for localizing critical regions, and provides a strategy for identifying new candidate genes. To date, no large-scale study has been conducted on subtelomeric aberrations in DD/MR patients in mainland China.
This study included 451 Chinese children with moderate to severe clinically unexplained DD/MR. The subtelomere-MLPA (multiplex ligation dependent probe amplification) and Affymetrix human SNP array 6.0 were used to determine the subtelomeric copy number variations. The exact size and the breakpoint of each identified aberration were well defined.
The submicroscopic subtelomeric aberrations were identified in 23 patients, with a detection rate of 5.1%. 16 patients had simple deletions, 2 had simple duplications and 5 with both deletions and duplications. The deletions involved 14 different subtelomeric regions (1p, 2p, 4p, 6p, 7p, 7q, 8p, 9p, 10p, 11q, 14q, 15q, 16p and 22q), and duplications involved 7 subtelomeric regions (3q, 4p, 6q, 7p, 8p, 12p and 22q). Of all the subtelomeric aberrations found in Chinese subjects, the most common was 4p16.3 deletion. The sizes of the deletions varied from 0.6 Mb to 12 Mb, with 5-143 genes inside. Duplicated regions were 0.26 Mb to 11 Mb, with 6-202 genes inside. In this study, four deleted subtelomeric regions and one duplicated region were smaller than any other previously reported, specifically the deletions in 11q25, 8p23.3, 7q36.3, 14q32.33, and the duplication in 22q13. Candidate genes inside each region were proposed.
Submicroscopic subtelomeric aberrations were detected in 5.1% of Chinese children with clinically unexplained DD/MR. Four deleted subtelomeric regions and one duplicated region found in this study were smaller than any previously reported, which will be helpful for further defining the candidate dosage sensitive gene associated with DD/MR.
PMCID: PMC2892449  PMID: 20459802

Results 1-6 (6)