Yanhong Wang

Robert Wood Johnson University Hospital, New Brunswick, New Jersey, United States

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Publications (5)23.06 Total impact

  • Molecular Genetics and Metabolism - MOL GENET METAB. 01/2010; 99(2).
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    ABSTRACT: Diagnosis of lysosomal storage diseases (LSDs) can be problematic in atypical cases where clinical phenotype may overlap with other genetically distinct disorders. In addition, LSDs may result from mutations in genes not yet implicated in disease. Thus, there are individuals that are diagnosed with apparent LSD based upon clinical criteria where the gene defect remains elusive. The objective of this study was to determine whether comparative proteomics approaches could provide useful insights into such cases. Most LSDs arise from mutations in genes encoding lysosomal proteins that contain mannose 6-phosphate, a carbohydrate modification that acts as a signal for intracellular targeting to the lysosome. We purified mannose 6-phosphorylated proteins by affinity chromatography and estimated relative abundance of individual proteins in the mixture by spectral counting of peptides detected by tandem mass spectrometry. Our rationale was that proteins that are decreased or absent in patients compared with controls could represent candidates for the primary defect, directing biochemical or genetics studies. On a survey of brain autopsy specimens from 23 patients with either confirmed or possible lysosomal disease, this approach identified or validated the genetic basis for disease in eight cases. These results indicate that this protein expression approach is useful for identifying defects in cases of undiagnosed lysosomal disease, and we demonstrated that it can be used with more accessible patient samples, e.g. cultured cells. Furthermore this approach was instrumental in the identification or validation of mutations in two lysosomal proteins, CLN5 and sulfamidase, in the adult form of neuronal ceroid lipofuscinosis.
    Molecular &amp Cellular Proteomics 05/2009; 8(7):1708-18. · 7.25 Impact Factor
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    ABSTRACT: Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.
    Molecular &amp Cellular Proteomics 11/2006; 5(10):1942-56. · 7.25 Impact Factor
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    ABSTRACT: The lysosome is a membrane delimited cytoplasmic organelle that contains at least 50 hydrolytic enzymes and associated cofactors. The biomedical importance of these enzymes is highlighted by the many lysosomal storage disorders that are associated with mutations in genes encoding lysosomal proteins, and there is also evidence that lysosomal activities may be involved in more widespread human diseases. The aim of this study was to characterize the human brain lysosomal proteome with the goal of establishing a reference map to investigate human diseases of unknown etiology and to gain insights into the cellular function of the lysosome. Proteins containing mannose 6-phosphate (Man6-P), a carbohydrate modification used for targeting resident soluble lysosomal proteins to the lysosome, were affinity-purified using immobilized Man6-P receptor. Fractionation by two-dimensional electrophoresis resolved a complex mixture comprising approximately 800 spots. Constituent proteins in each spot were identified using a combination of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (both peptide mass fingerprinting and tandem mass spectrometry) [corrected] on in-gel tryptic digests and N-terminal sequencing. In a complementary analysis, we also analyzed a tryptic digest of the unfractionated mixture by liquid chromatography MS/MS. In total, 61 different proteins were identified. Seven were likely contaminants associated with true Man6-P glycoproteins. Forty-one were known lysosomal proteins of which 11 have not previously been reported to contain Man6-P. An additional nine proteins were either uncharacterized or proteins not previously reported to have lysosomal function. We found that the human brain Man6-P-containing lysosomal proteome is highly complex and contains more proteins with a much greater number of individual isoforms than found in previous studies of Man6-P glycoproteomes.
    PROTEOMICS 05/2005; 5(6):1520-32. · 4.13 Impact Factor
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    ABSTRACT: See original http://dx.doi.org/10.1002/pmic.200401054
    Proteomics 01/2005; 5(8):2272-2272. · 4.43 Impact Factor