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    Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomic studies

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    Authors
    Zhang, Ying
    Bottinelli, Dario
    Lisacek, Frederique
    Luban, Jeremy
    Strambio-De-Castillia, Caterina
    Varesio, Emmanuel
    Hopfgartner, Gerard
    UMass Chan Affiliations
    Program in Molecular Medicine
    Document Type
    Journal Article
    Publication Date
    2015-09-01
    Keywords
    LC–MS/MS
    Monocyte-derived dendritic cells
    Protein precipitation
    Protein solubilization
    Proteomics
    Sample preparation
    Amino Acids, Peptides, and Proteins
    Biochemistry
    Chemistry
    Genomics
    Molecular Biology
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    Link to Full Text
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732721/
    Abstract
    Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry (MS)-based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize MS coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilization and denaturation methods were compared. Mechanical disruption of DC cell pellets under cryogenic conditions, coupled with the use of RIPA (radioimmunoprecipitation assay) buffer, was shown to be the method of choice based on total protein extraction and on the solubilization and identification of nuclear proteins. Precipitation by acetone was found to be more efficient than that by 10% trichloroacetic acid (TCA)/acetone, allowing in excess of 28% more protein identifications. Although being an effective strategy to eliminate the detergent residue, the acetone wash step caused a loss of protein identifications. However, this potential drawback was overcome by adding 1% sodium deoxycholate into the dissolution buffer, which enhanced both solubility of the precipitated proteins and digestion efficiency. This in turn resulted in 6 to 11% more distinct peptides and 14 to 19% more total proteins identified than using 0.5M triethylammonium bicarbonate alone, with the greatest increase (34%) for hydrophobic proteins.
    Source

    Anal Biochem. 2015 Sep 1;484:40-50. doi: 10.1016/j.ab.2015.05.007. Epub 2015 May 15. Link to article on publisher's site

    DOI
    10.1016/j.ab.2015.05.007
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/44478
    PubMed ID
    25983236
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    Link to Article in PubMed

    ae974a485f413a2113503eed53cd6c53
    10.1016/j.ab.2015.05.007
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