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Flow cytometry‐assisted purification and proteomic analysis of the corticotropes dense‐core secretory granules
Author(s) -
Gauthier Daniel J.,
Sobota Jacqueline A.,
Ferraro Francesco,
Mains Richard E.,
Lazure Claude
Publication year - 2008
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200700969
Subject(s) - proteomics , organelle , proteome , cell fractionation , biology , protein purification , biochemistry , chemistry , chromatography , cell sorting , microbiology and biotechnology , cell , enzyme , gene
The field of organellar proteomics has emerged as an attempt to minimize the complexity of the proteomics data obtained from whole cell and tissue extracts while maximizing the resolution on the protein composition of a single subcellular compartment. Standard methods involve lengthy density‐based gradient and/or immunoaffinity purification steps followed by extraction, 1‐DE or 2‐DE, gel staining, in‐gel tryptic digestion, and protein identification by MS. In this paper, we present an alternate approach to purify subcellular organelles containing a fluorescent reporter molecule. The gel‐free procedure involves fluorescence‐assisted sorting of the secretory granules followed by gentle extraction in a buffer compatible with tryptic digestion and MS. Once the subcellular organelle labeled, this procedure can be done in a single day, requires no major modification to any instrumentation and can be readily adapted to the study of other organelles. When applied to corticotrope secretory granules, it led to a much enriched granular fraction from which numerous proteins could be identified through MS.