Flow cytometry-assisted purification and proteomic analysis of the corticotropes dense-core secretory granules.
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.
Gauthier
Daniel J
DJ
Neuropeptides Structure and Metabolism Research Unit, Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada.
Sobota
Jacqueline A
JA
Ferraro
Francesco
F
Mains
Richard E
RE
Lazure
Claude
C
D000818:Animals; D045744:Cell Line, Tumor; D052680:Corticotrophs / Q000378:metabolism*; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D006899:Mixed Function Oxygenases / Q000235:genetics; D009097:Multienzyme Complexes / Q000235:genetics; D020543:Proteome / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D022142:Secretory Vesicles / Q000378:metabolism*
D009097:Multienzyme Complexes; D020543:Proteome; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase