title: A mass spectrometry-based workflow for the proteomic analysis of in vitro cultured cell subsets isolated by means of laser capture microdissection.
abstract: This paper describes a microproteomic workflow that is useful for simultaneously identifying and quantifying proteins from a minimal number of morphotypically heterogeneous cultured adherent cells. The analytical strategy makes use of laser capture microdissection, an effective means of harvesting pure cell populations, and label-free mass spectrometry. We optimised the workflow with particular reference to cell fixation which is crucial for successful laser-based microdissection and also downstream molecular studies. In addition, we defined the minimum number of cells to be isolated and analysed for satisfactory proteome coverage. To set up this workflow, we choose human monocyte-derived macrophages spontaneously differentiated in vitro. These cells, under our culture conditions, show distinct morphotypes, reminiscent of the heterogeneity observed in tissues in various homeostatic and pathological states, e.g. atherosclerosis. This optimised workflow may provide new insights into biology and pathology of heterogeneous cell in culture, particularly when other cell selection approaches are not suitable.
authors.affiliation: Centro Cardiologico Monzino, IRCCS, Via Parea 4, 20138, Milan, Italy.
authors.forename: Maura
authors.initials: M
authors.lastname: Brioschi
authors.forename: Sonia
authors.initials: S
authors.lastname: Eligini
authors.forename: Mauro
authors.initials: M
authors.lastname: Crisci
authors.forename: Susanna
authors.initials: S
authors.lastname: Fiorelli
authors.forename: Elena
authors.initials: E
authors.lastname: Tremoli
authors.forename: Susanna
authors.initials: S
authors.lastname: Colli
authors.forename: Cristina
authors.initials: C
authors.lastname: Banfi
mesh_terms: D002478:Cells, Cultured; D006801:Humans; D061346:Laser Capture Microdissection / Q000379:methods*; D008264:Macrophages / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D057188:Workflow
chemical_list: D011506:Proteins