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 workflowwith 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.

A mass spectrometry-based workflow for the proteomic analysis of in vitro cultured cell subsets isolated by means of laser capture microdissection / M. Brioschi, S. Eligini, M. Crisci, S. Fiorelli, E. Tremoli, S. Colli, C. Banfi. - In: ANALYTICAL AND BIOANALYTICAL CHEMISTRY. - ISSN 1618-2642. - 406:12(2014 May), pp. 2817-2825. [10.1007/s00216-014-7724-9]

A mass spectrometry-based workflow for the proteomic analysis of in vitro cultured cell subsets isolated by means of laser capture microdissection

M. Brioschi
Primo
;
S. Fiorelli;E. Tremoli;S. Colli
Penultimo
;
C. Banfi
2014-05

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 workflowwith 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.
Heterogeneous cellmorphology; Label-freemass spectrometry; Laser capture microdissection; Proteomics
Settore BIO/10 - Biochimica
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/239619
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