Proteome Analysis of Serum-Containing Conditioned Medium from Primary Astrocyte Cultures

Annika Thorsell,Fredrik Blomstrand,Peter S Eriksson,Ann Westman-Brinkmalm,Michael Nilsson,Kaj Blennow,Jonas Faijerson

doi:10.4172/jpb.1000019

Annika Thorsell, Fredrik Blomstrand + Show 5 more

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https://doi.org/10.4172/jpb.1000019

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Abstract

Development of strategies that allow for identification of secreted factors in conditioned media is of significance for a wide range of research areas. Secreted factors are involved in intercellular communication and might also be biomarkers of potential clinical importance, used for early detection and diagnosis of disease. The aim of this study was to investigate whether metabolic labeling combined with mass spectrometry could be used to identify secreted proteins in serum-containing conditioned medium. Earlier proteomic studies of conditioned media have been performed on cells cultured in serum-free media. In the present study the fact that only the proteins derived from the cells contain the incorporated isotopically labeled amino acid was taken advantage of, making it possible to differentiate released proteins from medium proteins. The second objective was to examine whether any quantitative differences in the secretion profiles between primary astrocytes and astrocytes in a scratch injury model of reactive astrogliosis could be found. To our knowledge, this is the first study to identify secreted proteins in serum-containing medium using a proteomic approach involving stable isotope labeling by amino acids in cell cultures and mass spectrometry.

Highlights

Several studies have demonstrated that conditioned media contain secreted factors that have significant biological effects.Secreted factors have been proposed to be involved in intercellular communication
The present study evaluated whether secreted proteins can be identified and differentiated from the medium proteins in serum-containing conditioned media by a combination of SILAC and mass spectrometry
Conditioned media from primary astrocytes and astrocytes in a scratch injury model of reactive astrogliosis substituted with either normal or isotopically labeled arginine served as our model system (Figure 1)

Summary

Introduction

Secreted factors have been proposed to be involved in intercellular communication. They might be a source for early detection and diagnosis of disease, and identification of released factors, including proteins, might help to discover novel biomarkers of potential clinical significance. An autocrine/ paracrine survival factor for cultured neural stem/progenitor cells has been found to stimulate neurogenesis in vivo (Taupin et al, 2000). These findings demonstrate that the identification of secreted proteins in conditioned media is important, since these factors can have potent effects on endogenous cells

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