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J Lab Autom. 2013 Feb;18(1):19-29. doi: 10.1177/2211068212454738. Epub 2012 Aug 1.

Automated mass spectrometry-based functional assay for the routine analysis of the secretome.

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1
Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular Science, Clarkson University, Potsdam, NY 13699-5810, USA.

Abstract

The secretome represents the set of proteins secreted into the extracellular space of cells. These proteins have been shown to play a major role in cell-cell communication. For example, recent observations revealed the presence of diffusible factors with proliferative properties in the secretome of cancer cells. Thus, a qualitative and quantitative analysis of the secretome could lead to the identification of these factors and consequently to the development of new therapeutic strategies. Here, we provide an automated simple and effective strategy to identify novel targets in the secretome of specifically treated cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, we explore the supportive role of mass spectrometry (MS) in the development of functional assays of identified secreted target molecules. Simplicity is achieved by growing cells in medium free of serum, which eliminates the need to remove the most abundant serum proteins and at the same time reduces disturbing matrix effects. Upon identification of these factors, their validation and characterization will follow. Moreover, this approach can also lead to the identification of proteins abnormally secreted, shed, or oversecreted by cells as response to a stimulus. Furthermore, we also discuss the problems that one may encounter. Finally, we discuss the broad application of automated MS-based proteomics, particularly in cancer research, highlighting new horizons for the use of MS.

PMID:
22853965
DOI:
10.1177/2211068212454738
[Indexed for MEDLINE]
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