FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations
Understanding disease pathology often does not require an overall proteomic analysis of clinical samples but rather the analysis of different, often rare, subpopulations of cells in a heterogeneous mixture of cell types. For the isolation of pre-specified cellular subtypes, fluorescence activated ce...
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doaj-2c06fa4c96044c6f9d087c50d41406002020-11-25T03:47:07ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-09-01216557655710.3390/ijms21186557FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell PopulationsEvelyne Maes0Nathalie Cools1Hanny Willems2Geert Baggerman3Food & Bio-Based Products, AgResearch Ltd., Lincoln 7674, New ZealandLaboratory of Experimental Hematology, Faculty of Medicine and Health Sciences, Vaccine and Infectious Disease Institute (VaxInfectio), Antwerp University Hospital (UZA), University of Antwerp, 2020 Antwerpen, BelgiumCentre for Proteomics, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, BelgiumCentre for Proteomics, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, BelgiumUnderstanding disease pathology often does not require an overall proteomic analysis of clinical samples but rather the analysis of different, often rare, subpopulations of cells in a heterogeneous mixture of cell types. For the isolation of pre-specified cellular subtypes, fluorescence activated cell sorting (FACS) is commonly used for its ability to isolate the required cell populations with high purity, even of scarce cell types. The proteomic analysis of a limited number of FACS-sorted cells, however, is very challenging as both sample preparation inefficiencies and limits in terms of instrument sensitivity are present. In this study, we used CD14+CD15+ immune cells sorted out of peripheral blood mononuclear cells isolated from whole blood to improve and evaluate FACS-based proteomics. To optimize both the protein extraction protocol and the mass spectrometry (MS) data acquisition method, PBMCs as well as commercialized HeLa digest were used. To reflect the limited number of sorted cells in some clinical samples, different numbers of sorted cells (1000, 5000, 10,000, or 50,000) were used. This allowed comparing protein profiles across samples with limited protein material and provided further insights in the benefits and limitations of using a very limited numbers of cells.https://www.mdpi.com/1422-0067/21/18/6557FACSproteomicscellular heterogeneity |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Evelyne Maes Nathalie Cools Hanny Willems Geert Baggerman |
spellingShingle |
Evelyne Maes Nathalie Cools Hanny Willems Geert Baggerman FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations International Journal of Molecular Sciences FACS proteomics cellular heterogeneity |
author_facet |
Evelyne Maes Nathalie Cools Hanny Willems Geert Baggerman |
author_sort |
Evelyne Maes |
title |
FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations |
title_short |
FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations |
title_full |
FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations |
title_fullStr |
FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations |
title_full_unstemmed |
FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations |
title_sort |
facs-based proteomics enables profiling of proteins in rare cell populations |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1661-6596 1422-0067 |
publishDate |
2020-09-01 |
description |
Understanding disease pathology often does not require an overall proteomic analysis of clinical samples but rather the analysis of different, often rare, subpopulations of cells in a heterogeneous mixture of cell types. For the isolation of pre-specified cellular subtypes, fluorescence activated cell sorting (FACS) is commonly used for its ability to isolate the required cell populations with high purity, even of scarce cell types. The proteomic analysis of a limited number of FACS-sorted cells, however, is very challenging as both sample preparation inefficiencies and limits in terms of instrument sensitivity are present. In this study, we used CD14+CD15+ immune cells sorted out of peripheral blood mononuclear cells isolated from whole blood to improve and evaluate FACS-based proteomics. To optimize both the protein extraction protocol and the mass spectrometry (MS) data acquisition method, PBMCs as well as commercialized HeLa digest were used. To reflect the limited number of sorted cells in some clinical samples, different numbers of sorted cells (1000, 5000, 10,000, or 50,000) were used. This allowed comparing protein profiles across samples with limited protein material and provided further insights in the benefits and limitations of using a very limited numbers of cells. |
topic |
FACS proteomics cellular heterogeneity |
url |
https://www.mdpi.com/1422-0067/21/18/6557 |
work_keys_str_mv |
AT evelynemaes facsbasedproteomicsenablesprofilingofproteinsinrarecellpopulations AT nathaliecools facsbasedproteomicsenablesprofilingofproteinsinrarecellpopulations AT hannywillems facsbasedproteomicsenablesprofilingofproteinsinrarecellpopulations AT geertbaggerman facsbasedproteomicsenablesprofilingofproteinsinrarecellpopulations |
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1724503311413936128 |