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Label-Free Assay of Protein Tyrosine Phosphatase Activity in Single Cells.
- Source :
-
Analytical chemistry [Anal Chem] 2019 Oct 15; Vol. 91 (20), pp. 13206-13212. Date of Electronic Publication: 2019 Sep 26. - Publication Year :
- 2019
-
Abstract
- Populations of cells exhibit variations in biochemical activity, resulting from many factors including random stochastic variability in protein production, metabolic and cell-cycle states, regulatory mechanisms, and external signaling. The development of methods for the analysis of single cells has allowed for the measurement and understanding of this inherent heterogeneity, yet methods for measuring protein activities on the single-cell scale lag behind their genetic analysis counterparts and typically report on expression rather than activity. This paper presents an approach to measure protein tyrosine phosphatase (PTP) activity in individual cells using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. Using flow cytometry, individual cells are first sorted into a well plate containing lysis buffer and a phosphopeptide substrate. After lysis and incubation-during which the PTP enzymes act on the peptide substrate-the reaction substrate and product are immobilized onto arrays of self-assembled monolayers, which are then analyzed using mass spectrometry. PTP activities from thousands of individual cells were measured and their distributions analyzed. This work demonstrates a general method for measuring enzyme activities in lysates derived from individual cells and will contribute to the understanding of cellular heterogeneity in a variety of contexts.
- Subjects :
- Cell Line, Tumor
Ethylene Glycols chemistry
Flow Cytometry
HEK293 Cells
Humans
Membranes, Artificial
Phosphopeptides chemistry
Protein Tyrosine Phosphatases chemistry
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Sulfhydryl Compounds chemistry
Enzyme Assays methods
Protein Tyrosine Phosphatases analysis
Single-Cell Analysis methods
Subjects
Details
- Language :
- English
- ISSN :
- 1520-6882
- Volume :
- 91
- Issue :
- 20
- Database :
- MEDLINE
- Journal :
- Analytical chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 31536703
- Full Text :
- https://doi.org/10.1021/acs.analchem.9b03640