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Label-Free Assay of Protein Tyrosine Phosphatase Activity in Single Cells
- Source :
- Anal Chem
- 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 (SAMDI-MS). 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 :
- Phosphopeptides
Lysis
Protein tyrosine phosphatase
010402 general chemistry
01 natural sciences
Article
Analytical Chemistry
Flow cytometry
Cell Line, Tumor
Lysis buffer
medicine
Humans
Sulfhydryl Compounds
Enzyme Assays
medicine.diagnostic_test
Phosphopeptide
Chemistry
010401 analytical chemistry
HEK 293 cells
Membranes, Artificial
Flow Cytometry
0104 chemical sciences
Matrix-assisted laser desorption/ionization
HEK293 Cells
Cell culture
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Biophysics
Ethylene Glycols
Protein Tyrosine Phosphatases
Single-Cell Analysis
Subjects
Details
- ISSN :
- 15206882
- Volume :
- 91
- Issue :
- 20
- Database :
- OpenAIRE
- Journal :
- Analytical chemistry
- Accession number :
- edsair.doi.dedup.....80ffd6884c1eda3fc1e6688787a0902e