Label-Free Assay of Protein Tyrosine Phosphatase Activity in Single Cells
| Autor: | Milan Mrksich, Elamar Hakim Moully, Eric J. Berns |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
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 |
| Zdroj: | Anal Chem |
| ISSN: | 1520-6882 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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