ArticleAnalytical chemistry2012
Development of a peptidase-resistant substrate for single-cell measurement of protein kinase B activation.
Article in Analytical chemistry, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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Who cites it
21 citing papers in PubMed.
- Subtimizer: Computational Workflow for Structure-Guided Design of Potent and Selective Kinase Peptide Substrates.Journal of chemical information and modeling · 2026Article
- Cross-Species Applications of Peptide Substrate Reporters to Quantitative Measurements of Kinase Activity.ACS measurement science au · 2024Article
- CPProtectides: Rapid uptake of well-folded β-hairpin peptides with enhanced resistance to intracellular degradation.Peptide science (Hoboken, N.J.) · 2019Article
- Assays for tyrosine phosphorylation in human cells.Methods in enzymology · 2019Article
- Selection and optimization of enzyme reporters for chemical cytometry.Methods in enzymology · 2019Article
- Design and Application of Sensors for Chemical Cytometry.ACS chemical biology · 2018Review
- N-Gemini peptides: cytosolic protease resistance via N-terminal dimerization of unstructured peptides.Chemical communications (Cambridge, England) · 2017Article
- Development of β-Hairpin Peptides for the Measurement of SCF-Family E3 Ligase Activity in Vitro via Ornithine Ubiquitination.ACS omega · 2017Article
- Development of a protease-resistant reporter to quantify BCR-ABL activity in intact cells.The Analyst · 2016Article
- Single Cell Chemical Cytometry of Akt Activity in Rheumatoid Arthritis and Normal Fibroblast-like Synoviocytes in Response to Tumor Necrosis Factor α.Analytical chemistry · 2016Article
- Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity.ACS chemical biology · 2016Article
- Identification of a p53-based portable degron based on the MDM2-p53 binding region.The Analyst · 2016Article
- Separation of peptide fragments of a protein kinase C substrate fused to a β-hairpin by capillary electrophoresis.Analytical and bioanalytical chemistry · 2015Article
- Pronase E-Based Generation of Fluorescent Peptide Fragments: Tracking Intracellular Peptide Fate in Single Cells.Analytical chemistry · 2015Article
- Single-cell sphingosine kinase activity measurements in primary leukemia.Analytical and bioanalytical chemistry · 2014Article
- Measurement of protein kinase B activity in single primary human pancreatic cancer cells.Analytical chemistry · 2014Article
- Response of single leukemic cells to peptidase inhibitor therapy across time and dose using a microfluidic device.Integrative biology : quantitative biosciences from nano to macro · 2014Article
- Measuring activity in the ubiquitin-proteasome system: from large scale discoveries to single cells analysis.Cell biochemistry and biophysics · 2013Review
- Microfluidic chemical cytometry of peptide degradation in single drug-treated acute myeloid leukemia cells.Analytical chemistry · 2013Article
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Authors and funding
4 authors.
Funding
Abstract
An iterative design strategy using three criteria was utilized to develop a peptidase-resistant substrate peptide for protein kinase B. Libraries of peptides possessing non-native amino acids were screened for time to 50% phosphorylation, degradation half-life within a lysate, and appearance of a dominant fragment. The lead peptide possessed a half-life of 92 ± 7 and 16 ± 2 min in HeLa and LNCaP cytosolic lysates, respectively, representing a 4.6- and 2.7-fold lifetime improvement over that of the starting peptide. The redesigned peptide possessed a 4.5-fold improvement in phosphorylation efficiency compared to the starting peptide. The same peptide fragments were formed when the lead peptide was incubated in a lysate or loaded into single cells although the fragments formed in significantly different ratios suggesting that distinct peptidases metabolized the peptide in the two preparations. The rate of peptide degradation and phosphorylation was on average 0.1 ± 0.2 zmol pg(-1) s(-1) and 0.04 ± 0.08 zmol pg(-1) s(-1), respectively, for single LNCaP cells loaded with 4 ± 8 μM of peptide. Peptidase-resistant kinase substrates should find widespread utility in both lysate-based and single-cell assays of kinase activity.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.