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An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
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Simplest Chemosensor Array for Phosphorylated Saccharides.

Yui Sasaki1, Éric Leclerc1,2, Vahid Hamedpour1

  • 1Institute of Industrial Science, The University of Tokyo , 4-6-1 Komaba, Meguro-ku , Tokyo , 153-8505 , Japan.

Analytical Chemistry
|November 13, 2019
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Summary
This summary is machine-generated.

A simple, two-molecule fluorescence sensor array accurately detects and quantifies phosphorylated saccharides. This system monitors glucose and fructose-6-phosphate levels in human stem cells, demonstrating high accuracy for modified saccharide detection.

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Materials Science

Background:

  • Phosphorylated saccharides play crucial roles in cellular metabolism and signaling.
  • Accurate detection of these molecules is vital for understanding biological processes and disease states.
  • Existing sensing methods can be complex and lack specificity for certain saccharide modifications.

Purpose of the Study:

  • To develop a simple, highly accurate "turn-on" fluorescence chemosensor array for phosphorylated saccharides.
  • To achieve simultaneous qualitative and quantitative detection of multiple phosphorylated saccharides.
  • To demonstrate the application of the sensor array in monitoring biologically relevant saccharides in human induced pluripotent stem (hiPS) cells.

Main Methods:

  • Fabrication of a chemosensor array using readily available reagents (esculetin, 4-methylesculetin, 3-nitrophenylboronic acid).
  • Utilized indicator displacement assay combined with photoinduced electron transfer (PeT) for fluorescence-based sensing.
  • Employed linear discriminant analysis for pattern recognition and classification of saccharide mixtures.

Main Results:

  • Achieved 100% classification rate for simultaneous recognition of 14 types of saccharides, including glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P).
  • Successfully performed quantitative analysis of glucose (Glc), G6P, and F6P mixtures, mimicking pseudoglycolysis.
  • Indirectly monitored Glc and F6P levels in hiPS cell supernatants, correlating with cell activity.

Conclusions:

  • The developed chemosensor array offers a simple yet effective platform for detecting phosphorylated and similarly modified saccharides.
  • The system demonstrates high accuracy and potential for real-time monitoring of metabolic pathways in biological samples.
  • This approach provides a valuable tool for biochemical research and diagnostics.