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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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Single-Cell Protein Assays in Context: From 2D to 3D and In Situ Analysis.

Jocelyn S Baker, Neica I Joseph, Alison Lao

¹Department of Bioengineering, Rice University, Houston, Texas, USA;

Annual Review of Analytical Chemistry (Palo Alto, Calif.)

|December 10, 2025

Summary

Single-cell proteomics (scP) offers deeper cellular insights than transcriptomics by analyzing proteins directly. This review covers modern scP technologies and their applications in various biological models for clinical translation.

Keywords:

3D tissue models cytometry imaging mass spectrometry sample preparation single-cell proteomics spatial proteomics

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Area of Science:

Proteomics
Cell Biology
Biotechnology

Background:

Transcriptomics has limitations as RNA levels do not always correlate with protein levels.
Key cellular details like protein isoforms, posttranslational modifications, and complexes are missed by transcriptomics.
Single-cell proteomics (scP) complements transcriptomics for deeper cellular analysis.

Purpose of the Study:

To review modern single-cell proteomics technologies.
To discuss the application of these technologies in various biological samples and models.
To identify future directions for spatially resolved scP in clinical settings.

Main Methods:

Flow cytometry
Mass cytometry
Single-cell mass spectrometry
Immunohistochemistry
Cyclic imaging
Imaging mass cytometry

Main Results:

scP technologies can be applied to dissociated and spatially preserved samples.
Techniques are adaptable to organ-on-a-chip, organoids, spheroids, and intact tissues.
Advances in spatial resolution and multiplexing are highlighted.

Conclusions:

Modern scP technologies provide crucial insights into cellular heterogeneity, disease mechanisms, and therapeutic vulnerabilities.
Trade-offs between throughput, spatial fidelity, and protein selectivity exist across platforms.
Future research should focus on addressing measurement gaps for spatially resolved scP clinical translation.