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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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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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Subcellular Fractionation01:32

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The homogenate obtained after cell lysis contains various membrane-bound organelles that can be further separated into pure fractions by subcellular fractionation. These isolates are used to study specific cellular components, analyze localized protein activity, and are even employed in diagnostics. Fractionation is typically achieved using centrifugation methods, the most common being density-gradient and differential centrifugation.
Differential Centrifugation
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Related Experiment Video

Updated: Sep 12, 2025

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
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Localization of Organelle Proteins Using Data-Independent Acquisition (DIA-LOP).

K J A McCaskie1, C Hutchings1, R Feret1

  • 1Department of Biochemistry, Cambridge Centre for Proteomics, University of Cambridge, Cambridge, UK.

Molecular & Cellular Proteomics : MCP
|August 9, 2025
PubMed
Summary

We developed DIA-LOP, a high-throughput method for spatial proteome mapping. This approach provides in-depth subcellular resolution, identifying thousands of proteins across cellular compartments for biochemical discovery.

Keywords:
data-independent acquisitionlocalisation of organelle proteinsmachine learningmass spectrometrysubcellular spatial proteomics

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Area of Science:

  • Proteomics
  • Cellular Biology
  • Biochemistry

Background:

  • Subcellular localization of proteins is crucial for cellular functions.
  • High-throughput methods for mapping the entire proteome's subcellular locations are challenging to develop.

Purpose of the Study:

  • To present DIA-LOP, a novel approach for high-throughput spatial proteome mapping with subcellular resolution.
  • To compare differential ultracentrifugation (DC) with detergent-based fractionation using DIA and DDA mass spectrometry.
  • To demonstrate the utility of DIA-LOP in identifying disease-related proteins for clinical applications.

Main Methods:

  • Integration of differential ultracentrifugation (DC) with ion-mobility-based data-independent acquisition (DIA) mass spectrometry.
  • Utilizing DIA-NN for data processing and pRoloc for spatial analysis.
  • Comparison of DC fractionation with detergent-based protocols using DIA and data-dependent acquisition (DDA) mass spectrometry.

Main Results:

  • Generation of the largest DIA-based subcellular proteome map to date, identifying 8242 proteins across 13 organellar compartments in U-2 OS cells.
  • Demonstration that DC fractionation offers superior subcellular resolution compared to detergent-based methods.
  • Highlighting enhanced proteome coverage achieved with DIA compared to DDA mass spectrometry.
  • Identification and mapping of disease-related proteins within an osteosarcoma cell model.

Conclusions:

  • DIA-LOP is a straightforward, high-throughput tool for biochemical discovery, offering significant proteome coverage and subcellular resolution.
  • The study provides guidance on optimal workflows for subcellular proteomics using biochemical fractionation.
  • DIA-LOP facilitates the identification of disease-related proteins, informing clinical studies.