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Related Concept Videos

Subcellular Fractionation01:32

Subcellular Fractionation

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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.
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Related Experiment Video

Updated: Dec 27, 2025

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry
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Comparative Analysis of Quantitative Mass Spectrometric Methods for Subcellular Proteomics.

Abla Tannous1, Marielle Boonen2, Haiyan Zheng1

  • 1Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, United States.

Journal of Proteome Research
|March 6, 2020
PubMed
Summary

Quantitative mass spectrometry combined with subcellular fractionation effectively maps cellular proteomes. Tandem mass tag (TMT)-MS2 offers the highest proteome coverage and lowest missing data for protein localization studies.

Keywords:
isobaric labelinglabel freequantitative mass spectrometrysubcellular fractionation

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Area of Science:

  • Proteomics
  • Cell Biology
  • Biochemistry

Background:

  • Understanding protein intracellular location is crucial for elucidating protein function and organelle roles.
  • Global cellular mapping integrates subcellular fractionation with quantitative mass spectrometry.
  • Evaluating mass spectrometry techniques is essential for accurate proteome analysis.

Purpose of the Study:

  • To assess various mass spectrometry approaches for quantitative proteomic analysis of subcellular fractions.
  • To compare the performance of tandem mass tag (TMT) isobaric labeling (MS2 and MS3) and label-free methods (MS1 and data-independent acquisition - DIA).
  • To determine the optimal method for high-coverage, accurate protein localization in cellular maps.

Main Methods:

  • Rat liver subcellular fractions were generated using differential centrifugation and Nycodenz density gradients.
  • Proteomic analysis employed TMT isobaric labeling with MS2 and MS3 reporter ion quantification.
  • Label-free quantification utilized MS1 intensity-based and data-independent acquisition (DIA) approaches.
  • Protein localization accuracy was assessed using a protein clustering approach with reference proteins.

Main Results:

  • TMT-MS2 achieved the highest proteome coverage and lowest percentage of missing quantifiable data.
  • Ratio compression in TMT-MS2 narrowed the accurate dynamic range compared to TMT-MS3, MS1, and DIA.
  • All evaluated methods demonstrated good data quality for protein localization, with isobaric labeling yielding superior results.
  • TMT-MS2 provided comparable protein localization accuracy to other methods despite dynamic range limitations.

Conclusions:

  • TMT-MS2 is a valuable method for subcellular proteome mapping, offering extensive coverage and minimal missing values.
  • While TMT-MS2 exhibits ratio compression, its performance in protein localization and data completeness is highly competitive.
  • The study validates mass spectrometry-based approaches for generating comprehensive cellular maps through subcellular fractionation.