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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.
Differential Centrifugation
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Mass Spectrometry-Compatible Subcellular Fractionation for Proteomics.

Takeshi Masuda1, Naoyuki Sugiyama2, Masaru Tomita3

  • 1Department of Pharmaceutical Microbiology, Faculty of Life Sciences , Kumamoto University , Chuo-ku, Kumamoto 862-0973 , Japan.

Journal of Proteome Research
|October 11, 2019
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Summary

Nuclear envelopes resist surfactants with ethylene glycol (EG). A new EG-RIPA buffer method efficiently isolates cellular fractions for proteomics, identifying phosphorylation sites that regulate protein localization.

Keywords:
ethylene glycolmass spectrometry-compatible methodphase transfer surfactantphosphoproteomicssubcellular fractionation

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Area of Science:

  • Cell Biology
  • Proteomics
  • Biochemistry

Background:

  • Nuclear envelopes are crucial for cellular structure and function.
  • Traditional subcellular fractionation methods can be time-consuming and require ultracentrifugation.
  • Existing methods may use mass spectrometry (MS)-incompatible chemicals, necessitating additional sample processing steps.

Purpose of the Study:

  • To develop a novel, efficient subcellular fractionation protocol for proteomics.
  • To identify phosphorylation sites that regulate protein subcellular localization.
  • To characterize cellular fractions without ultracentrifugation.

Main Methods:

  • Developed a novel RIPA buffer containing ethylene glycol (EG) and phase transfer surfactants for cell fractionation.
  • Separated cells into cytoplasm, organelles, and nucleus fractions without ultracentrifugation.
  • Applied the fractions directly to shotgun proteomics, phosphoproteomics, and proteomics.

Main Results:

  • The EG-RIPA buffer method effectively stabilizes nuclear envelopes against surfactants.
  • Achieved successful subcellular fractionation using as few as 3.5 × 10^5 cells.
  • Identified 59 phosphorylation sites on 42 phosphopeptides and 32 proteins potentially regulating subcellular localization, including specific sites on CD44 and lamin A/C.

Conclusions:

  • The novel EG-RIPA buffer protocol provides an efficient and direct method for subcellular fractionation compatible with shotgun proteomics.
  • This approach facilitates the identification of phosphorylation sites that control protein localization.
  • The protocol significantly reduces sample preparation time and complexity.