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SDS-PAGE01:27

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Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
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Enhanced S-Palmitoylated Protein Detection by Mild Nonionic Detergent in Proteomic Workflow.

Hyojung Kim1, Jiraphorn Issara-Amphorn2, SungHwan Yoon2

  • 1Section on Structural and Chemical Biology, Neurosciences and Cellular and Structural Biology Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892, United States.

Journal of the American Society for Mass Spectrometry
|December 2, 2025
PubMed
Summary

Supplementing resolubilization with n-dodecyl-β-d-maltopyranoside (DDM) significantly improves hydrophobic protein recovery in proteomics. This method enhances identification of membrane proteins and S-palmitoylation candidates, addressing a key challenge in proteomic analysis.

Keywords:
Hydrophobic peptidesHydrophobic proteomeImmunoproteomicsMacrophagesMembrane proteinsProtein resolubilizationS-Palmitoylationn-Dodecyl-β-D-maltopyranoside (DDM)

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Area of Science:

  • Proteomics
  • Biochemistry
  • Cell Biology

Background:

  • Hydrophobic proteins, crucial for cellular function and disease, are often under-represented in bottom-up proteomics.
  • This limitation particularly affects the analysis of S-palmitoylation, a modification on membrane-proximal cysteines.

Purpose of the Study:

  • To evaluate the efficacy of n-dodecyl-β-d-maltopyranoside (DDM) in enhancing hydrophobic protein recovery during post-precipitation resolubilization in proteomics.
  • To assess DDM's impact on both global proteomics and targeted acyl-biotin exchange (ABE) proteomics for S-palmitoylation analysis.

Main Methods:

  • Supplementation with n-dodecyl-β-d-maltopyranoside (DDM) during the post-precipitation resolubilization step was compared to standard 8 M urea resolubilization.
  • Experiments were conducted using immortalized bone marrow-derived macrophages (iBMDMs).
  • Global proteomics and acyl-biotin exchange (ABE) proteomics were employed to assess protein and peptide identifications and recovery.

Main Results:

  • DDM supplementation significantly improved peptide and protein identifications, especially for membrane proteins.
  • In global proteomics, 539 proteins were uniquely identified with DDM, enriched in mitochondrial and membrane-bounded organelle components.
  • For ABE proteomics, DDM enhanced recovery of palmitoylated proteins, with 223 proteins consistently requiring DDM for identification and contributing to the discovery of 336 new S-palmitoylation candidates.

Conclusions:

  • DDM-assisted resolubilization effectively overcomes the challenge of hydrophobic protein loss in proteomics workflows.
  • This approach enables more comprehensive characterization of hydrophobic and lipid-modified proteomes, including S-palmitoylated proteins.
  • DDM supplementation offers a simple yet powerful modification to existing proteomics protocols for improved proteome coverage.