Enhanced S-Palmitoylated Protein Detection by Mild Nonionic Detergent in Proteomic Workflow

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.