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

Loss of hydrophobic peptides and proteins remains a significant
challenge in bottom-up proteomics, resulting in under-representation
of membrane and membrane-associated proteins that are critical for
understanding cellular function and disease. This limitation is particularly
acute for targeted applications such as S-palmitoylation analysis,
where modifications occur preferentially on membrane-proximal cysteines.
This study evaluated supplementation by n-dodecyl-β-d-maltopyranoside (DDM), a mild detergent widely used in structural
biology but not proteomics, during the postprecipitation resolubilization
step to enhance hydrophobic protein recovery. Using immortalized bone
marrow-derived macrophages (iBMDMs), we compared standard resolubilization
(8 M urea in 50 mM ammonium bicarbonate) with DDM-supplemented conditions.
In global proteomics, DDM supplementation improved peptide and protein
identifications, with particularly pronounced benefits for membrane
protein recovery. The 539 proteins uniquely identified with DDM were
enriched for mitochondrial components, protein complexes, and membrane-bounded
organelles. For acyl-biotin exchange (ABE) proteomics targeting palmitoylated
proteins, DDM supplementation enhanced recovery of proteins, with
223 proteins consistently requiring DDM for identification. These
DDM-dependent proteins showed enrichment for transport and localization
functions characteristic of palmitoylated proteins. Comparison with
the SwissPalm database revealed 336 previously unreported S-palmitoylation
candidates, with DDM conditions contributing more novel identifications
than urea alone. These findings demonstrate that DDM-assisted resolubilization
addresses a key bottleneck in proteomics workflows, enabling more
comprehensive characterization of hydrophobic and lipid-modified proteomes
without requiring extensive protocol modifications.