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 ...

Journal of the American Society for Mass SpectrometryDOI: 10.1021/jasms.5c00186

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.

To identify modules significantly associated with NAFLD, we performed WGCNA on the GSE89632 dataset. Initially, we conducted hierarchical clustering analysis on the samples and detected two outlier samples, GSM2385767 and GSM2385782. These outliers were removed by setting the cutHeight to...

Reversible S-palmitoylation facilitates the regulation of a plethora of protein functions. Here, we present a protocol for enriching lipidated proteins with click-chemistry labeling and immunoprecipitation using biotin-azide-streptavidin interaction where protein activity assays are possible. We describe steps for performing the copper-based reaction for high-scale applications in milligram ranges, sample preparation, and the elution of palmitoylated-biotinylated proteins. Further, we detail pro...

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