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N-Dodecyl-β-D-maltopyranoside (DDM)-Assisted Resolubilization Enhances S-Palmitoylation Proteomics

ABSTRACT: This study demonstrates that adding the non-ionic surfactant n-dodecyl-β-D-maltopyranoside (DDM) during protein resolubilization improves the identification of hydrophobic peptides and proteins in bottom-up proteomics. DDM's effectiveness in overcoming the challenge of losing hydrophobic proteins after precipitation from strong detergent lysis buffers was investigated. Using immortalized mouse macrophages (IMMs), DDM (0.05% or 0.1%) to resolubilization buffers, both with and without 8M urea in 50 mM ammonium bicarbonate (ABC) were added, for global and acyl-biotin exchange (ABE) proteomics (targeting S-palmitoylation). Key findings revealed that the addition of 0.1% DDM increased the identification of total peptides, proteins, and especially membrane-associated proteins in global proteomics compared to urea alone. Gene Ontology (GO) analysis showed that proteins uniquely identified with DDM were enriched in critical cellular processes. In ABE proteomics, 0.1% DDM enhanced the identification of hydrophobic and candidate S-palmitoylated peptides. DDM also increased the number of ABE-enriched proteins, with those uniquely identified by DDM being involved in cellular transport and localization known functions of S-palmitoylated proteins. In conclusion, incorporating DDM during protein resolubilization is a simple and effective method to enhance the coverage of hydrophobic proteomes in various bottom-up proteomics applications.

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Macrophage

SUBMITTER: Aleksandra Nita-Lazar

LAB HEAD: Aleksandra Nita-Lazar

PROVIDER: PXD068353 | Pride | 2025-12-16

REPOSITORIES: Pride

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Project description:Mutations in Ppt1, which is a depalmitoylation enzyme result in early onset neurodegeneration, therefore, we hypothesized that the protein content of brain membranes from mutant newborn mice will differ from those derived from wild-type. This unbiased analysis revealed that the majority of the differentially identified proteins are palmitoylated and their upstream pathway analysis point to neurodegenerative diseases. One of the differentially expressed proteins was Rab3IP, which is a direct downstream effector of Rab11 and affects the guanine nucleotide-exchange activity of Rab3IP toward Rab8. Rab3IP, Rab11 and Rab8 are known to be involved in ciliogenesis. Rab3IP distribution in cilia differed between the wild type and the mutant cells. Our analysis showed that these three proteins are palmitoylated. Site-directed mutagenesis of the palmitoylation sites of Rab11 affects its intracellular localization. Most importantly, in MEFs, neuroblasts, neurons and brain preparations, longer cilia were detected in Ppt1-/-. Collectively, our studies suggest that cilia abnormalities need to be considered as part of the pathophysiology of CLN1. Mutations in Ppt1, which is a depalmitoylation enzyme result in early onset neurodegeneration, therefore, we hypothesized that the protein content of brain membranes from mutant newborn mice will differ from those derived from wild-type. This unbiased analysis revealed that the majority of the differentially identified proteins are palmitoylated and their upstream pathway analysis point to neurodegenerative diseases. One of the differentially expressed proteins was Rab3IP, which is a direct downstream effector of Rab11 and affects the guanine nucleotide-exchange activity of Rab3IP toward Rab8. Rab3IP, Rab11 and Rab8 are known to be involved in ciliogenesis. Rab3IP distribution in cilia differed between the wild type and the mutant cells. Our analysis showed that these three proteins are palmitoylated. Site-directed mutagenesis of the palmitoylation sites of Rab11 affects its intracellular localization. Most importantly, in MEFs, neuroblasts, neurons and brain preparations, longer cilia were detected in Ppt1-/-. Collectively, our studies suggest that cilia abnormalities need to be considered as part of the pathophysiology of CLN1. Mutations in Ppt1 result in early onset neurodegeneration, therefore, we hypothesized that the levels of proteins extracted from purified brain membranes of newborn mice will differ. One of the main effects of palmitoylation is changing the hydrophobic index of proteins and thus affecting these proteins interactions with different membranal domains. Assuming that PPT1 substrates are over palmitoylated in Ppt1-/- brains it is possible that its localization to specific membranal domain is different than in the wild-type brains. We isolated Triton X-100 soluble membranes from three wild-type and three Ppt1-/- brains and identified the proteins by PalmPISC. Many of the identified proteins were found to be palmitoylated. A second feature, which was noted is that many of the differential proteins (62 out of 88, 70%) can be detected in the cilia proteome (http://v3.ciliaproteome.org/cgi-bin/index.php). This observation led to test whether ciliary proteins differ between wild-type and Ppt1-/-. Cilia were prepared from newborn dissociated brain cell cultures and analyzed by MS

			Action	DRS
	1_HEK_ABE_N_1_Urea_8.raw	Raw
	1_HEK_ABE_N_2_DDM_005.raw	Raw
	1_HEK_ABE_N_3_DDM_01.raw	Raw
	1_HEK_ABE_N_4_DDM_02.raw	Raw
	1_HEK_ABE_P_1_Urea_8.raw	Raw

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N-Dodecyl-β-D-maltopyranoside (DDM)-Assisted Resolubilization Enhances S-Palmitoylation Proteomics

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