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N-Dodecyl-β-D-maltopyranoside (DDM)-Assisted Resolubilization Enhances Hydrophobic Peptide and Protein Identification in Bottom-Up 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.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Macrophage

SUBMITTER: Aleksandra Nita-Lazar

LAB HEAD: Aleksandra Nita-Lazar

PROVIDER: PXD064279 | Pride | 2025-12-16

REPOSITORIES: Pride

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			Action	DRS
	2_IMM_BR1_ABE_N_1_Urea_8.raw	Raw
	2_IMM_BR1_ABE_N_2_DDM_0_0_5.raw	Raw
	2_IMM_BR1_ABE_N_3_DDM_0_1.raw	Raw
	2_IMM_BR1_ABE_N_4_Urea_8_DDM_0_0_5.raw	Raw
	2_IMM_BR1_ABE_N_5_Urea_8_DDM_0_1.raw	Raw

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Project description:Disruptions of protein homeostasis in the endoplasmic reticulum (ER) elicit activation of the unfolded protein response (UPR), a translation- and transcription-coupled proteostatic stress response pathway. The primary translational control arm of the UPR is initiated by the PERK-dependent phosphorylation of eIF2Î±, leading to a large-scale reprogramming of translation and subsequent activation of an ATF4-mediated transcriptional program. It has remained challenging, however, to accurately evaluate the contribution of each component of the eIF2Î±/ATF4 pathway to the remodelling of transcription and translation. Here, we have used mouse embryonic fibroblasts containing either a knock-in of the non-phosphorylatable eIF2Î± S51A mutant or knock-out for ATF4 by ribosome profiling and mRNA-seq to define the specific contributions of eIF2Î± phosphoryation and ATF4 in controlling the translational and transcriptional components of the UPR. These studies show that the transcriptional and translational targets of each P-eIF2Î±, ATF4, and the other UPR gene expression programs overlapped extensively, leading to a core set of UPR genes whose robust expression in response to ER stress is driven by multiple mechanisms. The identification of other, more factor-specific targets illustrated the potential for functional specialization of the UPR. As the UPR progressed temporally, cells employed distinct combinations of transcriptional and translational mechanisms, initiated by different factors, to adapt to ongoing stress. These effects were accompanied by a buffering effect where changes in mRNA levels were coupled to opposing changes in ribosome loading, a property which makes cooperation between transcription and translation essential to confer robust protein expression. Translational analysis by ribosome profiling and mRNA-seq of PERK pathways mutants during the UPR. Mouse embryonic fibroblasts (MEFs) lacking components of the PERK pathway (eIF2a phosphorylation and ATF4) were subjected to ER stress and analyzed by ribosome profiling.

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N-Dodecyl-β-D-maltopyranoside (DDM)-Assisted Resolubilization Enhances Hydrophobic Peptide and Protein Identification in Bottom-Up Proteomics

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