Proteomics

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Application of liquid-liquid extraction for N-terminal myristoylation proteomics


ABSTRACT: Proteins can be modified by lipids in various ways, for example by myristoylation, palmitoylation, farnesylation, and geranylgeranylation—these processes are collectively referred to as lipidation. Current chemical proteomics using alkyne lipids has enabled the identification of lipidated protein candidates but does not identify endogenous lipidation sites and is not readily applicable to in vivo systems. Here, we introduce a proteomic methodology for global analysis of endogenous protein N-terminal myristoylation sites that combines liquid-liquid extraction of hydrophobic lipidated peptides with liquid chromatography-tandem mass spectrometry using a gradient program of acetonitrile in the high concentration range. We applied this method to explore myristoylation sites in HeLa cells, and identified a total of 75 protein N-terminal myristoylation sites, which is more than the number of high-confidence myristoylated proteins identified by myristic acid analog-based chemical proteomics. Isolation of myristoylated peptides from HeLa digests prepared with different proteases enabled the identification of different myristoylated sites, extending the coverage. Finally, we analyzed in vivo myristoylation sites in mouse tissues and found that the lipidation profile is tissue-specific. This simple method (not requiring chemical labeling or affinity purification) should be a promising tool for global profiling of protein N-terminal myristoylation.

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Koshi Imami 

PROVIDER: PXD041499 | JPOST Repository | Thu Nov 09 00:00:00 GMT 2023

REPOSITORIES: jPOST

Dataset's files

Source:
Action DRS
20220909_kt_lipidpept_EtoAc_1.raw Raw
20220909_kt_lipidpept_EtoAc_2.raw Raw
20220909_kt_lipidpept_EtoAc_3.raw Raw
20220909_kt_lipidpept_heptanol_1.raw Raw
20220909_kt_lipidpept_heptanol_2.raw Raw
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Publications

Application of Liquid-Liquid Extraction for N-terminal Myristoylation Proteomics.

Tsumagari Kazuya K   Isobe Yosuke Y   Ishihama Yasushi Y   Seita Jun J   Arita Makoto M   Imami Koshi K  

Molecular & cellular proteomics : MCP 20231109 12


Proteins can be modified by lipids in various ways, for example, by myristoylation, palmitoylation, farnesylation, and geranylgeranylation-these processes are collectively referred to as lipidation. Current chemical proteomics using alkyne lipids has enabled the identification of lipidated protein candidates but does not identify endogenous lipidation sites and is not readily applicable to in vivo systems. Here, we introduce a proteomic methodology for global analysis of endogenous protein N-ter  ...[more]

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