Dataset Information

Super-resolution proximity labeling method with enhanced direct identification of biotinylation sites

ABSTRACT: Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proteins within membranous organelles. Applying this method to the processing body (PB), a non-membranous organelle, successfully allowed unbiased identification of PB core proteins, including novel candidates. We anticipate that our newly developed method will replace the conventional method for identifying biotinylated proteins labeled by promiscuous labeling enzymes.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Sanghee Shin

LAB HEAD: Jong-Seo Kim

PROVIDER: PXD047979 | Pride | 2024-05-22

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	0_JACS_modificationSpecificPeptides.txt	Txt
	0_onbead_proteinGroups.txt	Txt
	172-1_J100_NAQ448_20180719.raw	Raw
	172-2_J100_NAQ448_20180719.raw	Raw
	172-3_J100_NAQ448_20180719.raw	Raw

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Publications

Super-resolution proximity labeling with enhanced direct identification of biotinylation sites.

Shin Sanghee S Lee Song-Yi SY Kang Myeong-Gyun MG Jang Dong-Gi DG Kim Jeesoo J Rhee Hyun-Woo HW Kim Jong-Seo JS

Communications biology 20240509 1

Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed ...[more]

PMID: 38724559

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Project description:Background: Cell surface proteins perform critical functions related to immune response, signal transduction, cell-cell interactions, and cell migration. Expression of specific cell surface proteins can determine cell-type identity, and can be altered in diseases including infections, cancer and genetic disorders. Identification of the cell surface proteome remains a challenge despite the development of several enrichment methods exploiting their biochemical and biophysical properties. Methods: We report a novel method for enrichment of proteins localized to the cell surface. We developed surface Biotinylation Site Identification Technology (sBioSITe) by adapting our previously published method for direct identification of biotinylated peptides. The primary amine groups of lysines on cell surface proteins in live PC3 cells in culture were first labeled with biotin with subsequent enrichment of biotinylated peptides with anti-biotin antibodies. The enriched peptides containing biotin were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Biotinylated peptides identified in triplicate were mapped to proteins. Results: By direct detection of biotinylated lysines using sBioSITe, we identified 5,851 peptides biotinylated on the cell surface derived from 1,409 proteins. 533 of these proteins have been previously shown or predicted to be localized to the cell surface, or secreted into the extracellular space. Importantly, several of the identified markers have known associations with prostate cancer and metastasis including CD59, epithelial growth factor receptor (EGFR), 4F2 cell-surface antigen heavy chain (SLC3A2) and Adhesion G protein-coupled receptor E5 (ADGRE5) thus highlighting the robustness of this method. Conclusions: Detection of biotinylation sites on cell surface proteins modified in vitro provides a reliable method for the identification of cell surface proteins. This method is an excellent tool that complements existing methods for the analysis of surface expressed proteins in qualitative as well as comparative studies.