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Structural surfaceomics of Nomo-1 using PhoX cross-linker with timsTOF

ABSTRACT: The project aimed to profile the cell surface proteins of Nomo-1 (AML cell line) using structural surfaceomics for identification of protein conformation-based cancer antigens thereby expanding the toolkit for cancer target discovery for immunotherapeutic targeting. To achieve the goal, cell surface capture (CSC) was integrated with cross-linking mass spectrometry (XL-MS). PhoX was used as a cross-linker to freeze the structural conformations of protein in three-dimensional space, followed by biotinylation of cell surface proteins to enable enrichment of cell surface proteins to allow focused XL-MS analysis of those enriched proteins. PhoX having in-built phosphonate-based IMAC handle which allowed additional enrichment of cross-linked peptides.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Myeloid Cell Line, Myeloid Cell

DISEASE(S): Acute Myeloid Leukemia

SUBMITTER: Kamal Mandal

LAB HEAD: Dr. Arun P. Wiita

PROVIDER: PXD035591 | Pride | 2023-10-04

REPOSITORIES: Pride

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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.

			Action	DRS
	F09_RG1_1_2994.d.zip	Other
	F10_RG2_1_2995.d.zip	Other
	F11_RG3_1_2996.d.zip	Other
	F12_RG4_1_2997.d.zip	Other
	F13_RG5_1_2998.d.zip	Other

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Structural surfaceomics of Nomo-1 using PhoX cross-linker with timsTOF

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