Identification and quantification of aminophospholipid molecular species on the surface of apoptotic and activated cells

Nature Protocols
Christopher P ThomasValerie B O'Donnell

Abstract

This protocol measures externalization of aminophospholipids (APLs) to the outside of the plasma membrane using mass spectrometry (MS). APL externalization occurs in numerous events, and it is relevant for transplant medicine, immunity and cancer. In this protocol, externalized APLs are chemically modified by using a cell-impermeable reagent (sulfo-NHS-biotin), and then they are isolated via a liquid:liquid extraction and quantified by reverse-phase liquid chromatography tandem MS (LC-MS/MS) against in-house-generated standards. This protocol describes a complementary method to existing assays that are not quantitative (e.g., annexin V flow cytometry), and it is applicable to the study of membrane reorganization in all cell types during apoptosis (e.g., during development, cancer, psychiatric disorders and other conditions, aging, vesiculation and cell division). The protocol takes ∼2-4 d, including the generation of standards.

References

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Mar 27, 2013·Proceedings of the National Academy of Sciences of the United States of America·Stephen R ClarkValerie B O'Donnell

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Citations

Mar 13, 2015·Nature Communications·Anupam BandyopadhyayJianmin Gao
Apr 25, 2019·Nano Convergence·Melanie A Fuller, Ingo Köper
Jun 2, 2017·The Journal of Experimental Medicine·Stefan UderhardtGerhard Krönke
Apr 5, 2019·Proceedings of the National Academy of Sciences of the United States of America·Keith Allen-RedpathValerie B O'Donnell

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Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis