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

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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.

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Figure 1: Structures of the molecular species of PE and PS externalized by human platelets on activation, apoptosis or during aging.
Figure 2: Externalization of APLs by apoptotic neutrophils.
Figure 3: Schematic representation of the detection of externalized APLs.
Figure 4
Figure 5: Negative MS/MS of biotinylated standards showing fragmentation pattern.
Figure 6: Analysis of APLs by using direct infusion and LC-MS/MS.

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Acknowledgements

The studies were funded by the Wellcome Trust (V.B.O., V.J.H., P.W.C.). C.P.T. is a Marie Curie International Outgoing Fellow.

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  1. Christopher P Thomas and Stephen R Clark: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, UK

    Christopher P Thomas, Stephen R Clark, Victoria J Hammond, Maceler Aldrovandi, Peter W Collins & Valerie B O'Donnell

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  1. Christopher P Thomas
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  2. Stephen R Clark
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  3. Victoria J Hammond
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  4. Maceler Aldrovandi
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  6. Valerie B O'Donnell
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Contributions

C.P.T., S.R.C., V.J.H. and M.A. conducted the experiments; V.B.O. and C.P.T. wrote the paper; and P.W.C. edited the paper. V.B.O., S.R.C., C.P.T. and P.W.C. designed the protocols.

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Correspondence to Valerie B O'Donnell.

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The authors declare no competing financial interests.

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Thomas, C., Clark, S., Hammond, V. et al. Identification and quantification of aminophospholipid molecular species on the surface of apoptotic and activated cells. Nat Protoc 9, 51–63 (2014). https://doi.org/10.1038/nprot.2013.163

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