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Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

Nature Protocols volume 10, pages 149–168 (2015)Cite this article

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Abstract

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein CrkL, a major substrate of the oncogenic tyrosine kinase BCR-ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <104 cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-CrkL and the protein tyrosine phosphatase PTPRC/CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents.

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Figure 1: Workflow from patient sample collection to data analysis.
Figure 2: Assay plate design.
Figure 3: Assay development process for CrkL antibody.
Figure 4: Assay development process for pTyr207-CrkL antibody.
Figure 5: Chemiluminescence data for DAS and IM drug treatments displaying phosphorylation profile shifts from control.

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Acknowledgements

This work was supported by Leukaemia and Lymphoma Research UK (08004 to F.P., M.T.S. and T.L.H.), Cancer Research UK (C11074/A11008 to F.P., M.T.S. and T.L.H.) and the Chief Scientist Office (CZB/4/690 to F.P., M.T.S. and T.L.H.), and also by the Glasgow and Manchester Experimental Cancer Medicine Centres, which are funded by Cancer Research UK and the Chief Scientist Office in Scotland.

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Authors and Affiliations

  1. Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK

    Mark Aspinall-O'Dea, Andrew Pierce, Andrew J Williamson, Michael J Walker & Anthony D Whetton

  2. Paul O'Gorman Leukaemia Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK

    Francesca Pellicano, Mary T Scott & Tessa L Holyoake

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  1. Mark Aspinall-O'Dea
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Contributions

A.D.W. and T.L.H. devised the study; M.A.-O. performed the experimental and study design and wrote the manuscript with A.D.W.; A.P. contributed to study design and instrument setup; F.P. and M.T.S. prepared clinical material; A.J.W. provided the Src inhibitor; and all authors contributed experimental methods and reviewed the manuscript.

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Correspondence to Anthony D Whetton.

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Integrated supplementary information

Supplementary Figure 1 CD45 assay development.

Representative trace for total-CD45 (black) and pCD45 (red). b, LOD curve for CD45. c, UV titration for CD45 using total antibody, error bars S.E.M (n=3 technical replicate).

Supplementary Figure 2 Lambda phosphatase treatment of HL60 and K562 cell lysates.

Assay plate design and results created using Compass software assay and analysis perspectives respectively. a, layout of samples, antibodies and reagents in 384-well plate format b, detailed description of samples and antibodies applied to plate designed in (a). c, representative differences in total CrkL spectra resulting from lambda phosphatase (λp) treatment, K562-NDC (black), K562-NDC-λp (green), K562-IM 5µM-λp (blue) and K562-DAS 150nM-λp (red).

Supplementary Figure 3 Spectra mismatching and poor ladder detection.

Assay data showing total CrkL antibody probed against K562 cell lysate with no TKI reatment. Spectra shown represent technical replicates from the same sample. a, mismatched data example; correct standard ladder pI associations applied to the black trace, and incorrect associations applied to the blue trace. Red dotted lines indicate matching peaks and the extent of peak offset in the blue trace as a result of poor ladder detection. b, manual alteration of this error results in perfect alignment.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 (PDF 927 kb)

Poor separation; sample salt content > 150 mM.

Video showing the effect on protein separation with cIEF when using clinical samples prepared in a high ionic strength buffer (n = 4 biological samples, loaded in triplicate). (MOV 1916 kb)

Good separation; sample salt content <150 mM.

Video showing the effect on protein separation with cIEF when using clinical samples prepared in a low ionic strength buffer (n = 4 biological samples, loaded in triplicate). (MOV 1402 kb)

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Aspinall-O'Dea, M., Pierce, A., Pellicano, F. et al. Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines. Nat Protoc 10, 149–168 (2015). https://doi.org/10.1038/nprot.2015.007

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