title: Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines.
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 <10(4) 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.
authors.affiliation: Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Aspinall-O'Dea
authors.affiliation: Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Pierce
authors.affiliation: Paul O'Gorman Leukaemia Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK.
authors.forename: Francesca
authors.initials: F
authors.lastname: Pellicano
authors.affiliation: Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Williamson
authors.affiliation: Paul O'Gorman Leukaemia Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK.
authors.forename: Mary T
authors.initials: MT
authors.lastname: Scott
authors.affiliation: Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Walker
authors.affiliation: Paul O'Gorman Leukaemia Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK.
authors.forename: Tessa L
authors.initials: TL
authors.lastname: Holyoake
authors.affiliation: Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK.
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Whetton
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000906:Antibodies / Q000378:metabolism*; D015153:Blotting, Western; D005434:Flow Cytometry; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism*; D017493:Leukocyte Common Antigens / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; C082869:CRKL protein; D009687:Nuclear Proteins; D011506:Proteins; D014443:Tyrosine; D016044:Fusion Proteins, bcr-abl; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human