Robert Popp (Presenter)
UVic - Genome BC Proteomics Centre
Bio: Robert Popp is a 3rd year PhD student in Dr. Christoph Borchers lab at the University of Victoria - Genome BC Proteomics Center in Victoria, British Columbia, Canada. He is working on developing diagnostic and predictive immuno-MALDI assays for protein quantitation from patient specimens such as plasma or tissue lysates with the goal of transferring the assays to a clinical setting.
Authorship: Robert Popp (1), Huiyan Li (1), Yassene Mohammed (1, 2), Adriana Aguilar-Mahecha (3), Andrew G. Chambers (1), Cathy Lan (3), Oliver Pötz (4), Mark Basik (3), Gerald Batist (3), Christoph H. Borchers (1, 3, 5)
(1) University of Victoria - Genome BC Proteomics Centre, Victoria, CA (2) Leiden University Medical Center, Leiden, NL (3) Jewish General Hospital, McGill University, Montreal, CA (4) Natural and Medical Sciences Institute (NMI), Reutlingen, DE (5) University of Victoria, Victoria, CA
Novel anti-cancer drugs only works in a minority of patients, and reliable methods to quantify signaling pathway activity are lacking. We therefore set out to develop immuno-MALDI (iMALDI) assays to determine expression levels and stoichiometry of critical phosphorylation sites in AKT1 (P31749) and AKT2 (P31751) in cancer cells and tumours. We quantified the target peptides from breast and colon cancer cell lines (MDA-231, SW480 and HCT116), as well as breast cancer and HCT116 mouse xenograft colon-cancer fresh frozen and FFPE tumor samples (~50 µg protein/sample). Target peptide levels ranged from ~ 0.1-1 fmol/µg protein lysate with phosphorylation levels of 0-~50%. The assay has a linear range of 0.5-10 fmol of peptide on the MALDI plate with CVs <10%. In conclusion, we have developed iMALDI AKT1 and AKT2 assays for quantitation of non-phosphorylated AKT1 and AKT2.
Breast cancer and colorectal cancer are responsible for 0.52 and 0.69 million deaths worldwide, respectively. Aberrations in PI3K/AKT/mTOR play a critical role in the development and progression of these cancers and are targeted by novel therapeutic agents. However, these targeted treatments only work in a minority of patients. Additionally, predictive protein biomarkers are lacking or only partially successful, and currently there are no reliable methods to quantify the activity of these pathways. We therefore set out to develop immuno-matrix assisted laser desorption/ionization (iMALDI) assays based on our phosphatase-based phosphopeptide quantitation (PPQ) method to determine expression levels of AKT1 (P31749) and AKT2 (P31751), as well as to measure the pathway activity by accurately and precisely quantitating stoichiometry of phosphorylation sites critical for protein function (S473 and S474, respectively) in cancer cells and tumors.
As a first step, lysates of cancer cell lines or tumor tissues undergo tryptic digestion, followed by addition of stable isotope-labelled standard (SIS) peptides corresponding to the tryptic C-terminal AKT1 and AKT2 peptides (466RPHFPQFSYSASGTA480 and 468THFPQFSYSASIRE481, respectively). The solution is split into two aliquots, of which one is treated with alkaline phosphatase. The non-phosphorylated target peptides of both aliquots are then captured by anti-target peptide antibodies coupled to magnetic Protein G Dynabeads®. The beads are washed and spotted directly onto a MALDI plate. Addition of the acidic HCCA-MALDI matrix elutes the captured peptides from the beads. A Bruker Microflex LRF MALDI-TOF instrument is used for absolute quantitation of non-phosphorylated target peptide in both aliquots, which allows determination of the degree of phosphorylation (stoichiometry) of the target phosphopeptides in the sample.
We quantified the target peptides from colon-cancer (SW480, HCT116) and breast-cancer (MDA-231) cell lines, as well as flash-frozen and formalin-fixed paraffin-embedded (FFPE) breast-cancer and HCT116 mouse xenograft colon-cancer tissues. Per sample, ~50 µg protein per sample are required (10 µg protein per capture). Target peptide levels ranged from ~ 0.1-1 fmol/µg protein lysate with phosphorylation levels of 0-50%. The lower limit of detection was 100 amol of peptide, with a linear range of 0.5-10 fmol of peptide on the MALDI plate. CVs for iMALDI assays were consistently <10%. All liquid handling steps have been automated on an Agilent Bravo liquid handling robot.
We have developed iMALDI AKT1 and AKT2 assays for quantitation of non-phosphorylated AKT1 and AKT2 in fresh frozen and FFPE tumor samples which will be validated in a currently on-going study.
References & Acknowledgements:
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