Björn Fröhlich (Presenter)
UVic Genome BC Proteomics Centre
Bio: From 2009 to 2013, Björn Fröhlich attended Hochschule Fresenius, University of Applied Sciences, and obtained a bachelor’s degree in Applied Chemistry. He continued his education at this university and graduated with a master’s degree in Bio-and Pharmaceutical Analysis in 2014. Before starting his PhD at the University of Victoria in May 2015, he worked as an academic assistant at the department of Chemistry and Biology at Hochschule Fresenius, organizing workshops in cooperation with Physikalisch-Technische Bundesanstalt (PTB) regarding strengthening food safety analysis skills in south Caucasian countries. Currently, he is a PhD student at the University of Victoria under Dr. Christoph Borchers.
Authorship: Björn Fröhlich (1,2) , Robert Popp (1,2) , Adriana Aguilar-Mahecha (3,4) , Mark Basik (5) , André LeBlanc (6) , Oliver Poetz (7) , Christoph H Borchers (1,2,5,6)
(1) University of Victoria - Genome British Columbia Proteomics Centre, Victoria British Columbia, Canada (2) Department of Biochemistry and Microbiology, University of Victoria, Victoria British Columbia, Canada (3) Goodman Cancer Research Centre, McGill University, Montréal Quebec, Canada (4) Department of Biochemistry, McGill University, Montréal Quebec, Canada (5) Gerald Bronfman Department of Oncology, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, Montréal Quebec, Canada (6) Proteomics Centre, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montréal Quebec, Canada (7) Natural and Medical Sciences Institute (NMI) at the University of Tübingen, Reutlingen Baden-Württemberg, Germany
| Short Abstract Colorectal cancer is one of the most common cancers in incidence and cancer-related deaths. The PI3K/AKT/mTOR pathway is commonly upregulated in colorectal cancer and is the target of many anti-cancer therapies. Immuno-MALDI (iMALDI) was used to quantify the expression levels and phosphorylation status of key proteins in this pathway, which could be useful for patient stratification. Cancer cell lysates were digested using trypsin, stable-isotope labeled standards added, enriched from the sample using anti-peptide antibodies and analysed using MALDI-TOS MS. Quantification of AKT I+II expression-and-phosphorylation levels was performed on cell lysates and tumor tissue. Endogenous PI3K p110a and PTEN was detected in cell lysate. The next steps of this project will include additional method validation and a higher degree of multiplexing. |
Long Abstract
Introduction
Colorectal cancer is one of the most common cancers in both incidence and cancer-related deaths[1], The PI3K/AKT/mTOR pathway is commonly upregulated in colorectal cancer and the target of many anti-cancer therapies[2], but current patient stratification methods for targeted therapy are based mostly on genomic data.
The goal of this project was to develop immuno-MALDI mass spectrometry (iMALDI) assays to quantify the expression levels and phosphorylation levels of proteins in the PI3K/AKT/mTOR pathway. In combination with genomic data, these levels can be used to build a predictive model for a patient’s response to targeted therapy.
Methods
iMALDI is a method for protein quantification which combines antibody enrichment with detection by MALDI-TOF mass spectrometry. After enzymatic digestion of the sample, analyte-specific endogenous peptides (END peptides), together with analogous stable-isotope labeled peptides (SIS peptides), are enriched using antibodies immobilized to magnetic beads. The beads are separated from the sample using a magnet, washed, and spotted on a MALDI target. After matrix addition, the target peptides are analyzed by MALDI-TOF MS. Protein quantification is achieved using the ratio of the END-and-SIS peptides. Quantification of phosphorylation levels is achieved by splitting the sample into two aliquots. Both samples are subjected to enzymatic digestion, but one of the aliquots is also treated with a phosphatase prior to peptide enrichment[3]. The increase in the amount of unphosphorylated END peptide due to removal of the phosphoryl group is equal to the amount of peptide that was originally phosphorylated.
Results
PI3K p110α, PTEN, and AKT I and II were selected as targets for assay development. Unique tryptic peptides containing the cancer-related phosphorylation sites were selected, confirmed experimentally, and used to raise polyclonal antibodies.
Quantification of AKT I and II expression-and-phosphorylation levels and were achieved in various cancer cell lines, as well as in flash-frozen and formalin fixed tumour tissue samples, using 10 µg cell lysate. Endogenous PI3K p110α and PTEN were detected in 25 µg MDA-MB-231 breast cancer cell lysate.
Conclusions & Discussion
Methods for quantifying AKT I and II as well as PTEN and PI3K p110α were developed. The next steps of this project include combining the PI3K p110 α, PTEN, and AKT assays into a single multiplexed assay, as well as adding additional protein targets from the PI3K/PI3K/mTOR pathway. Further method validation will be performed using different cancer cell lysates and tumour tissue samples. After the method has been fully validated, it will be used for the analysis of colorectal cancer xenograft mice and patient samples. Together with genomic data, a predictive algorithm for patient stratification for targeted therapies will be build.
References & Acknowledgements:
[1] Siegel RL, Miller KD, Jemal A, CA Cancer J Clin. 2015 Jan-Feb;65(1):5-29.
[2] Papadatos-Pastos D et al, Crit Rev Oncol Hematol. 2015 Apr;94(1):18-30
[3] Domanski D, Murphy LC, Borchers CH, Anal Chem. 2010 Jul 1;82(13):5610-20
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