Robert Popp(1), Andrew Chambers(1), Adriana Aguilar-Mahecha(2), Oliver Pötz(3), Mark Basik(2), Christoph Borchers(1,2)
(1)University of Victoria - Genome BC Proteomics Centre, (2)Jewish General Hospital - McGill University, (3)Natural and Medical Sciences Institute (NMI) at the University of Tübingen
Targeted treatment of colorectal cancer (CRC) 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 non-phosphorylated Akt1 from 100 µg protein of breast and CRC cells (MDA-231, SW480 and HCT116), and breast cancer tumours. Non-phosphorylated Akt2 has been quantified in 100 µg MDA-231 breast cancer cells. Recently, we improved sensitivity by 10-fold, which allowed decreasing the sample amount from 100 µg to 10 µg. The current lower limit of detection is 0.35 pg Akt1/µg lysate protein. We have developed iMALDI Akt1 and Akt2 assays for quantitation of non-phosphorylated Akt1 and Akt2.
Colorectal cancer (CRC) is the 4th most common cause of death from cancer. Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) signaling pathways play a critical role in colorectal cancer (CRC) 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-MALDI (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 tumours.
The iMALDI approach is based on tryptic digestion of the endogenous target proteins, followed by affinity-enrichment of specific tryptic peptides and analogue stable-isotope labeled standard (SIS) peptides, and absolute quantitation using MALDI-TOF.
Tryptic digestion is performed on cell or tumour tissue lysates. Post-digestion, a stable isotope-labeled standard (SIS) peptide corresponding to the tryptic target peptide is added as internal standard. The solution is split into two aliquots, one of which 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 target. 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 non-phosphorylated Akt1 in 100 µg of EGF-induced and non-induced MDA-231 breast cancer cell lines, SW480 and HCT116 colon cancer cell lines, and in 100 µg breast cancer tumor lysates. All samples analyzed fell within the linear range (4.4 – 1114 pg Akt1/µg) of the Akt1 assay. Precision for triplicate digests was found to be consistently <10%. In addition, non-phosphorylated Akt2 has been quantified in 100 µg MDA-231 breast cancer cell lysate. In recent efforts, we were able to improve sensitivity by a factor of 10, which allowed decreasing the sample amount from 100 µg protein to 10 µg. This is important when considering analyzing needle core biopsies, which yield low protein amounts. The current lower limit of detection is 0.35 pg Akt1/µg lysate protein.
We have developed iMALDI Akt1 and Akt2 assays for quantitation of non-phosphorylated Akt1 and Akt2. The next steps will involve optimization of the dephosphorylation step of the PPQ method, determination of phosphorylation stoichiometry in cancer cell lines and tumor samples, and automation of the sample preparation.