Immuno-MALDI (iMALDI) for quantifying AKT1 and AKT2 in breast and colorectal cancer cell lines and tumors.

Anal Chem. 2017 Aug 30. doi: 10.1021/acs.analchem.7b02934. [Epub ahead of print]
Popp R, Li H, LeBlanc A, Mohammed Y, Aguilar-Mahecha A, Chambers AG, Lan C, Poetz O, Basik M, Batist G, Borchers CH.

The PI3K/AKT/mTOR pathway is one of the most commonly dysregulated signaling pathways that is linked to cancer development and progression, and its quantitative protein analysis holds the promise to facilitate patient stratification for targeted therapies. Whereas immunohistochemistry (IHC) and immunoassays are routinely used for clinical analysis of signaling pathways, mass spectrometry-based approaches such as liquid chromatography electrospray ionization-multiple reaction monitoring (LC/ESI-MRM) are more commonly used in clinical research. Both technologies have certain disadvantages, namely the non-specificity of IHC and immunoassays, and potentially long analysis times per sample of LC/ESI-MRM-MS. To create a robust, fast, and sensitive protein quantification tool, we developed immuno-Matrix Assisted Laser Desorption/Ionization (iMALDI) assays with automated liquid handling. The assays are able to quantify AKT1 and AKT2 from breast cancer and colon cancer cell lines and flash-frozen tumor lysates with a linear range of 0.05 to 2.0 fmol/µg total lysate protein, and with CVs <15%. Compared to other mass spectrometric methods, the developed assays require less sample per analysis -- only 25 µg total protein -- and are therefore suitable for the analysis of needle biopsies. Furthermore, the presented iMALDI technique is the first MS-based method for absolute quantitation of AKT peptides from cancer tissues. This study demonstrates the suitability of iMALDI for low limit-of-detection and reproducible quantitation of signaling pathway members using a benchtop MALDI mass spectrometer within approximately 6-7 hours.