gms | German Medical Science

Patients with similar types of cancers may respond very differently to standard therapy because each patient´s tumor may be driven by distinct molecular alterations. Protein microarrays are an emerging class of nanotechnologies for tracking many different biomarkers simultaneously. Most researchers think that routinely processed formalin fixed tissues are not suitable for proteome analysis. We aimed to establish a robust method for extraction of full length proteins from archival tissues for quantitative analysis using protein microarrays. Our ultimate goal is to develop a technology for the characterization of specific protein abundances for individual patients. From single 10 µm sections of formalin fixed cancer samples, tissue areas of interest were microdissected. A special protein extraction buffer and protocol were developed. Protein lysates were analysed using standard Western blot. In addition, the lysates were manually or automatically spotted onto nitrocellulose or polyvinylidine fluoride (PVDF) membranes to generate protein microarrays. Our novel protein extraction technology from formalin fixed tissues yielded high amounts of proteins at the expected molecular weight. For a poof of principle we identified E-cadherin and its repressor Snail, HER2, beta-actin, and alpha-tubulin by Western blot analysis. After spotting onto different solid surfaces, the extracted proteins could be detected using appropriate antibodies. Dilution curves confirmed the complete solubilization of the proteins analysed. Western blots and protein microarrays correlated excellently. The protein yield was similar from formalin fixed and non-fixed cells. We succeeded in the isolation of full length, soluble and immunoreactive proteins from formalin fixed tissues routinely processed in our hospital. Using protein microarrays our technique can be used to quantify known molecular markers and to identify novel disease markers in high throughput and in an automatic fashion for the individualization of cancer therapy.