Abstract

The existing biobanks of remnant tissue from clinically indicated kidney biopsies are the attractive potential reservoirs for quantification of clinically relevant human tissue proteins by quantitative proteomics. However, a significant caveat of this strategy is that the tissues are often preserved in Optimal Cutting Temperature (OCT) medium. While OCT is an effective method of preserving the morphological and immunohistological characteristics of tissues for later study, it significantly impacts efforts to quantify protein expression by LC-MS/MS methods. We report here a simple, reproducible and cost-effective procedure to extract proteins from OCT-embedded tissue samples. Briefly, the excess frozen OCT medium was immediately scraped before thawing from the tissue specimens stored at -80 °C for ~3 months. The tissue samples were homogenized and diethyl ether:methanol extraction was performed to remove the remaining OCT medium. The recovered protein was denatured, reduced and alkylated. The second step of protein extraction and desalting was performed by chloroform:methanol:water extraction of denatured proteins. The resultant protein pellet was trypsin digested and the marker proteins of various kidney cellular compartments were quantified using targeted selective reaction monitoring (SRM) proteomics. When comparing protein signals from OCT-embedded tissue to flash frozen tissue from the same donors, both individual protein quantities, and their inter-individual variability were similar. Therefore, the approach reported here can be applied to use clinical reservoirs of OCT-preserved kidney tissue for quantitative proteomics studies of clinically relevant proteins expressed in different parts of kidney (including drug transporters and metabolizing enzymes).