title: An Optimized Method for Protein Extraction from OCT-Embedded Human Kidney Tissue for Protein Quantification by LC-MS/MS Proteomics.
abstract: The existing biobanks of remnant tissue from clinically indicated kidney biopsies are 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. Although OCT is an effective method of preserving the morphologic and immunohistological characteristics of tissues for later study, it significantly impacts efforts to quantify protein expression by liquid chromatography-tandem mass spectrometry 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 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 by targeted selective reaction monitoring proteomics. Upon comparison of peptide signals from OCT-embedded tissue and flash-frozen tissue from the same donors, both individual protein quantities, and their interindividual variabilities, were similar. Therefore, the approach reported here can be applied to clinical reservoirs of OCT-preserved kidney tissue to be used for quantitative proteomics studies of clinically relevant proteins expressed in different parts of the kidney (including drug transporters and metabolizing enzymes).
authors.affiliation: Department of Pharmaceutics (M.V., B.P.) and Kidney Research Institute and Division of Nephrology, Department of Medicine (A.G., M.A.), University of Washington, Seattle, Washington.
authors.forename: Marc
authors.initials: M
authors.lastname: Vrana
authors.affiliation: Department of Pharmaceutics (M.V., B.P.) and Kidney Research Institute and Division of Nephrology, Department of Medicine (A.G., M.A.), University of Washington, Seattle, Washington.
authors.forename: Anne
authors.initials: A
authors.lastname: Goodling
authors.affiliation: Department of Pharmaceutics (M.V., B.P.) and Kidney Research Institute and Division of Nephrology, Department of Medicine (A.G., M.A.), University of Washington, Seattle, Washington.
authors.forename: Maryam
authors.initials: M
authors.lastname: Afkarian
authors.affiliation: Department of Pharmaceutics (M.V., B.P.) and Kidney Research Institute and Division of Nephrology, Department of Medicine (A.G., M.A.), University of Washington, Seattle, Washington bhagwat@uw.edu.
authors.forename: Bhagwat
authors.initials: B
authors.lastname: Prasad
mesh_terms: D000368:Aged; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D011506:Proteins / Q000302:isolation & purification*; D053719:Tandem Mass Spectrometry; D013696:Temperature
chemical_list: D011506:Proteins