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).

PAR2 (Protease-Activated Receptor 2) Deficiency Attenuates Atherosclerosis in Mice

“…Briefly, frozen sections (n = 5 patient samples, 25 sections per patient) were purchased and protein (20 combined sections/patient) and mRNA (5 combined sections/patient) were obtained utilizing a previously established method by Vrana and colleagues and an RNAqueous ™ -Micro total RNA isolation kit (Thermo Fisher/Ambion; catalog #AM1931), respectively. 29…”

Comparative analysis of differentially abundant proteins quantified by LC–MS/MS between flash frozen and laser microdissected OCT-embedded breast tumor samples

“…In the present study, we compared proteomic profiles obtained from two standard storage and processing methods [ 1 , 13 – 16 , 34 ] for frozen tissue specimens: (1) FF samples lysed via homogenization and (2) laser microdissected OCT-embedded specimens lysed via incubation at 37 °C, followed by protein extraction from both using the same isolation kit. Our goal was to determine if MS data from these two different sample types can be combined and analyzed together in the same study.…”

“…Both FF and OCT-embedded specimens yield high-quality nucleic acids and proteins for downstream molecular analyses. For proteomic analyses, however, FF has been the preferred storage method for protein extraction and processing as OCT can interfere with the performance of liquid chromatography-tandem mass spectrometry (LC–MS/MS) [ 1 ]. OCT contains the water-soluble synthetic polymers polyvinyl alcohol (PVA) and polyethylene glycol (PEG) that can compete with peptides during mass spectrometry (MS) analysis to impede chromatographic separation and suppress ion formation, leading to decreased sensitivity and fewer peptide identifications [ 2 – 5 ].…”

“…OCT contains the water-soluble synthetic polymers polyvinyl alcohol (PVA) and polyethylene glycol (PEG) that can compete with peptides during mass spectrometry (MS) analysis to impede chromatographic separation and suppress ion formation, leading to decreased sensitivity and fewer peptide identifications [ 2 – 5 ]. Consequently, several methods to remove OCT have been published that have resulted in an improved number of protein identifications by MS [ 1 – 5 ].…”

Cryo-Gel embedding compound for renal biopsy biobanking

“…Currently the most common medium in which biopsy material is snap frozen is optimal cutting temperature (OCT) compound, a cryopreservative medium composed of polyethylene glycol (PEG), polyvinyl alcohol (PVA) and nonreactive ingredients 12 . OCT stabilizes tissue allowing easy positioning of tissue samples in the microtome.…”