Chemicals.

The ProteoExtract Native Membrane Protein Extraction Kit was procured from Calbiochem/MerckMillipore (Darmstadt, Germany). The protein quantification bicinchoninic acid (BCA) assay kit, sequencing-grade trypsin, iodoacetamide, and dithiothreitol were purchased from Pierce Biotechnology (Rockford, IL). Synthetic heavy peptides for all the surrogate peptides (Supplemental Table 1S) were obtained as internal standards from Thermo Fisher Scientific (Rockford, IL). Synthetic light peptides for the renal transporters OAT1, OAT3, OCT2, and P-glycoprotein (P-gp) were obtained as calibrators from New England Peptides (Boston, MA). Chloroform, ethyl ether, high-performance liquid chromatography (HPLC)-grade acetonitrile/methanol, and formic acid were purchased from Fischer Scientific (Fair Lawn, NJ). Ammonium bicarbonate (98% purity) and sodium deoxycholate (DOC, 98% purity) were obtained from Thermo Fisher Scientific (Rockford, IL) and MP Biomedicals (Santa Ana, CA), respectively.

Sample Procurement, OCT Removal, and Trypsin Digestion.

Histologically normal sections of kidney tissue from healthy donors (n = 5, 3 male, 2 female, 53–67 years old) undergoing clinically indicated partial nephrectomy (e.g., removal of a kidney mass) were obtained after informed consent (Proteogenex, Culver City, CA). Specimens weighed 450–700 mg and were divided into two equal sections, with one section immediately frozen in liquid nitrogen and stored at –80°C. The second half was embedded in the OCT medium using a protocol followed by the University of Washington Department of Pathology; samples were subsequently transferred to –80°C. After 3 months of storage at –80°C, excess OCT around the tissue block was removed with a scalpel without compromising the embedded tissue. Two milliliters of extraction buffer I and 10 μl of protease inhibitor (Calbiochem ProteoExtract Native Protein Extraction Kit) were added to both sets of tissue samples. The samples were homogenized until completely suspended and then incubated on ice for 20 minutes on a rocker table to prevent the formation of cell clumps. The samples were then sonicated for 60 seconds and centrifuged at 16,000g, 4°C, for 15 minutes. The supernatant (the cytosolic fraction) was removed without disturbing the pellet and transferred to a new tube and set aside. One milliliter of extraction buffer II plus 5 μl of protease inhibitor was added to the pellet, which was then resuspended by pipetting up and down ten times or until the pellet was completely broken apart. The sample was incubated on ice for 30 minutes on a rocker table to prevent cell clumps from forming, then centrifuged at 16,000g, 4°C, for 15 minutes. The supernatant (membrane fraction) was removed without disturbing the pellet and transferred completely to a new tube. Total protein was quantified using BCA assay kit (Pierce Biotechnology) and the sample was diluted to 2 mg/ml before analysis.

To remove OCT contamination a previously developed method was modified (Weston and Hummon, 2013). First, 800 μl of diethyl ether and 200 μl of methanol were added to 50 μl of diluted protein sample (100 μg total protein). The sample was vortexed for 5 minutes and then centrifuged at 10,000g for 10 minutes. The upper and lower liquid layers were removed carefully so as not to disturb the interfacial pellet, and air dried. The pellet was resuspended in 80 μl of ammonium bicarbonate, 20 μl of DOC (3%), and 15 μl of 250 mM dithiothreitol and incubated for 5 minutes at 95°C. iodoacetamide (IAA) (18 μl of 500 mM) was added to the samples, and then they were incubated in the dark for 20 minutes. Beyond the previously reported method, a second cleaning step was added to decrease ion suppression in liquid chromatography–tandem mass spectrometry (LC-MS/MS) and to remove any contamination that could affect trypsinization. Five-hundred microliters of methanol, 100 μl of chloroform, and 400 μl of water were added and the samples were vortexed for 1 minute. The samples were centrifuged at 6000g, 4°C, for 5 minutes. The upper and lower liquid layers were removed without disturbing the interfacial layer. The pellet was washed with 1 ml of methanol followed by centrifugation at 6000g for 5 minutes. Then the methanol was completely removed and the sample was air dried. The pellet was completely resuspended in 20 μl of sodium DOC (3%) and 40 μl of 100 mM ammonium bicarbonate. Twenty microliters of 0.16 μg/μl trypsin was added to the sample, which was then incubated for 18 hours at 37°C, with gentle shaking to assist trypsin digestion. The reaction was quenched by adding 30 μl of heavy peptide cocktail in 80% acetonitrile containing 0.1% formic acid. The sample was centrifuged at 3000g for 2 minutes and 100 μl of the supernatant was transferred into LC-MS vials for analysis. The absolute quantification of OAT1, OAT3, OCT2, and P-gp was performed by using light surrogate peptides as calibrators per published protocol (Prasad et al., 2016).

LC-MS/MS Analysis.

LC-MS/MS was performed to quantify 23 structural proteins expressed in different sections of the nephron, the functional unit of the kidney (Table 1). The surrogate peptides of the markers of various cellular compartments of kidney were quantified using triple-quadrupole LC-MS instruments [Xevo TQ-S coupled to ACQUITY UPLC (Waters, Milford, MA)] in positive electrospray ionization mode. Approximately 10 μg of the trypsin digest (5 μl) was injected onto the column (Waters 2.1 μm, C18 100A; 150 × 2.1 mm; Phenomenex, Torrance, CA) and eluted at 0.3 ml/min. A mobile phase consisting of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) was employed. A flow rate of 0.3 ml/min was used in a gradient manner (Supplemental Table 1S). MS/MS analysis was performed by monitoring the surrogate peptides and the internal standards using instrument parameters provided in Supplemental Table 1S.

Data Analysis.

LC-MS/MS data were processed by integrating the peak areas generated from the reconstructed ion chromatograms for the surrogate peptides and the internal standards using MassLynx 4.1 (Waters, Hertfordshire, UK). The peak response for two to three transitions from each peptide was averaged for quantification of samples or standards. Paired Student’s t test was used to compare peptide recovery between flash-frozen and OCT-embedded samples.