1. The renal nephron is composed of many cell types. Studying a specific cell response to a physiological stress such as metabolic acidosis can be difficult due to the effects that tissue heterogeneity impart on the assayed samples. The aim of this study is to characterize the response to metabolic acidosis that occurs specifically within the apical membrane of the proximal convoluted tubule. P1: Mitochondria, whole cells, basolateral membrane vesicles, etc.. S1 S4 BBMV P3 P2 S2: Cytosol S3 Mg 2+, Soft Spin Mg 2+, Soft Spin Hard Spin Hard Spin Homogenized PCT or Cortex Cortex Cortex PCT BBMV BBMV Peptides Peptides LC/MS analysis, spectral identification, etc….. To accomplish this analysis, two methods to prepare apical membranes were compared. Using Mg 2+ /EGTA precipitation (Biber, et al. 2007), brush border membranes were isolated from kidney cortex (BBMV CTX ) or Percoll gradient purified proximal convoluted tubules (BBMV PCT ) (n=2). Mg 2+ /EGTA Precipitation Study Design Introduction The samples were assayed for activity of  glutamyl transpeptidase (  GT). The results indicated a decrease in the specific activity of  GT for the BBMV PCT when compared to the BBMV CTX samples consistent with its greater abundance in the proximal straight tubule * . Western Blots indicated that contaminating apical membranes were co-purified with the BBMV CTX sample when using the standard precipitation procedure. Further analysis revealed a reduction in these contaminants when precipitating the BBMV from isolated proximal tubules. However, Western blots do indicate that the precipitation technique preferentially enriches for the apical membrane. Biochemical Analysis  GT Specific Activity Relative to Cortex Sample Assayed (n=3 / group) U  GT / mg protein (umol  NA min -1 mg -1 ) BBMV PCT BBMV CTX Reducing Tissue Heterogeneity for the Specific Proteomic Analysis of Proximal Convoluted Tubule Apical Membranes “ Beat CU” Scott Walmsley* 1 , Corey Broeckling 2 , Jessica Prenni 2 , Norman Curthoys 1 1 Cell and Molecular Biology Interdisciplinary Program, Department of Biochemistry and Molecular Biology, 2 Proteomics and Metabolomics Facility Colorado State University, Fort Collins, CO * A  GT assay of (BBMV PCT ) from acidotic rats indicated a significant increase in specific activity validating our suggestion that heterogeneity of the sample assayed influences the result. Taken together, the data indicate that reducing tissue heterogeneity will improve the identification of proteins present in the apical membrane of the PCT and will aid in the proteomic analysis of the response to chronic metabolic acidosis by proximal convoluted tubule cells. The improved method will allow for the characterization of the Proximal Convoluted Tubule response to chronic acidosis. Proteomic Analysis and Spectral Counting Proteomic analysis revealed proteins unique to the BBMV PCT sample group. Each sample was solubilized, reduced and alkylated, then treated with trypsin prior to SCX fractionation. The fractionated peptides were then multiply injected (n=3) for 21 total MS injections per sample. Analysis of the normalized spectral counts revealed several proteins enriched in the BBMV PCT . The heat maps at left are representative of the change of spectral counts per protein across biological samples. The proteins identified in the BBMV PCT samples included those involved in villus structure, protein trafficking, solute transport, signal transduction, and enzymes of glucose metabolism. Conclusion The G test compared spectral counts (SC) between samples. G test scores that pass the cutoff from a contingency table are considered significant (colored points). Sample Assayed (n=3 / group) Acidosis versus control Fold Increase  GT Specific Activity relative to CTX Control BBMV CTX BBMV PCT G Test: BBMV CTX vs BBMV CTX Average SC SC G Test BBMV CTX vs BBMV PCT SC Relative SC of Selected Markers Comparing Multiple MS/MS Injections BBMV CTX and BBMV PCT compared (n=6) NHE3 (PT) NKCC2 (TAL) TSC (DT) AQP2 (CD) Cortex BBMV CTX PCT BBMV PCT