In-Depth Proteomic Profiling of the Normal Human Kidney Glomerulus Using Two-Dimensional Protein Prefractionation in Combination with Liquid Chromatography-Tandem Mass Spectrometry

Abstract

The kidney glomerulus plays a pivotal role in ultrafiltration of plasma into urine and also is the locus of kidney disease progressing to chronic renal failure. We have focused proteomic analysis on the glomerulus that is most proximal to the disease locus. In the present study, we aimed to provide a confident, in-depth profiling of the glomerulus proteome. The glomeruli were highly purified from the kidney cortex from a male, 68-year-old patient who underwent nephroureterectomy due to ureter carcinoma. The patient was normal in clinical examinations including serum creatinine and urea levels and liver function, and did not receive any chemotherapy and radiotherapy. The cortical tissue was histologically normal, and no significant deposition of immunoglobulins and complement C3 was observed. We employed a novel strategy of protein separation using 1D (SDS-PAGE) and 2D (solution-phase IEF in combination with SDS-PAGE) prefractionation prior to the shotgun analysis with LC-MS/MS. The protein prefractionation produced 90 fractions, and eventually provided a confident set of identified proteins consisting of 6686 unique proteins (3679 proteins with two or more peptide matches and 3007 proteins with one peptide match), representing 2966 distinct genes. All the identified proteins were annotated and classified in terms of molecular function and biological process, compiled into 1D and 2D protein arrays, consisting of 15 and 75 sections, corresponding to the protein fractions which were defined by MW and pI range, and deposited on a Web-based database (http://www.hkupp.org). The most remarkable feature of the glomerulus proteome was a high incidence of identification of cytoskeleton-related proteins, presumably reflecting the well-developed, cytoskeletal organization of glomerular cells related to their physiological functions.

Global Analysis Reveals the Complexity of the Human Glomerular Extracellular Matrix

“…Only proteins identified in at least two of three biologic replicate analyses were included in the dataset, and identified ECM proteins were further categorized as basement membrane proteins (Table 1), other structural ECM proteins (Table 2), or ECM-associated proteins (Supplemental Table 2). There are a number of published glomerular proteomic studies, [21][22][23][24][25] and a comparison was made with protein identifications in our dataset ( Figure 3). Of two published studies for which complete MS datasets were available, 22,25 neither study enriched for glomerular ECM or analyzed more than one biologic replicate.…”

Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine

“…17, 18 We performed a comprehensive proteomic analysis of this subfraction enriched in GMVs (apical membrane vesicles) and then assessed the overlap of our GMV proteome with the published glomerular tissue and urine exosome proteomes. 19 We studied the post-translational processing of a number of known glomerular disease proteins for the first time in vivo and provide these data in a searchable database.…”

Quantification of Norepinephrine and Its Metabolites in the Plasma of Renal Failure Models

“…Control of the P1 and P2 promoters would influence the expression of MB- and S-COMT isozymes. In fact, proteome analyses of kidney indicated that COMT is expressed in the glomerulus [23,24]. Oxidative stress was shown to inhibit the activity of S-COMT [25].…”