Comprehensive analysis of the mouse renal cortex using two-dimensional HPLC - tandem mass spectrometry.
BACKGROUND
Proteomic methodologies increasingly have been applied to the kidney to map the renal cortical proteome and to identify global changes in renal proteins induced by diseases such as diabetes. While progress has been made in establishing a renal cortical proteome using 1-D or 2-DE and mass spectrometry, the number of proteins definitively identified by mass spectrometry has remained surprisingly small. Low coverage of the renal cortical proteome as well as our interest in diabetes-induced changes in proteins found in the renal cortex prompted us to perform an in-depth proteomic analysis of mouse renal cortical tissue.


RESULTS
We report a large scale analysis of mouse renal cortical proteome using SCX prefractionation strategy combined with HPLC - tandem mass spectrometry. High-confidence identification of ~2,000 proteins, including cytoplasmic, nuclear, plasma membrane, extracellular and unknown/unclassified proteins, was obtained by separating tryptic peptides of renal cortical proteins into 60 fractions by SCX prior to LC-MS/MS. The identified proteins represented the renal cortical proteome with no discernible bias due to protein physicochemical properties, subcellular distribution, biological processes, or molecular function. The highest ranked molecular functions were characteristic of tubular epithelium, and included binding, catalytic activity, transporter activity, structural molecule activity, and carrier activity. Comparison of this renal cortical proteome with published human urinary proteomes demonstrated enrichment of renal extracellular, plasma membrane, and lysosomal proteins in the urine, with a lack of intracellular proteins. Comparison of the most abundant proteins based on normalized spectral abundance factor (NSAF) in this dataset versus a published glomerular proteome indicated enrichment of mitochondrial proteins in the former and cytoskeletal proteins in the latter.


CONCLUSIONS
A whole tissue extract of the mouse kidney cortex was analyzed by an unbiased proteomic approach, yielding a dataset of ~2,000 unique proteins identified with strict criteria to ensure a high level of confidence in protein identification. As a result of extracting all proteins from the renal cortex, we identified an exceptionally wide range of renal proteins in terms of pI, MW, hydrophobicity, abundance, and subcellular location. Many of these proteins, such as low-abundance proteins, membrane proteins and proteins with extreme values in pI or MW are traditionally under-represented in 2-DE-based proteomic analysis.
Zhao
Yingxin
Y
Department of Internal Medicine, The University of Texas Medical Branch, Galveston, USA. yizhao@utmb.edu
Denner
Larry
L
Haidacher
Sigmund J
SJ
LeJeune
Wanda S
WS
Tilton
Ronald G
RG