The Details of Human Glomerulus Protein Database

Source Infomation

Glomerulus was highly purified from renal cortex with no pathologic manifestations obtained from a kidney removed by nephrectomy because of ureter tumor.

A phase-constrast micrograph of highly purified glomeruli.

Identification Strategy

Proteins extracted from highly purified glomeruli were cysteine-alkylated. A part of protein (20μg) extract was directly separated by SDS-PAGE (1D prefractionation) and the rest (2 mg) was separated by solution phase IEF into 5 fractions, and each fraction was separated by SDS-PAGE (2D prefractionation). Each lane on SDS-PAGE were sliced15 sections and proteins were in-gel digested with trypsin, and analyzed by nanoflow-LC- iontrap MS/MS. The protein identifications were conducted by searching IPI human protein database by using Spectrum Mill as a search engine.

Acquisition of MS Data

Mass spectra data were obtained by nano-LC-IT-MS/MS (1100 LC/ MSD Trap XCTUltra, Agilent Technologies) with a HPLC nanospray Chip. Each sample was run twice for 70 minutes each with 2 blank runs between the sample runs.

Protein Identification and Criteria for Confidence of Identification

Protein database for search IPI Human, release 3.18 Search engine Spectrum Mill (ver.A.03.12.060) with the following parameters Precursor mass tolerance +2.5 to -2.5 Da Product mass tolerance +0.7 to -0.7 Da Max missed cleavage 2 Modification Carbamidomethylation(cys) Validation Auto+Manual validation by Reverse-Forward Database search (Rank 1-2) *Table1 False-positive rate: 1.8% by searching a random database created with IPI Human 3. *Table1. Summary of Protein Identifications by 1D and 2D Protein Prefractionation Strategy Protein Prefractionation Number of Identified Proteins High Confidence *1 Lower Confidence *2 Total Number of Iidentified Proteins *3 1D Prefractionation *4 2630 797 3427 2D Prefractionation *5 Fr.1(pH 3-4.6) 992 1088 2080 Fr.2(pH 4.6-5.4) 1422 1155 2577 Fr.3(pH 5.4-6.2) 1140 1144 2284 Fr.4(pH 6.2-7.0) 1721 1289 3010 Fr.5(pH 7.0-10.0) 1309 1177 2486 Total Number 3653 2684 6337 Total Number of Distinct Proteins *6 3679 3007 6686 Total Number of Distinct Genes *7 1592 1374 2966   The number of proteins identified with two or more peptide matches  *1. The number of proteins identified with one peptide match  *2. The number of proteins identified with one or more peptide matches  *3. Glomerular proteins separated by 1D-SDS-PAGE  *4. Glomerular proteins separated by 2D prefractionation(solution phase IEF and SDS-PAGE)  *5. The number of distinct proteins identified by 1D and 2D prefractionation strategies  *6. The number of distinct genes identified by 1D and 2D prefractionation

Back to previous page.