The Details of Our DB
Source Infomation
Glomerulus was highly purified from renal cortex with no pathologic manifestations obtained from a kidney removed by nephrectomy because of ureter tumor.
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 Da, Product mass tolerance; ±0.7 Da, Max missed cleavage; 2, Modification; Carbamidomethylation (cys)
- Validation:
- Auto+Manual validation by Reverse-Forward Database search (Rank 1-2): Table I.
- False-positive rate: 1.8% by searching a random database created with IPI Human 3.
Table 1. Summary of Protein Identifications by 1D and 2D Protein Prefractionation Strategy
Protein Prefractionation |
Number of Identified Proteins |
||
High Confidence1 |
Lower Confidence2 |
Total Number of Iidentified Proteins3 |
|
1D Prefractionation4 |
2630 |
797 |
3427 |
2D Prefractionation5 |
|||
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 Proteins6 |
3679 |
3007 |
6686 |
Total Number of Distinct Genes7 |
1592 |
1374 |
2966 |
- The number of proteins identified with one peptide match
- The number of proteins identified with one or more peptide matches
- Glomerular proteins separated by 1D-SDS-PAGE
- Glomerular proteins separated by 2D prefractionation (solution phase IEF and SDS-PAGE)
- The number of distinct proteins identified by 1D and 2D prefractionation strategies
- The number of distinct genes identified by 1D and 2D prefractionation