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The Details of Human Glomerulus Protein Database
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db1Source Infomation
Glomerulus was highly purified from renal cortex with no pathologic manifestations obtained from a kidney removed by nephrectomy because of ureter tumor. glo
A phase-constrast micrograph of highly purified glomeruli.

db1Identification Strategy
Proteins extracted from highly purified glomeruli were cysteine-alkylated. A part of protein (20g) 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. idn

db1Acquisition 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.

db1Protein Identification and Criteria for Confidence of Identification
Protein database for searchIPI Human, release 3.18
Search engineSpectrum 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 cleavage2
ModificationCarbamidomethylation(cys)
ValidationAuto+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 PrefractionationNumber of Identified Proteins
High
Confidence *1
Lower
Confidence *2
Total Number of
Iidentified Proteins *3
1D Prefractionation *426307973427
2D Prefractionation *5
Fr.1(pH 3-4.6)99210882080
Fr.2(pH 4.6-5.4)142211552577
Fr.3(pH 5.4-6.2)114011442284
Fr.4(pH 6.2-7.0)172112893010
Fr.5(pH 7.0-10.0)130911772486
Total Number365326846337
Total Number of
Distinct Proteins *6
367930076686
Total Number of
Distinct Genes *7
159213742966
 
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
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