Human Kidney Glomerulus Proteome and Proposition of a Method for Native Protein Profiling A.Tsugita, K. Miyazaki, Y. Yoshida and T. Yamamoto NEC Proteome Research Center And Niigata Univ. Medical Faculty, Japan
Schematic Structure of Nephron 1 Glomerulus 2 Proximal convoluted tubule 3 Proximal straight tubule 4 Thin descending loop of Henle 5 Thin ascending loop of Henle 6 Thick ascending loop of Henle 7 Juxtaglomerular appatatus 8 Distal convoluted tubule 9 Distal straight tubule 10 Collecting tubule
Preparation of 2-DE Samples Kidney cortex with normal appearance from patients under nephrectomy due to renal tumor A standard sieving method Partially purified glomeruli Picking-up under a phase-contrast microscopy Highly purified glomeruli Solubilized with lysis solution Lysis solution : 9.8 M urea, 2 % NP-40, 2 % Pharmalyte, pH 3-10, 100 mM DTT, 0.5 µg/ml E-64, 0.5 mM PMSF, 40 µg/ml TLCK, 1 µg/ml aprotinin, 10 µg/ml cymostatin, 0.5 mM EDTA 2-DE sample
Protein identification Silver stain without glutaraldehyde Pick out spots In gel digestion (Trypsin) Voyager Spec #1[BP = 842.5, 9265] 842.4997 9.3E+3 100 90 80 70 MALDI-TOF MS 60 % Intensity 50 40 2212.0469 30 1182.5798 1752.8214 882.5471 1051.6854 20 1377.6101 1104.5689 1605.7765 850.5129 1331.6562 1028.5579 1566.7133 894.5313 10 2284.1260 1089.5465 1248.5789 839.0846 1448.8851 1734.8168 1894.8798 1073.5256 1283.6578 1473.7080 2234.0448 2808.2439 1645.7710 1809.8538 1991.0203 2535.2396 0 0 799.0 1339.4 1879.8 2420.2 2960.6 3501.0 Mass (m/z) PMF database search
Subject Subject Serial no. Sex Age Pathologic diagnosis anonymous code No glomerular lesions 1 male 50 except for slight age- OQBfN779 related changes* No glomerular lesions 2 male 68 except for slight age- apXME367 related changes* No glomerular lesions 3 male 50 except for slight age- NMXFp768 related changes* Normal (no apparent 4 male 38 abnormal lesions) VpxZu206 * Slight age-related changes : a slight widening of mesangium; a small number of collapsed glomeruli; focal interstitial infiltration of lymphocytes; tubular atrophy ; hyaline degeneration
2-DE gel images of highly purified glomeluli from 4 subjects 1 2 Valid spot count:984 Valid spot count:1015 3 4 Valid spot count:944 Valid spot count:834
Silver-stained 2-DE Pattern of Human Glomerular Proteins (Subject 1, 50-years-old) Isoelectric point 4.0 5.0 6.0 7.0 8.0 9.0 60 16101612 1609 1567 1619 1546 1521 Molecular weight, kDa 1546 1568 1584 15131514 1537 1609 1521 1583 1539 1536 1585 1582 1518 1512 1545 1528 1471 1509 1471 141c 1452 50 1519 1450 1454 1470 1426 1433 1490 140d 1494 141f 1427 1460 1434 1459 140q 1425 140h 1428 1453 142f 1463 1492 141d 1443 1455 141t141w 1430 141g 140j 141a 1458 1486 40 1461 134h 1394 142d 131e 1493 130e 140n 141b 134n 134u 1393 131f 130l 132n 1358 132c 1382 134r 130j 133x 1360 131b 134z 1364 1399 133u 132j 1392 134y 130m 1380 134m 1395 130c 131d 134b 30 1383 130f 1348 133m 1359 131c 135a 1361 1294 1356 120d 124p 123n 120a 1377 120w 1275 123r 1291 120f 123k 1253 120c 1295 123w 123z 1297 1281 123m 120h 122k 121h 120k 1251 122a 122d 1282 123t 120n 120z 122k 123v 1275 124o 123e 1227 1250 20 124r 1145 124t 110r 113j 1168 1121 1130 1181 114v 1174 1126 113e 1166 110l 113v 1177 110y 1102 1167 1125 1176 1119 111o 114d 1142 1165 110s 111y 111p 114m 1120 113s 15 112m 113h 1169 110x 114r 1124 113c 110e 1128 114h 1170 110p 113k 1109 1127 112i 114q 1179 112c 110m 113i 1111 114a 110o 113l 111d 1139 113o 110w
Human Glomerulus Proteome Database Example 1 Observed Theoretical DNA Sequence Access Gene Number Protein Reference coverage number name Mw Mw Access No pI pI (kDa) (kDa) QHKG1165 Cell division control 4,81 16.4 8.8 26.8 27 pir: S43199 protein CDC46 QHKG1166 Gamma actin 4.82 18.2 5.3 41.9 25 pir: JC5818 QHKG1167 Unknown protein* 4.82 17.5 5.3 41.9 14 gb: AAH12854 QHKG1168 Myosin VIIA 4.82 19.3 9.1 256.6 13 ref: NP_000251 MYO7A NM_00260 QHKG1169 Beta galactosidase 4.84 14.9 5.3 14.9 52 ref: NP_002296 LGALS1 NM_002305 soluble lectin QHKG1170 Beta galactosidase 4.86 14.6 5.3 14.9 49 ref: NP_002296 LGALS1 NM_002305 soluble lectin QHKG1174 Unknown protein* 4.93 18.4 5.7 41.5 20 gb: AAH04223 BC004223 QHKG1177 sudD homolog 5.03 17.5 5.5 59.6 13 ref: NP_003822 SUDO NM_003831
Human Glomerulus Proteome Database Example 2 Observed Theoretical DNA Sequence Access Gene Number Protein Reference coverage number name Mw Mw Access No pI pI (kDa) (kDa) QHKG1179 Immunoglobulin 5.08 13.8 9.1 47.7 14 gb: AAB59395 IgH J00221 epsilon chain QHKG1181 KIAA0472 protein 5.14 19.2 7.1 26.2 24 ref: XP_050147 KIAA0472 XM_050147 QHKG1182 Unknown protein 5.14 19.5 5.2 18.0 38 gb: AAH09544 BC009544 QHKG110e Protein disulfide- 5.73 15.3 6.0 57.2 38 pir: JC5704 D83485 isomerase QHKG111d Endocytosis protein 6.36 13.2 6.7 100.6 14 gb: AAK76362 AF380833 HSTNB* QHKG1250 Hypothetical protein 4.78 22.7 8.6 39.6 26 ref: XP_012952 LOC57761 XM_012952 XP_012952 QHKG1251 Suppression of 4.79 25.0 9.7 127.7 16 emb: CAC35387 ST5 AJ400879 tumorigenecity protein QHKG1253 Hypothetical protein 4.82 28.6 5.9 42.8 20 ref: XP_015485 FLJ22313
Human Glomerulus Proteome Database Example 3 Observed Theoretical DNA Sequence Access Gene Number Protein Reference coverage number name Mw Mw Access No pI pI (kDa) (kDa) QHKG1275 Keratin 1 5.30 23.5 8.3 66.2 32 ref: XP_049904 LOC144511 XM_049902 QHKG1278 Gamma actin 5.34 29.8 5.6 26.2 23 gb: AAA51580 (frangment) QHKG1282 Thiol specific 5.44 25.4 5.2 18.5 24 emb: CAA57764 TSA X82321 antioxidant QHKG1291 Vimentin 5.80 29.7 5.1 53.7 28 ref: NP_003371 VIM NM_003380 QHKG120r Hypothetical 6.31 29.1 7.9 26.5 30 ref: XP_030811 XM_030811 protein XP_030811* QHKG120w Hypothetical 6.40 29.5 7.2 214.4 11 ref: XP_030392 RNAH XM_030392 protein XP_030392* QHKG121h orfx* 6.59 25.7 9.3 40.2 34 emb: CAB03630 Z81330 QHKG1348 Zinc finger protein 4.73 31.7 6.6 141.9 12 emb: CAB41399 SALL1 Y18264 SALL1
Human Glomerulus Proteome Database Example 4 Observed Theoretical DNA Sequence Access Gene Number Protein Reference coverage number name Mw Mw Access No pI pI (kDa) (kDa) QHKG1359 Gamma actin 5.07 30.4 5.3 41.9 45 pir: JC5818 QHKG1360 Unknown protein* 5.10 37.5 5.2 74.2 24 gb: AAH15620 BC015620 QHKG1361 Ro alternative 5.15 30.4 9.6 23.6 39 gb: AAB81552 U44388 protein 60e2* QHKG1425 KIAA0336 gene 4.79 46.2 5.1 185.6 18 ref: XP_017779 KIAA0336 XM_017779 product QHKG1426 ATP synthase beta 4.84 49.3 5.3 56.5 34 sp: P06576 ATP5B NM_001686 chain QHKG1427 Discoidin receptor 4.91 46.2 6.0 98.0 12 ref: NP_001945 DDR1 NM_001945 tyrosine kinase isoform b QHKG1429 Vimentin 4.99 46.4 5.1 53.7 34 ref: NP_003380 NM_003380 QHKG1423 Gamma Actin 5.16 47.0 5.3 42.1 27 ref: NP_001614 ACYG1 NM_001604
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