Human Kidney Glomerulus Proteome and Proposition of a Method for - - PowerPoint PPT Presentation

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

Collecting tubule

10

Distal straight tubule

9

Distal convoluted tubule

8

Juxtaglomerular appatatus

7

Thick ascending loop of Henle

6

Thin ascending loop of Henle

5

Thin descending loop of Henle

4

Proximal straight tubule

3

Proximal convoluted tubule

2

Glomerulus

1

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 Highly purified glomeruli

Picking-up under a phase-contrast microscopy

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)

MALDI-TOF MS PMF database search

Subject

Normal (no apparent abnormal lesions)

38 male

VpxZu206

4

No glomerular lesions except for slight age- related changes*

50 male

NMXFp768

3

No glomerular lesions except for slight age- related changes*

68 male

apXME367

2

No glomerular lesions except for slight age- related changes*

50 male

OQBfN779

1 Pathologic diagnosis Age Sex Subject

anonymous code

Serial no.

*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 2 1

Valid spot count:984 Valid spot count:1015

3 4

Valid spot count:834 Valid spot count:944

Silver-stained 2-DE Pattern of Human Glomerular Proteins (Subject 1, 50-years-old)

Isoelectric point Molecular weight, kDa

15 20 30 40 50 60 4.0 5.0 6.0 7.0 8.0 9.0

1165 1166 1174 1167 1168 1169 1170 1177 1179 1181 110e 110l 110m 110o 110p 110r 110s 110w 110x 110y 111d 111o 111p 111y 112c 112i 112m 113c 113e 113h 113i 113j 113k 113l 113o 113s 113v 114a 114d 114h 114m 114q 114r 114v 123k 1227 1250 1251 1253 1275 1275 1281 1282 1291 1294 1295 1297 120a 120c 120d 120f 120h 120k 120n 120w 120z 121h 122a 122d 122k 122k 123e 123m 123n 123r 123t 123v 123w 123z 124o 124p 124r 124t 1348 1356 1358 1359 1360 1361 1364 1377 1380 1382 1383 1392 1393 1394 1395 1399 130c 130e 130f 130j 130l 130m 131b 131c 131d 131e 131f 132c 132j 132n 133m 133u 133x 134b 134m 134n 134r 134u 134y 134z 135a 1425 1426 1427 1428 1430 1433 1434 1443 1450 1452 1453 1454 1455 1458 1459 1460 1461 1463 1470 1471 1486 1490 1492 1493 1494 1471 140d 140h 140j 140n 140q 141a 141b 141c 141d 141f 141g 134h 141t141w 142d 142f 1509 1512 15131514 1518 1519 1521 1521 1536 1537 1539 1545 1546 1546 1528 1567 1568 1582 1583 1584 1585 1609 1609 16101612 1619 1102 1109 1111 1119 1120 1121 1124 1125 1126 1127 1128 1130 1139 1142 1145 1176

Human Glomerulus Proteome Database Example 1

NM_003831 BC004223 NM_002305 NM_002305 NM_00260 DNA Reference Access No SUDO LGALS1 LGALS1 MYO7A Gene name ref: NP_003822 13 59.6 5.5 17.5 5.03 sudD homolog QHKG1177 gb: AAH04223 20 41.5 5.7 18.4 4.93 Unknown protein* QHKG1174 ref: NP_002296 49 14.9 5.3 14.6 4.86 Beta galactosidase soluble lectin QHKG1170 ref: NP_002296 52 14.9 5.3 14.9 4.84 Beta galactosidase soluble lectin QHKG1169 ref: NP_000251 13 256.6 9.1 19.3 4.82 Myosin VIIA QHKG1168 gb: AAH12854 14 41.9 5.3 17.5 4.82 Unknown protein* QHKG1167 pir: JC5818 25 41.9 5.3 18.2 4.82 Gamma actin QHKG1166 pir: S43199 27 26.8 8.8 16.4 4,81 Cell division control protein CDC46 QHKG1165 Mw (kDa) pI Mw (kDa) pI Access number Sequence coverage Theoretical Observed Protein Number

Human Glomerulus Proteome Database Example 2

AJ400879 XM_012952 AF380833 D83485 BC009544 XM_050147 J00221 DNA Reference Access No ST5 LOC57761 KIAA0472 IgH Gene name ref: XP_015485 20 42.8 5.9 28.6 4.82 Hypothetical protein FLJ22313 QHKG1253 emb: CAC35387 16 127.7 9.7 25.0 4.79 Suppression of tumorigenecity protein QHKG1251 ref: XP_012952 26 39.6 8.6 22.7 4.78 Hypothetical protein XP_012952 QHKG1250 gb: AAK76362 14 100.6 6.7 13.2 6.36 Endocytosis protein HSTNB* QHKG111d pir: JC5704 38 57.2 6.0 15.3 5.73 Protein disulfide- isomerase QHKG110e gb: AAH09544 38 18.0 5.2 19.5 5.14 Unknown protein QHKG1182 ref: XP_050147 24 26.2 7.1 19.2 5.14 KIAA0472 protein QHKG1181 gb: AAB59395 14 47.7 9.1 13.8 5.08 Immunoglobulin epsilon chain QHKG1179 Mw (kDa) pI Mw (kDa) pI Access number Sequence coverage Theoretical Observed Protein Number

Human Glomerulus Proteome Database Example 3

Y18264 Z81330 XM_030392 XM_030811 NM_003380 X82321 XM_049902 DNA Reference Access No SALL1 RNAH VIM TSA LOC144511 Gene name emb: CAB41399 12 141.9 6.6 31.7 4.73 Zinc finger protein SALL1 QHKG1348 emb: CAB03630 34 40.2 9.3 25.7 6.59

• rfx*

QHKG121h ref: XP_030392 11 214.4 7.2 29.5 6.40 Hypothetical protein XP_030392* QHKG120w ref: XP_030811 30 26.5 7.9 29.1 6.31 Hypothetical protein XP_030811* QHKG120r ref: NP_003371 28 53.7 5.1 29.7 5.80 Vimentin QHKG1291 emb: CAA57764 24 18.5 5.2 25.4 5.44 Thiol specific antioxidant QHKG1282 gb: AAA51580 23 26.2 5.6 29.8 5.34 Gamma actin (frangment) QHKG1278 ref: XP_049904 32 66.2 8.3 23.5 5.30 Keratin 1 QHKG1275 Mw (kDa) pI Mw (kDa) pI Access number Sequence coverage Theoretical Observed Protein Number

Human Glomerulus Proteome Database Example 4

NM_001604 NM_003380 NM_001945 NM_001686 XM_017779 U44388 BC015620 DNA Reference Access No ACYG1 DDR1 ATP5B KIAA0336 Gene name ref: NP_001614 27 42.1 5.3 47.0 5.16 Gamma Actin QHKG1423 ref: NP_003380 34 53.7 5.1 46.4 4.99 Vimentin QHKG1429 ref: NP_001945 12 98.0 6.0 46.2 4.91 Discoidin receptor tyrosine kinase isoform b QHKG1427 sp: P06576 34 56.5 5.3 49.3 4.84 ATP synthase beta chain QHKG1426 ref: XP_017779 18 185.6 5.1 46.2 4.79 KIAA0336 gene product QHKG1425 gb: AAB81552 39 23.6 9.6 30.4 5.15 Ro alternative protein 60e2* QHKG1361 gb: AAH15620 24 74.2 5.2 37.5 5.10 Unknown protein* QHKG1360 pir: JC5818 45 41.9 5.3 30.4 5.07 Gamma actin QHKG1359 Mw (kDa) pI Mw (kDa) pI Access number Sequence coverage Theoretical Observed Protein Number