Introduction

References

Methods Results – Normal Cornea Results - Keratoconus Results – Corneal Edema

Results

Keratoconus is a non-inflammatory, bilateral, progressive, often asymmetric, primary ectasia, associated with irregular astigmatism and decreased visual acuity. It results in progressive dissolution of Bowman’s membrane, the layer lying between the corneal epithelium and stroma. Cellular and structural changes in the cornea affect the integrity and lead to protrusion and scarring. Reported biochemical abnormalities in corneas of patients with keratoconus have included decreased protease inhibitors, increased keratocyte catalase activity, decreased alcohol dehydrogenase, decreased matrix metalloproteinase 8 (MMP-8, collagenase), increased MMP-1, increased reactive oxygen/nitrogen species, higher hydrogen peroxide levels, and increased cathepsin K activity. Other abnormalities include absence of nerve growth factor receptor and absence of aquaporin 5. Since proteins are key players which execute nearly all downstream processes, proteomic analysis provides functional assessment of cellular processes. A recent report characterized the proteome of the normal human cornea and identified 141 distinct proteins; 86 of them were recognized in cDNA libraries from the corneas of patients with keratoconus. Furthermore, the bioinformatic analyses indicated the import of plasma proteins into the cornea. It is hypothesized that dysregulation of proteins in the corneas of patients with Keratoconus causes altered proteomic profile when compared to normal corneas.

Normal and Keratoconus corneas

Under an IRB approved protocol surgically discarded and de-identified normal donor and keratoconic corneas were obtained. The endothelium-Descemet’s membrane was removed from the cornea. The endothelium-free cornea was soaked in prewarmed 20mM EDTA in PBS solution for 30 minutes at 37º C. Forceps were used to separate the epithelial layer from the stroma. The samples were kept frozen until they are homogenized. The samples were homogenized using celLysis buffer (Sigma) and ultrasonicated for 30 seconds on ice. The homogenates were centrifuged at 10,000 rpm for 10 minutes and the supernatant was aliquoted and frozen at -70ºC. Protein quantification of the samples was done by modified Lowry’s technique. The protein content of the samples was adjusted to 1mg/ml concentration. Corneal homogenates in the amount of 40µg were used to perform proteomic profiling by using Gold (Au) ProteinChips. Profiles were analyzed for protein peaks according to molecular weight using Ciphergen ProteinChip software.

.

ConclusionsGold (Au) ProteinChip array using SELDI-TOF-MS is a valuable technique to perform proteomic profiling of normal and keratoconic epithelial and stromal layers. The altered proteomic profile shows absence of peaks in the 20-150 Kd range in the keratoconic epithelium. However, there are distinct peaks in Keratoconus at 10.8, 12.7, 13.1, 14.6, 15.8 kDa which are absent in normals. Further studies are warranted to validate these results. The Normal corneal stroma contain peaks at 9.9, 14.5 in the <20kDa range; as well as 22.8, 43.7, 67.4, 97.2 and 1.12E5 in the >20kDa which are absent in Keratoconus.

1. US National Eye Institute, Facts about the Cornea and Corneal Disease Keratoconus. (http://www.nei.nih.gov/health/cornealdisease/index.asp#h). Accessed Feb 12 2006.

2. Ashwin PT, McDonnell. Collagen cross-linkage: a comprehensive review and directions for future research. Br J Ophthalmol 94:965-970, 2010.

3. Karring H, Thogersen IB, Klintworth GK, et al. The human cornea proteome: Bioinformatic analyses indicate import of plasma proteins into the cornea. Molecular Vision 12:451-460, 2006.

4. Attilano SR et al. Accumulation of mitochondrial DNA damage in keratoconus corneas. Invest Ophthalmol Vis Sci 46:1256-63, 2005.

Acknowledgements: The Richard A. Perritt Charitable Foundation

The normal corneal epithelium displayed a distinct proteomic profile in the <25Kd molecular weight range. Mass spectrometry with the Gold (Au) chip shows prominent peaks at 5.0, 7.0, 7.7, 8.5, 10.1, 11.6, 12.3, 15 and 17.9 Kd. The stromal layer showed prominent peaks at 8.5, 9.4, 9.8 and 11.0 Kd. However, the peaks in the 20-150 Kd range which were present in the normals were not observed in keratoconic epithelial layer. The epithelial layer from corneal edematous cornea showed similar peaks to that of normals at >20Kd. However, unique peaks were observed at 10.1, 10.7 and 11.6 Kd

SELDI-TOF-MS & Protein Chip

THE PROTEOMIC PROFILE OF NORMAL AND KERATOCONIC EPITHELIAL AND STROMAL LAYERS BY PROTEINCHIP® USING SELDI-TOF-MS

Omer Iqbal2, MD, Daniel Kahn2, BS, Shawn Aranha2, MD, Samir Vira1, MD, George Fisher3, MS4, Bruce Gaynes1, OD, PharmD,Jawed Fareed2, PhD, Charles Bouchard1, MD

Departments of Ophthalmology1 and Pathology2, Loyola University Chicago, Stritch School of Medicine3, Maywood, IL

5,053

5,824

7,089

7,770

8,561

9,158

10,103

11,191

11,654

12,360

13,920

15,083

15,91516,922

17,232

17,905

18,434

5,000 6,000 7,000 8,000 9,000 10,000 11,000 12,000 13,000 14,000 15,000 16,000 17,000 18,000 19,000

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

20,964

23,272

25,208

26,123

27,849

30,805

34,069

36,037

36,651

38,69141,830

47,286

48,605

50,516

51,856

55,117

58,08363,221

68,304

69,760

80,993 94,90098,1471.02E51.06E5 1.17E5

20,000 30,000 40,000 50,000 60,000 70,000 80,000 90,000 1E5 1.1E5 1.2E5 1.3E5 1.4E5

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

5

5.2

5.4

5.6

5.8

Normal - Epithelium MW range: 5-20 kDa;

Intensity: 30

MW range: 20-150 kDa; Intensity: 6

5,4295,829

7,4408,578

10,106

10,362

10,849

11,198

11,658

12,36112,705

13,169

14,65114,991

15,814 16,820 17,91218,442

5,000 6,000 7,000 8,000 9,000 10,000 11,000 12,000 13,000 14,000 15,000 16,000 17,000 18,000 19,000

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

20,24320,954

22,752

27,879

36,049

38,673

41,900

47,290

50,230

54,80557,136 63,34468,433

20,000 30,000 40,000 50,000 60,000 70,000 80,000 90,000 1E5 1.1E5 1.2E5 1.3E5 1.4E5

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

5

5.2

5.4

5.6

5.8

Keratoconus - Epithelium MW range: 5-20 kDa;

Intensity: 30

MW range: 20-150 kDa; Intensity: 6

10,108 11,08911,651

5,000 6,000 7,000 8,000 9,000 10,000 11,000 12,000 13,000 14,000 15,000 16,000 17,000 18,000 19,000

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

20,000 30,000 40,000 50,000 60,000 70,000 80,000 90,000 1E5 1.1E5 1.2E5 1.3E5 1.4E5

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

5

5.2

5.4

5.6

5.8

Corneal Edema - Epithelium MW range: 5-20 kDa;

Intensity: 30

MW range: 20-150 kDa; Intensity: 6

5,676

6,808

8,568

9,193

9,951

11,076

14,590

5,000 6,000 7,000 8,000 9,000 10,000 11,000 12,000 13,000 14,000 15,000 16,000 17,000 18,000 19,000

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

22,820

43,726

67,440

97,222

1.12E5

20,000 30,000 40,000 50,000 60,000 70,000 80,000 90,000 1E5 1.1E5 1.2E5 1.3E5 1.4E5

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

Normal - StromaMW range: 5-20 kDa; Intensity: 5

MW range: 20-150 kDa; Intensity: 5

8,544

5,000 6,000 7,000 8,000 9,000 10,000 11,000 12,000 13,000 14,000 15,000 16,000 17,000 18,000 19,000

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

20,000 30,000 40,000 50,000 60,000 70,000 80,000 90,000 1E5 1.1E5 1.2E5 1.3E5 1.4E5

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

Keratconus - Stroma MW range: 5-20 kDa;

Intensity: 5

MW range: 20-150 kDa; Intensity: 5

5,000 6,000 7,000 8,000 9,000 10,000 11,000 12,000 13,000 14,000 15,000 16,000 17,000 18,000 19,000

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

57,107

20,000 30,000 40,000 50,000 60,000 70,000 80,000 90,000 1E5 1.1E5 1.2E5 1.3E5 1.4E5

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

Corneal Edema - Stroma MW range: 5-20 kDa;

Intensity: 5

MW range: 20-150 kDa; Intensity: 5