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Proteomic analysis of the tear film in patients with keratoconus.

Lema I, Brea D, Rodríguez-González R, Díez-Feijoo E, Sobrino T - Mol. Vis. (2010)

Bottom Line: Finally, results were validated by western-blot.IGKC protein, ZAG, and lactoferrin are under-expressed in the tears of patients diagnosed with bilateral KC compared with healthy subjects.These differences could contribute to the knowledge of the pathophysiology of this disease.

View Article: PubMed Central - PubMed

Affiliation: Instituto Galego de Oftalmoloxía, Servizo Galego de Saúde, University of Santiago de Compostela, Santiago de Compostela, Spain. mariaisabel.lema@usc.es

ABSTRACT

Purpose: To identify proteins differentially expressed between the tear film of keratoconus (KC) patients and control subjects using two dimensional electrophoresis (2-DE) and mass spectrometry-based techniques.

Methods: Twenty two patients (44 eyes) diagnosed with bilateral KC and 22 control subjects (44 eyes) were studied in a prospective case-control study. Keratoconus screening programs and Orbscan II topographies were performed on all participants. Tear samples were collected by the Schirmer I method using filter paper. Proteins were extracted from the Schirmer strips and separated by 2-DE. Comparison of protein patterns was performed using PDQuest Software and protein differences were identified by mass spectrometry. Finally, results were validated by western-blot.

Results: Four spots were identified to be differentially expressed between KC patients and control subjects. Three of them were more expressed in healthy subjects and they were identified as zinc-α2-glycoprotein (ZAG), lactoferrin, and IGKC (immunoglobulin kappa chain). The other spot was more expressed in KC patients and it was identified as ZAG. Differences in ZAG seem controversial in two different spots because different posttranslational modifications, however, analysis of both spots revealed that globally, ZAG is overexpressed in healthy subjects. Founded differences in ZAG, lactoferrin, and IGKC expression were subsequently validated by western blot.

Conclusions: IGKC protein, ZAG, and lactoferrin are under-expressed in the tears of patients diagnosed with bilateral KC compared with healthy subjects. These differences could contribute to the knowledge of the pathophysiology of this disease.

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Related in: MedlinePlus

A typical profile of molecular mass of tryptic fragments of zinc- α2-glycoprotein (ZAG) during analysis by MALDI-TOF mass spectrometry.
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f3: A typical profile of molecular mass of tryptic fragments of zinc- α2-glycoprotein (ZAG) during analysis by MALDI-TOF mass spectrometry.

Mentions: An average of 532 spots was observed in both groups (Figure 1). Gels were highly reproducible (r2=0.95) between groups. No qualitative differences have been found, however three spots were found to be expressed at least twofold in healthy subjects than in KC patients and one spot was found twofold in KC patients than in healthy subjects (Figure 2). The expression levels are shown in Table 2. Furthermore these differences were statistically significant (p<0.001). Mass spectrometry analysis revealed that quantitative differences more expressed in healthy subjects were; IGKC (Immunoglobulin Kappa Chain) protein, zinc-α2-glycoprotein (ZAG; Figure 3), and lactoferrin and quantitative difference more expressed in KC patients was identified as ZAG.


Proteomic analysis of the tear film in patients with keratoconus.

Lema I, Brea D, Rodríguez-González R, Díez-Feijoo E, Sobrino T - Mol. Vis. (2010)

A typical profile of molecular mass of tryptic fragments of zinc- α2-glycoprotein (ZAG) during analysis by MALDI-TOF mass spectrometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2965569&req=5

f3: A typical profile of molecular mass of tryptic fragments of zinc- α2-glycoprotein (ZAG) during analysis by MALDI-TOF mass spectrometry.
Mentions: An average of 532 spots was observed in both groups (Figure 1). Gels were highly reproducible (r2=0.95) between groups. No qualitative differences have been found, however three spots were found to be expressed at least twofold in healthy subjects than in KC patients and one spot was found twofold in KC patients than in healthy subjects (Figure 2). The expression levels are shown in Table 2. Furthermore these differences were statistically significant (p<0.001). Mass spectrometry analysis revealed that quantitative differences more expressed in healthy subjects were; IGKC (Immunoglobulin Kappa Chain) protein, zinc-α2-glycoprotein (ZAG; Figure 3), and lactoferrin and quantitative difference more expressed in KC patients was identified as ZAG.

Bottom Line: Finally, results were validated by western-blot.IGKC protein, ZAG, and lactoferrin are under-expressed in the tears of patients diagnosed with bilateral KC compared with healthy subjects.These differences could contribute to the knowledge of the pathophysiology of this disease.

View Article: PubMed Central - PubMed

Affiliation: Instituto Galego de Oftalmoloxía, Servizo Galego de Saúde, University of Santiago de Compostela, Santiago de Compostela, Spain. mariaisabel.lema@usc.es

ABSTRACT

Purpose: To identify proteins differentially expressed between the tear film of keratoconus (KC) patients and control subjects using two dimensional electrophoresis (2-DE) and mass spectrometry-based techniques.

Methods: Twenty two patients (44 eyes) diagnosed with bilateral KC and 22 control subjects (44 eyes) were studied in a prospective case-control study. Keratoconus screening programs and Orbscan II topographies were performed on all participants. Tear samples were collected by the Schirmer I method using filter paper. Proteins were extracted from the Schirmer strips and separated by 2-DE. Comparison of protein patterns was performed using PDQuest Software and protein differences were identified by mass spectrometry. Finally, results were validated by western-blot.

Results: Four spots were identified to be differentially expressed between KC patients and control subjects. Three of them were more expressed in healthy subjects and they were identified as zinc-α2-glycoprotein (ZAG), lactoferrin, and IGKC (immunoglobulin kappa chain). The other spot was more expressed in KC patients and it was identified as ZAG. Differences in ZAG seem controversial in two different spots because different posttranslational modifications, however, analysis of both spots revealed that globally, ZAG is overexpressed in healthy subjects. Founded differences in ZAG, lactoferrin, and IGKC expression were subsequently validated by western blot.

Conclusions: IGKC protein, ZAG, and lactoferrin are under-expressed in the tears of patients diagnosed with bilateral KC compared with healthy subjects. These differences could contribute to the knowledge of the pathophysiology of this disease.

Show MeSH
Related in: MedlinePlus