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Proteomics of Fuchs' endothelial corneal dystrophy support that the extracellular matrix of Descemet's membrane is disordered.

Poulsen ET, Dyrlund TF, Runager K, Scavenius C, Krogager TP, Højrup P, Thøgersen IB, Sanggaard KW, Vorum H, Hjortdal J, Enghild JJ - J. Proteome Res. (2014)

Bottom Line: The first quantitation method, based on the areas of the extracted ion chromatograms, quantified the 51 and 48 most abundant proteins of the Descemet's membrane/endothelial layer in patient and control tissues, respectively, of which 10 were significantly regulated.The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been shown to be implicated in familial early-onset forms of the disease.These results support that the morphological changes observed in FECD are caused in part by an aberrant assembly of the extracellular matrix within the Descemet's membrane/endothelial layer.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics and ‡Interdisciplinary Nanoscience Center, Aarhus University , 8000 Aarhus, Denmark.

ABSTRACT
Fuchs' endothelial corneal dystrophy (FECD) is a major corneal disorder affecting the innermost part of the cornea, leading to visual impairment. As the morphological changes in FECD are mainly observed in the extracellular matrix of the Descemet's membrane/endothelial layer, we determined the protein profiles of diseased and control tissues using two relative quantitation MS methods. The first quantitation method, based on the areas of the extracted ion chromatograms, quantified the 51 and 48 most abundant proteins of the Descemet's membrane/endothelial layer in patient and control tissues, respectively, of which 10 were significantly regulated. The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been shown to be implicated in familial early-onset forms of the disease. Using the second relative quantitation method, iTRAQ, we identified 22 differentially regulated proteins, many of which are extracellular proteins known to be involved in proper assembly of the basement membrane in other tissues. In total, 26 differentially regulated proteins were identified, of which 6 proteins were regulated in both methods. These results support that the morphological changes observed in FECD are caused in part by an aberrant assembly of the extracellular matrix within the Descemet's membrane/endothelial layer.

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Related in: MedlinePlus

Experimental setup. The Descemet’s membrane/endotheliallayer from 10 FECD patients and 4 controls were subjected to two differentquantitative LC–MS/MS analyses: area-based extracted ion chromatogram(XIC) and isobaric tags for relative and absolute quantification (iTRAQ).Skyline was used to perform area-based XIC quantification on all 56LC–MS/MS analyses to provide the relative protein abundancesin individual tissue samples (left). The iTRAQ-based quantificationwas used to find protein regulations between patients and controls(right). Five sets of iTRAQ labeling were performed to cover all tissuesamples. Each iTRAQ set contained a Mix sample constituting tissuefrom all FECD and control samples (Mix). The Mix sample was used tonormalize between the five iTRAQ sets (SCX 1–5). Labeling wasdone according to the schema and fractionated using SCX (bracketsindicate labeling tag in the iTRAQ 4plex). Five SCX runs were performed(SCX 1–5), and the collected fractions from each run were analyzedby LC–MS/MS and searched against all human sequences in theSwiss-Prot database using the Mascot search engine. Finally, iTRAQdata were parsed using MS Data Miner (MDM). All raw data were depositedto the ProteomeXchange consortium.
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fig1: Experimental setup. The Descemet’s membrane/endotheliallayer from 10 FECD patients and 4 controls were subjected to two differentquantitative LC–MS/MS analyses: area-based extracted ion chromatogram(XIC) and isobaric tags for relative and absolute quantification (iTRAQ).Skyline was used to perform area-based XIC quantification on all 56LC–MS/MS analyses to provide the relative protein abundancesin individual tissue samples (left). The iTRAQ-based quantificationwas used to find protein regulations between patients and controls(right). Five sets of iTRAQ labeling were performed to cover all tissuesamples. Each iTRAQ set contained a Mix sample constituting tissuefrom all FECD and control samples (Mix). The Mix sample was used tonormalize between the five iTRAQ sets (SCX 1–5). Labeling wasdone according to the schema and fractionated using SCX (bracketsindicate labeling tag in the iTRAQ 4plex). Five SCX runs were performed(SCX 1–5), and the collected fractions from each run were analyzedby LC–MS/MS and searched against all human sequences in theSwiss-Prot database using the Mascot search engine. Finally, iTRAQdata were parsed using MS Data Miner (MDM). All raw data were depositedto the ProteomeXchange consortium.

Mentions: Isobaric tags for relative and absolute quantification (iTRAQ) analysiswas performed using 10 μg of in-solution digested sample fromeach patient and control. Samples were labeled with iTRAQ 4plex (ABSCIEX, Framingham, MA, USA) according to the manufacturer’sprotocol, resulting in a total of five sets of 4plex experiments (Figure 1). A mixed sample containing 5 μg of samplefrom each of the patients and controls was labeled with the iTRAQ114-tag in all five experiments and used to normalize between experiments.Each 4plex experiment was diluted 10 times in buffer A (0.5% formicacid and 5% acetonitrile) and separated on a Higgins Analytical strongcation exchange (SCX) column (PL-SCX 1000 Å 5 μm 20 ×2.1 mm column, Higgins Analytical, Rengstorff, CA, USA) equilibratedin buffer A and connected to an Ettan LC system (GE Healthcare, Wauwatosa,WI, USA). The peptides were eluted using a 100 μL/min flow rateand a 1% B/min linear gradient from buffer A to buffer B (buffer Bcontaining 1 M NaCl). The 15 collected fractions were lyophilized,redissolved in 100 μL 0.1% formic acid, and desalted using POROS50 R2 material packed in GELoader tips. The desalted samples werelyophilized and resuspended in 12 μL 0.1% formic acid and storedat −20 °C.


Proteomics of Fuchs' endothelial corneal dystrophy support that the extracellular matrix of Descemet's membrane is disordered.

Poulsen ET, Dyrlund TF, Runager K, Scavenius C, Krogager TP, Højrup P, Thøgersen IB, Sanggaard KW, Vorum H, Hjortdal J, Enghild JJ - J. Proteome Res. (2014)

Experimental setup. The Descemet’s membrane/endotheliallayer from 10 FECD patients and 4 controls were subjected to two differentquantitative LC–MS/MS analyses: area-based extracted ion chromatogram(XIC) and isobaric tags for relative and absolute quantification (iTRAQ).Skyline was used to perform area-based XIC quantification on all 56LC–MS/MS analyses to provide the relative protein abundancesin individual tissue samples (left). The iTRAQ-based quantificationwas used to find protein regulations between patients and controls(right). Five sets of iTRAQ labeling were performed to cover all tissuesamples. Each iTRAQ set contained a Mix sample constituting tissuefrom all FECD and control samples (Mix). The Mix sample was used tonormalize between the five iTRAQ sets (SCX 1–5). Labeling wasdone according to the schema and fractionated using SCX (bracketsindicate labeling tag in the iTRAQ 4plex). Five SCX runs were performed(SCX 1–5), and the collected fractions from each run were analyzedby LC–MS/MS and searched against all human sequences in theSwiss-Prot database using the Mascot search engine. Finally, iTRAQdata were parsed using MS Data Miner (MDM). All raw data were depositedto the ProteomeXchange consortium.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4227554&req=5

fig1: Experimental setup. The Descemet’s membrane/endotheliallayer from 10 FECD patients and 4 controls were subjected to two differentquantitative LC–MS/MS analyses: area-based extracted ion chromatogram(XIC) and isobaric tags for relative and absolute quantification (iTRAQ).Skyline was used to perform area-based XIC quantification on all 56LC–MS/MS analyses to provide the relative protein abundancesin individual tissue samples (left). The iTRAQ-based quantificationwas used to find protein regulations between patients and controls(right). Five sets of iTRAQ labeling were performed to cover all tissuesamples. Each iTRAQ set contained a Mix sample constituting tissuefrom all FECD and control samples (Mix). The Mix sample was used tonormalize between the five iTRAQ sets (SCX 1–5). Labeling wasdone according to the schema and fractionated using SCX (bracketsindicate labeling tag in the iTRAQ 4plex). Five SCX runs were performed(SCX 1–5), and the collected fractions from each run were analyzedby LC–MS/MS and searched against all human sequences in theSwiss-Prot database using the Mascot search engine. Finally, iTRAQdata were parsed using MS Data Miner (MDM). All raw data were depositedto the ProteomeXchange consortium.
Mentions: Isobaric tags for relative and absolute quantification (iTRAQ) analysiswas performed using 10 μg of in-solution digested sample fromeach patient and control. Samples were labeled with iTRAQ 4plex (ABSCIEX, Framingham, MA, USA) according to the manufacturer’sprotocol, resulting in a total of five sets of 4plex experiments (Figure 1). A mixed sample containing 5 μg of samplefrom each of the patients and controls was labeled with the iTRAQ114-tag in all five experiments and used to normalize between experiments.Each 4plex experiment was diluted 10 times in buffer A (0.5% formicacid and 5% acetonitrile) and separated on a Higgins Analytical strongcation exchange (SCX) column (PL-SCX 1000 Å 5 μm 20 ×2.1 mm column, Higgins Analytical, Rengstorff, CA, USA) equilibratedin buffer A and connected to an Ettan LC system (GE Healthcare, Wauwatosa,WI, USA). The peptides were eluted using a 100 μL/min flow rateand a 1% B/min linear gradient from buffer A to buffer B (buffer Bcontaining 1 M NaCl). The 15 collected fractions were lyophilized,redissolved in 100 μL 0.1% formic acid, and desalted using POROS50 R2 material packed in GELoader tips. The desalted samples werelyophilized and resuspended in 12 μL 0.1% formic acid and storedat −20 °C.

Bottom Line: The first quantitation method, based on the areas of the extracted ion chromatograms, quantified the 51 and 48 most abundant proteins of the Descemet's membrane/endothelial layer in patient and control tissues, respectively, of which 10 were significantly regulated.The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been shown to be implicated in familial early-onset forms of the disease.These results support that the morphological changes observed in FECD are caused in part by an aberrant assembly of the extracellular matrix within the Descemet's membrane/endothelial layer.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics and ‡Interdisciplinary Nanoscience Center, Aarhus University , 8000 Aarhus, Denmark.

ABSTRACT
Fuchs' endothelial corneal dystrophy (FECD) is a major corneal disorder affecting the innermost part of the cornea, leading to visual impairment. As the morphological changes in FECD are mainly observed in the extracellular matrix of the Descemet's membrane/endothelial layer, we determined the protein profiles of diseased and control tissues using two relative quantitation MS methods. The first quantitation method, based on the areas of the extracted ion chromatograms, quantified the 51 and 48 most abundant proteins of the Descemet's membrane/endothelial layer in patient and control tissues, respectively, of which 10 were significantly regulated. The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been shown to be implicated in familial early-onset forms of the disease. Using the second relative quantitation method, iTRAQ, we identified 22 differentially regulated proteins, many of which are extracellular proteins known to be involved in proper assembly of the basement membrane in other tissues. In total, 26 differentially regulated proteins were identified, of which 6 proteins were regulated in both methods. These results support that the morphological changes observed in FECD are caused in part by an aberrant assembly of the extracellular matrix within the Descemet's membrane/endothelial layer.

Show MeSH
Related in: MedlinePlus