Limits...
iTRAQ proteomic analysis of extracellular matrix remodeling in aortic valve disease.

Martin-Rojas T, Mourino-Alvarez L, Alonso-Orgaz S, Rosello-Lleti E, Calvo E, Lopez-Almodovar LF, Rivera M, Padial LR, Lopez JA, de la Cuesta F, Barderas MG - Sci Rep (2015)

Bottom Line: Thus, a better characterization of the role of ECM proteins in this disease would increase our understanding of the underlying molecular mechanisms.The results showed an altered expression of 13 ECM proteins of which 3 (biglycan, periostin, prolargin) were validated by Western blotting and/or SRM analyses.These findings are substantiated by our previous results demonstrating differential ECM protein expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Vascular Physiopathology, Hospital Nacional de Parapléjicos, SESCAM, Toledo, Spain.

ABSTRACT
Degenerative aortic stenosis (AS) is the most common worldwide cause of valve replacement. The aortic valve is a thin, complex, layered connective tissue with compartmentalized extracellular matrix (ECM) produced by specialized cell types, which directs blood flow in one direction through the heart. There is evidence suggesting remodeling of such ECM during aortic stenosis development. Thus, a better characterization of the role of ECM proteins in this disease would increase our understanding of the underlying molecular mechanisms. Aortic valve samples were collected from 18 patients which underwent aortic valve replacement (50% males, mean age of 74 years) and 18 normal control valves were obtained from necropsies (40% males, mean age of 69 years). The proteome of the samples was analyzed by 2D-LC MS/MS iTRAQ methodology. The results showed an altered expression of 13 ECM proteins of which 3 (biglycan, periostin, prolargin) were validated by Western blotting and/or SRM analyses. These findings are substantiated by our previous results demonstrating differential ECM protein expression. The present study has demonstrated a differential ECM protein pattern in individuals with AS, therefore supporting previous evidence of a dynamic ECM remodeling in human aortic valves during AS development.

No MeSH data available.


Related in: MedlinePlus

(A) Example of one of the proteins identified after iTRAQ labeling and LC-MS/MS analysis. This protein was up-regulated in aortic stenosis (labeled with 115 and 117), with respect to control valves (labeled with 114 and 116). (1) MS/MS spectra after collision-induced dissociation (CID), (2) MS/MS fragmentation spectra after high-energy collision dissociation (HCD) for the analysis of low molecular masses, (3) detail of low molecular weight area after HCD fragmentation, showing the peaks that correspond to the reporter ions. (B) Quantification of reporter ion intensities for one of the proteins varied after iTRAQ label, biglycan. As can be observed, the ratio (114 + 116)/(115 + 117), in this case corresponding to controls/AS, is lower than the 0.71 cutoff, which means this protein is increased in AS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664895&req=5

f2: (A) Example of one of the proteins identified after iTRAQ labeling and LC-MS/MS analysis. This protein was up-regulated in aortic stenosis (labeled with 115 and 117), with respect to control valves (labeled with 114 and 116). (1) MS/MS spectra after collision-induced dissociation (CID), (2) MS/MS fragmentation spectra after high-energy collision dissociation (HCD) for the analysis of low molecular masses, (3) detail of low molecular weight area after HCD fragmentation, showing the peaks that correspond to the reporter ions. (B) Quantification of reporter ion intensities for one of the proteins varied after iTRAQ label, biglycan. As can be observed, the ratio (114 + 116)/(115 + 117), in this case corresponding to controls/AS, is lower than the 0.71 cutoff, which means this protein is increased in AS.

Mentions: For each experiment, we examined the original protein list generated by Protein Discoverer and filtered it according to the following criteria: a manual inspection of spectra was carried out wherever identification was supported among 90−95% confidence values. Fold changes were calculated by comparing iTRAQ reporter ion intensities of peptides derived from AS and control valves (representative peptide MS/MS spectra and reporter ions of some differentially expressed proteins are shown in Fig. 2). Using 1.4-0.71 fold ratio as a cutoff to assign up or down-regulated proteins, we observed 56 differential proteins present in the four runs (mixtures) of the experiment, 34 up-regulated and 19 down-regulated in stenotic aortic valves, in addition to 3 proteins (haptoglobin, peptidyl-prolyl cis trans isomerase A, L-lactate dehydrogenase A chain isoform 1) which were increased or decreased depending on the isoform identified (Tables 1 and 2).


iTRAQ proteomic analysis of extracellular matrix remodeling in aortic valve disease.

Martin-Rojas T, Mourino-Alvarez L, Alonso-Orgaz S, Rosello-Lleti E, Calvo E, Lopez-Almodovar LF, Rivera M, Padial LR, Lopez JA, de la Cuesta F, Barderas MG - Sci Rep (2015)

(A) Example of one of the proteins identified after iTRAQ labeling and LC-MS/MS analysis. This protein was up-regulated in aortic stenosis (labeled with 115 and 117), with respect to control valves (labeled with 114 and 116). (1) MS/MS spectra after collision-induced dissociation (CID), (2) MS/MS fragmentation spectra after high-energy collision dissociation (HCD) for the analysis of low molecular masses, (3) detail of low molecular weight area after HCD fragmentation, showing the peaks that correspond to the reporter ions. (B) Quantification of reporter ion intensities for one of the proteins varied after iTRAQ label, biglycan. As can be observed, the ratio (114 + 116)/(115 + 117), in this case corresponding to controls/AS, is lower than the 0.71 cutoff, which means this protein is increased in AS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664895&req=5

f2: (A) Example of one of the proteins identified after iTRAQ labeling and LC-MS/MS analysis. This protein was up-regulated in aortic stenosis (labeled with 115 and 117), with respect to control valves (labeled with 114 and 116). (1) MS/MS spectra after collision-induced dissociation (CID), (2) MS/MS fragmentation spectra after high-energy collision dissociation (HCD) for the analysis of low molecular masses, (3) detail of low molecular weight area after HCD fragmentation, showing the peaks that correspond to the reporter ions. (B) Quantification of reporter ion intensities for one of the proteins varied after iTRAQ label, biglycan. As can be observed, the ratio (114 + 116)/(115 + 117), in this case corresponding to controls/AS, is lower than the 0.71 cutoff, which means this protein is increased in AS.
Mentions: For each experiment, we examined the original protein list generated by Protein Discoverer and filtered it according to the following criteria: a manual inspection of spectra was carried out wherever identification was supported among 90−95% confidence values. Fold changes were calculated by comparing iTRAQ reporter ion intensities of peptides derived from AS and control valves (representative peptide MS/MS spectra and reporter ions of some differentially expressed proteins are shown in Fig. 2). Using 1.4-0.71 fold ratio as a cutoff to assign up or down-regulated proteins, we observed 56 differential proteins present in the four runs (mixtures) of the experiment, 34 up-regulated and 19 down-regulated in stenotic aortic valves, in addition to 3 proteins (haptoglobin, peptidyl-prolyl cis trans isomerase A, L-lactate dehydrogenase A chain isoform 1) which were increased or decreased depending on the isoform identified (Tables 1 and 2).

Bottom Line: Thus, a better characterization of the role of ECM proteins in this disease would increase our understanding of the underlying molecular mechanisms.The results showed an altered expression of 13 ECM proteins of which 3 (biglycan, periostin, prolargin) were validated by Western blotting and/or SRM analyses.These findings are substantiated by our previous results demonstrating differential ECM protein expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Vascular Physiopathology, Hospital Nacional de Parapléjicos, SESCAM, Toledo, Spain.

ABSTRACT
Degenerative aortic stenosis (AS) is the most common worldwide cause of valve replacement. The aortic valve is a thin, complex, layered connective tissue with compartmentalized extracellular matrix (ECM) produced by specialized cell types, which directs blood flow in one direction through the heart. There is evidence suggesting remodeling of such ECM during aortic stenosis development. Thus, a better characterization of the role of ECM proteins in this disease would increase our understanding of the underlying molecular mechanisms. Aortic valve samples were collected from 18 patients which underwent aortic valve replacement (50% males, mean age of 74 years) and 18 normal control valves were obtained from necropsies (40% males, mean age of 69 years). The proteome of the samples was analyzed by 2D-LC MS/MS iTRAQ methodology. The results showed an altered expression of 13 ECM proteins of which 3 (biglycan, periostin, prolargin) were validated by Western blotting and/or SRM analyses. These findings are substantiated by our previous results demonstrating differential ECM protein expression. The present study has demonstrated a differential ECM protein pattern in individuals with AS, therefore supporting previous evidence of a dynamic ECM remodeling in human aortic valves during AS development.

No MeSH data available.


Related in: MedlinePlus