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iTRAQ Quantitative Proteomic Comparison of Metastatic and Non-Metastatic Uveal Melanoma Tumors.

Crabb JW, Hu B, Crabb JS, Triozzi P, Saunthararajah Y, Tubbs R, Singh AD - PLoS ONE (2015)

Bottom Line: Protein identification utilized the Mascot search engine and the human Uni-Prot/Swiss-Protein database with false discovery ≤ 1%; protein quantitation utilized the Mascot weighted average method.Of 1644 proteins identified and quantified in 5 metastatic and 5 non-metastatic tumors, 12 proteins were found uniquely in ≥ 3 metastatic tumors, 28 were found significantly elevated and 30 significantly decreased only in metastatic tumors, and 31 were designated differentially expressed between metastatic and non-metastatic tumors.While sample size is limited and validation required, the results support collagen alpha-3(VI) and heat shock protein beta-1 as candidate biomarkers of uveal melanoma metastasis and establish a quantitative proteomic database for uveal melanoma primary tumors.

View Article: PubMed Central - PubMed

Affiliation: Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States of America; Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America; Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland Clinic, Cleveland, Ohio, United States of America; Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland Clinic, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Uveal melanoma is the most common malignancy of the adult eye. The overall mortality rate is high because this aggressive cancer often metastasizes before ophthalmic diagnosis. Quantitative proteomic analysis of primary metastasizing and non-metastasizing tumors was pursued for insights into mechanisms and biomarkers of uveal melanoma metastasis.

Methods: Eight metastatic and 7 non-metastatic human primary uveal melanoma tumors were analyzed by LC MS/MS iTRAQ technology with Bruch's membrane/choroid complex from normal postmortem eyes as control tissue. Tryptic peptides from tumor and control proteins were labeled with iTRAQ tags, fractionated by cation exchange chromatography, and analyzed by LC MS/MS. Protein identification utilized the Mascot search engine and the human Uni-Prot/Swiss-Protein database with false discovery ≤ 1%; protein quantitation utilized the Mascot weighted average method. Proteins designated differentially expressed exhibited quantitative differences (p ≤ 0.05, t-test) in a training set of five metastatic and five non-metastatic tumors. Logistic regression models developed from the training set were used to classify the metastatic status of five independent tumors.

Results: Of 1644 proteins identified and quantified in 5 metastatic and 5 non-metastatic tumors, 12 proteins were found uniquely in ≥ 3 metastatic tumors, 28 were found significantly elevated and 30 significantly decreased only in metastatic tumors, and 31 were designated differentially expressed between metastatic and non-metastatic tumors. Logistic regression modeling of differentially expressed collagen alpha-3(VI) and heat shock protein beta-1 allowed correct prediction of metastasis status for each of five independent tumor specimens.

Conclusions: The present data provide new clues to molecular differences in metastatic and non-metastatic uveal melanoma tumors. While sample size is limited and validation required, the results support collagen alpha-3(VI) and heat shock protein beta-1 as candidate biomarkers of uveal melanoma metastasis and establish a quantitative proteomic database for uveal melanoma primary tumors.

No MeSH data available.


Related in: MedlinePlus

Western and SDS-PAGE Analysis.(A) Western blot analysis of the indicated 8 proteins in 11 UM tumors (samples 02, 09, 13, 15, 19, 20, 21, 23, 25, 26, 30) and 9 control Bruch’s membrane choroid specimens. The intensity of immunoreactivity in the Western blot supports the average iTRAQ ratios for these proteins. (B) Coomassie blue stained SDS-PAGE (~10 μg/lane) is shown with the same samples and amounts used for the Western analyses in panel A. These results support equal protein amounts per lane and show that UM tumor specimens and normal Bruch’s/choroid control samples exhibit different SDS-PAGE profiles.
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pone.0135543.g002: Western and SDS-PAGE Analysis.(A) Western blot analysis of the indicated 8 proteins in 11 UM tumors (samples 02, 09, 13, 15, 19, 20, 21, 23, 25, 26, 30) and 9 control Bruch’s membrane choroid specimens. The intensity of immunoreactivity in the Western blot supports the average iTRAQ ratios for these proteins. (B) Coomassie blue stained SDS-PAGE (~10 μg/lane) is shown with the same samples and amounts used for the Western analyses in panel A. These results support equal protein amounts per lane and show that UM tumor specimens and normal Bruch’s/choroid control samples exhibit different SDS-PAGE profiles.

Mentions: Western blot analysis was used to independently evaluate the abundance of 8 proteins in 11 ocular tumors and 9 control samples. Immunoreactivity in the Western blots (Fig 2A) corroborate the iTRAQ quantitation and show that the primary tumor tissues, relative to the control, contain increased amounts of macrophage migration inhibitory factor, glyceraldehyde-3-phosphate dehydrogenase, glutathione S-transferase omega 1, and lactate dehydrogenase A and decreased amounts of A-kinase anchor protein 12, C9, metalloproteinase inhibitor 3, and vitronectin. As expected, UM tumor tissues exhibit different SDS-PAGE patterns than normal Bruch’s membrane/choriod control tissues (Fig 2B).


iTRAQ Quantitative Proteomic Comparison of Metastatic and Non-Metastatic Uveal Melanoma Tumors.

Crabb JW, Hu B, Crabb JS, Triozzi P, Saunthararajah Y, Tubbs R, Singh AD - PLoS ONE (2015)

Western and SDS-PAGE Analysis.(A) Western blot analysis of the indicated 8 proteins in 11 UM tumors (samples 02, 09, 13, 15, 19, 20, 21, 23, 25, 26, 30) and 9 control Bruch’s membrane choroid specimens. The intensity of immunoreactivity in the Western blot supports the average iTRAQ ratios for these proteins. (B) Coomassie blue stained SDS-PAGE (~10 μg/lane) is shown with the same samples and amounts used for the Western analyses in panel A. These results support equal protein amounts per lane and show that UM tumor specimens and normal Bruch’s/choroid control samples exhibit different SDS-PAGE profiles.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4549237&req=5

pone.0135543.g002: Western and SDS-PAGE Analysis.(A) Western blot analysis of the indicated 8 proteins in 11 UM tumors (samples 02, 09, 13, 15, 19, 20, 21, 23, 25, 26, 30) and 9 control Bruch’s membrane choroid specimens. The intensity of immunoreactivity in the Western blot supports the average iTRAQ ratios for these proteins. (B) Coomassie blue stained SDS-PAGE (~10 μg/lane) is shown with the same samples and amounts used for the Western analyses in panel A. These results support equal protein amounts per lane and show that UM tumor specimens and normal Bruch’s/choroid control samples exhibit different SDS-PAGE profiles.
Mentions: Western blot analysis was used to independently evaluate the abundance of 8 proteins in 11 ocular tumors and 9 control samples. Immunoreactivity in the Western blots (Fig 2A) corroborate the iTRAQ quantitation and show that the primary tumor tissues, relative to the control, contain increased amounts of macrophage migration inhibitory factor, glyceraldehyde-3-phosphate dehydrogenase, glutathione S-transferase omega 1, and lactate dehydrogenase A and decreased amounts of A-kinase anchor protein 12, C9, metalloproteinase inhibitor 3, and vitronectin. As expected, UM tumor tissues exhibit different SDS-PAGE patterns than normal Bruch’s membrane/choriod control tissues (Fig 2B).

Bottom Line: Protein identification utilized the Mascot search engine and the human Uni-Prot/Swiss-Protein database with false discovery ≤ 1%; protein quantitation utilized the Mascot weighted average method.Of 1644 proteins identified and quantified in 5 metastatic and 5 non-metastatic tumors, 12 proteins were found uniquely in ≥ 3 metastatic tumors, 28 were found significantly elevated and 30 significantly decreased only in metastatic tumors, and 31 were designated differentially expressed between metastatic and non-metastatic tumors.While sample size is limited and validation required, the results support collagen alpha-3(VI) and heat shock protein beta-1 as candidate biomarkers of uveal melanoma metastasis and establish a quantitative proteomic database for uveal melanoma primary tumors.

View Article: PubMed Central - PubMed

Affiliation: Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States of America; Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America; Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland Clinic, Cleveland, Ohio, United States of America; Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland Clinic, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Uveal melanoma is the most common malignancy of the adult eye. The overall mortality rate is high because this aggressive cancer often metastasizes before ophthalmic diagnosis. Quantitative proteomic analysis of primary metastasizing and non-metastasizing tumors was pursued for insights into mechanisms and biomarkers of uveal melanoma metastasis.

Methods: Eight metastatic and 7 non-metastatic human primary uveal melanoma tumors were analyzed by LC MS/MS iTRAQ technology with Bruch's membrane/choroid complex from normal postmortem eyes as control tissue. Tryptic peptides from tumor and control proteins were labeled with iTRAQ tags, fractionated by cation exchange chromatography, and analyzed by LC MS/MS. Protein identification utilized the Mascot search engine and the human Uni-Prot/Swiss-Protein database with false discovery ≤ 1%; protein quantitation utilized the Mascot weighted average method. Proteins designated differentially expressed exhibited quantitative differences (p ≤ 0.05, t-test) in a training set of five metastatic and five non-metastatic tumors. Logistic regression models developed from the training set were used to classify the metastatic status of five independent tumors.

Results: Of 1644 proteins identified and quantified in 5 metastatic and 5 non-metastatic tumors, 12 proteins were found uniquely in ≥ 3 metastatic tumors, 28 were found significantly elevated and 30 significantly decreased only in metastatic tumors, and 31 were designated differentially expressed between metastatic and non-metastatic tumors. Logistic regression modeling of differentially expressed collagen alpha-3(VI) and heat shock protein beta-1 allowed correct prediction of metastasis status for each of five independent tumor specimens.

Conclusions: The present data provide new clues to molecular differences in metastatic and non-metastatic uveal melanoma tumors. While sample size is limited and validation required, the results support collagen alpha-3(VI) and heat shock protein beta-1 as candidate biomarkers of uveal melanoma metastasis and establish a quantitative proteomic database for uveal melanoma primary tumors.

No MeSH data available.


Related in: MedlinePlus