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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

Identification of Differentially Expressed Proteins.Log ratios for proteins (n = 860) present in ≥ 2 specimens from both 5 metastatic and 5 non-metastatic tumors in the training set are plotted (o). Thirty-one proteins exhibiting quantitative differences between metastatic and non-metastatic tumors with p ≤ 0.05 (two sided t-test) were designated differentially expressed (●).
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pone.0135543.g003: Identification of Differentially Expressed Proteins.Log ratios for proteins (n = 860) present in ≥ 2 specimens from both 5 metastatic and 5 non-metastatic tumors in the training set are plotted (o). Thirty-one proteins exhibiting quantitative differences between metastatic and non-metastatic tumors with p ≤ 0.05 (two sided t-test) were designated differentially expressed (●).

Mentions: Finally, differentially expressed proteins were sought in the training set among 860 proteins present in ≥ 2 specimens from both metastatic and non-metastatic tumors (Fig 3). Thirty-one proteins were were designated differentially expressed (Table 5) based on quantitative differences with p ≤ 0.05 (two sided t-test). However, multiple bootstrap resampling could not statistically validate significant differences in protein levels (p ≤ 0.05) for any of the 31 proteins, therefore classification as differentially expressed must be considered tentative. A limitation of this study is the relatively small sample size. A larger sample size is required to validate differential expression status; power analysis suggests that 30 metastatic and 30 non-metastatic primary UM tumors would be sufficient to rigorously confirm about 30 differentially expressed proteins.


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)

Identification of Differentially Expressed Proteins.Log ratios for proteins (n = 860) present in ≥ 2 specimens from both 5 metastatic and 5 non-metastatic tumors in the training set are plotted (o). Thirty-one proteins exhibiting quantitative differences between metastatic and non-metastatic tumors with p ≤ 0.05 (two sided t-test) were designated differentially expressed (●).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135543.g003: Identification of Differentially Expressed Proteins.Log ratios for proteins (n = 860) present in ≥ 2 specimens from both 5 metastatic and 5 non-metastatic tumors in the training set are plotted (o). Thirty-one proteins exhibiting quantitative differences between metastatic and non-metastatic tumors with p ≤ 0.05 (two sided t-test) were designated differentially expressed (●).
Mentions: Finally, differentially expressed proteins were sought in the training set among 860 proteins present in ≥ 2 specimens from both metastatic and non-metastatic tumors (Fig 3). Thirty-one proteins were were designated differentially expressed (Table 5) based on quantitative differences with p ≤ 0.05 (two sided t-test). However, multiple bootstrap resampling could not statistically validate significant differences in protein levels (p ≤ 0.05) for any of the 31 proteins, therefore classification as differentially expressed must be considered tentative. A limitation of this study is the relatively small sample size. A larger sample size is required to validate differential expression status; power analysis suggests that 30 metastatic and 30 non-metastatic primary UM tumors would be sufficient to rigorously confirm about 30 differentially expressed proteins.

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