Quantitative Label-free LC-MS Proteomic Analysis of Uveal Melanoma Identifies Proteins Associated with Metastasis

Abstract

Purpose: To further the molecular biological understanding of the events governing the development of metastatic disease and to identify therapeutic targets for patients at risk of developing metastasis/patients with metastatic disease by comparing the protein expression profile between primary uveal melanoma (UM) tissues of patients who developed metastatic disease compared to those that did not.

Methods: 8 fresh frozen primary UM tissues of patients who developed metastasis vs. 8 who did not were subjected to quantitative, label-free LC-MS proteomic analysis. These patients had a minimum of 7 years follow-up. UM tissue samples were homogenised, lysed and proteins were digested into peptides using trypsin prior to mass spectrometry (MS) analysis. Progenesis LC-MS software was used to analyse the protein expression profile. Criteria applied to the data prior to exporting the MS output file for peptide identification were peptide features with p < 0.01, charge states +1 to +3 and > 3 isotopes per peptide. Peptides were identified with MASCOT searched against the UniProtKB-SwissProt database. Proteins with < 3 peptides and proteins with peptide conflicts were excluded. Only differentially expressed proteins with p < 0.05 between the two patient groups were considered.

Results: A total of 401 proteins were identified (global proteome). 50 proteins were found to be differentially expressed between metastatic and non-metastatic primary UM tissues. 28 proteins were upregulated and 22 were downregulated in UM tissues that developed metastasis. Increased expression of FABP3 and TPI1, and decreased expression of HSP-27, among other differentially expressed proteins were found. These proteins potentially are associated with the development of metastatic disease. A number of proteins of interest are currently being evaluated by immunohistochemistry using tissue microarrays and FFPE sections on patient groups.

Conclusions: Proteomic analysis of 8 metastatic vs. 8 non-metastatic primary uveal melanoma tissue has identified proteins that are associated with the development of metastatic disease.

Keywords: 744 tumors • 745 uvea