Identification of Novel Serum Proteins in Patients with Metastatic Uveal Melanoma Using Two-dimensional Gel Electrophoresis Coupled with Tandem Mass Spectrometry

Abstract

Abstract: : Purpose: Uveal melanoma is the most common intraocular malignancy with a propensity for liver metastasis. Currently there are no serologic tests to identify patients with UM who may harbor occult metastasis. The objective of this study was to identify novel serum proteins whose levels of expression may be associated with liver metastasis secondary to UM using two-dimensional gel electrophoresis coupled with tandem mass spectrometry. Methods: Serum samples were collected from three patients with biopsy proven liver metastasis secondary to uveal melanoma (patient 1,2, 3), one patient with uveal melanoma without clinical evidence of metastasis (patient 4), and one patient with uveal melanoma and cutaneous metastasis without clinical evidence of liver metastasis (patient 5). Five serum samples were obtained from age and sex-matched controls from the blood bank at the University of Texas M. D. Anderson Cancer Center. 20 ul of each plasma sample was loaded on a Citacron Blue bead column and eluted using 50mMTrisHCL, PH7.0. Samples were then centrifuged at 20,000 xg for 10 minutes. PH 4-7 gradient strips were loaded via in-gel rehydration. PH 6-11 gradient strips were loaded at the anode. First dimension isoelectric focusing was performed at 95,000 Vh. Second dimension separation was done using 8-18% T large format polyacrylamide slab gels, for 6 hours at 3mA/gel and 14 hours at 15 MA/gel. Gels were stained using SYPRO Ruby Fluorescent stain. All gel images were scanned and digitized and then compared using Z3 Image Analysis Software (Compugen). The 5 images from 5 replicate 2-D analysis of each plasma sample were used to compile a master reference gel composite. Results: Potential protein bands of interest were identified in serum from patients 1, 2, and 3 but were not present in the control serum samples or in serum collected from patients 4 and 5. Conclusion: We have identified several protein bands of interest that occurred more frequently in serum from patients with liver metastasis secondary to uveal melanoma. Future studies of larger number of patients are necessary to confirm our findings and to correlate the level of expression of these proteins with stage of uveal melanoma

Keywords: 611 uvea • 464 melanoma • 358 clinical laboratory testing