Mass Spectrometry-Based Identification of Proteins in Archival Malignant Melanoma Tissue Samples
ZP Elaba, K Rezaul, MJ Murphy, DK Han. Yale New Haven Hospital, New Haven, CT; University of Connecticut Health Center, Farmington, CT
Background: Genomic studies indicate that malignant melanoma (MM) has distinct molecular defects. However, it is difficult to predict the functional consequence of any particular gene mutation on tumor pathogenesis, progression or response to treatment. While genes contain the instructions for cellular assembly, it is through the actions of their encoded proteins that the functional characteristics of any tumor, including MM, are manifest. In addition, changes at the protein level do not always correlate with mRNA levels due to translational and post-translational modifications. Therefore, the identification of protein biomarkers that could refine the diagnostic and prognostic information gained from the clinical and histopathological features of MM, and aid in the development of novel targeted therapies is warranted. We aim to evaluate the feasibility of a mass spectrometry (MS)-based approach to discover proteins in formalin-fixed paraffin-embedded (FFPE) MM tissue samples.
Design: Proteins were extracted from a micro-dissected FFPE metastatic MM sample and analyzed by 1D SDS-PAGE separation coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Acquired MS/MS spectra was submitted for human protein database searching using SEQUEST algorithms, and identified proteins filtered, sorted, and analyzed by bioinformatics software tools. Validatory immunohistochemistry (IHC) was performed on the same FFPE sample.
Results: ∼250 μg of protein was extracted from six 10 μm tissue sections of a 0.8 x 0.8 cm tumor sample. 50μg of protein was analyzed by GeLC-MS/MS which identified 930 distinct proteins covering all sub-cellular localizations and a wide variety of biological functions. The false discovery rate was less than 1%. Expression of high abundance proteins was validated by IHC.
Conclusions: GeLC-MS/MS is a valid method to discover and characterize proteins in FFPE MM samples.
Monday, March 22, 2010 2:15 PM
Platform Session: Section F, Monday Afternoon