Quantitative Proteomic Analysis of API2-MALT1 Expression Signature by Isobaric Tags and High-Energy C-TRAP Dissociation Tandem Mass Spectrometry
SL Farmen, C Seiler, LM McAllister-Lucas, PC Lucas, KP Conlon, D Fermin, V Basrur, MS Lim, KSJ Elenitoba-Johnson. University of Michigan, Ann Arbor, MI
Background: Quantitative proteomic analysis by mass spectrometry (MS)-based approaches permits the interrogation of complex protein mixtures. API2-MALT1 chimeric fusion is the protein product of the t(11;18) aberration, which is the most common chromosomal alteration in extranodal marginal zone lymphoma. Despite its implication in lymphomagenesis, the signaling pathways modulated as a consequence of this chimeric fusion protein are unknown.
Design: We generated a human embryonic kidney (HEK-293) cell line that was stably transduced to express the API2-MALT1 fusion and another that was transduced with a vector-control. Equivalent amounts of protein from each cell type were labeled using using an isobaric (iTRAQ) tag that yields amine-derivatized peptides, and subjected to MS/MS using high energy fragmentation in the C-TRAP of an LTQ-Orbitrap XL tandem mass spectrometer. The MS/MS spectra were processed using the TransProteomic Pipeline equipped with X!Tandem for protein identification. Significantly expressed proteins were determined by beta-uniform mixture modeling approach with significant differential expression defined at 0.13 false discovery rate. Identified proteins were subjected to Ingenuity pathway analysis to reveal biological pathways deregulated as a consequence of API2-MALT1 expression, and several were validated with Western blotting.
Results: iTRAQ LC-MS/MS identified 27 proteins underexpressed (RR < 0.7) and 17 proteins overexpressed (RR >1.3) in cells overexpressing API2-MALT1. MALT1 was five-fold overexpressed in the experimental cells. Proteins reflective of a role for API2-MALT1 in deregulation of apoptosis, cytokine signaling, cell cycle progression and protein transport were illuminated by pathway analysis. Among others, iTRAQ data indicated that coatomer protein gamma, SEC22b, and REM1 were overexpressed in the API2-MALT1 positive cells, while GSPT1/eRF3, enolase, anamorsin, and serine/threonine kinase 24 were underexpressed. A subset of these were confirmed by Western blotting.
Conclusions: iTRAQ LC-MS/MS is a powerful tool for global quantitative proteomic analysis. Our studies reveal the ability of MS combined with bioinformatics-enabled discovery platforms to discover novel pathogenetic mechanisms and biomarkers that may be useful for facile identification of the disease entities.
Category: Special Category for 2009 - Pan-genomic/Pan-proteomic approaches to Cancer
Monday, March 9, 2009 9:30 AM
Poster Session I Stowell-Orbison/Autopsy Award # 240, Monday Morning