Abstract 1624: Identification and characterization of phospho-ORM-1 as a novel nicotinic acetylcholine receptor (NAChR)-associated protein and a potential serum marker for lung cancer detection

Abstract

Abstract Phosphorylation is a key regulating switch that plays a critical role in signaling pathways involved in oncogenesis. A high-throughput analysis of phosphoproteins could provide a useful tool for analysis of biological functions and signaling pathways associated with these molecular events. In this study, we developed an innovative functional proteomics platform using ProteinChip array-based SELDI-MS for high-throughput profiling and identification of phosphopeptides in human serum to identify specific phosphopeptides/phosphoproteins associated with human lung cancer. We performed phosphopeptide profiling on serum samples from human normal and lung cancer patients with varying stages and smoking histories. We used phospho-tyrosine antibody-conjugated super-paramagnetic beads to capture phosphopeptides generated in trypsin-digested serum samples. The captured phospho-tyrosine peptides (pYPs) were separated on a hybrid magnetic plate using a biological sample preparation robot. The affinity-enriched pYPs were then randomly loaded onto ProteinChips and analyzed by SELDI-TOF Mass Spectrometry. We used wavelets and the mean spectrum for peak detection and detected more than 600 pYP peaks spanning M/Z range from 50 to 5500 Dalton. For each peak, we recorded the p-value from an F-test and modeled the set of p-values using a beta-uniform mixture model to estimate the false discovery rate (FDR). We identified 39 pYP peaks with fold changes in intensity detected on SELDI-MS profiles to be significantly (at FDR = 10%) differentially expressed between the normal and lung cancer serum samples. The phosphopeptides detected on SELDI-MS spectra were further identified using a protein chip array-interfaced qSTAR-MS/MS. One of phospho-tyrosine containing peptides was identified as an Alpha-1-acid glycoprotein 1 precursor (A1AG1) or ORM-1 (Orosomucoid). The ORM-1 pYP showed a M/Z peak at 1752.3 Da and was significantly upregulated in lung cancer serum samples with more than 10-fold increase (P = 0.0024) in mass peak intensity. Computer-aided structural and functional analysis predicted the potential association of ORM-1 to the nicotinic acetyl choline receptor (nAChRs). We further validated phospho-ORM-1 protein expression in another set of lung cancer and control serum samples by Quantitative ELISA and confirmed the significantly upregulated expression of serum phospho-ORM-1 in lung cancer patients with ever-smoking history. We also identified protein interactions between the ORM-1 and subunits of nAchRs in lung cancer cell lines by immunoprecipitation and immune-blotting analysis. Our results suggest the role of the Phospho-ORM-1 peptide as a novel NAChR-associated protein in lung cancer pathogenesis and smoking-associated carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1624.