Abstract 5570: An affinity lectin chromatography approach to characterize N-linked glycoproteome in metastatic osteosarcoma

Abstract

Abstract Osteosarcoma (OS) is the most common bone cancer in children and adolescents. Approximately 20% of OS patients have clinically detectable metastasis at the time of diagnosis, which is the most consistent indicator of poor outcome. Therefore, an easy-to-use and sensitive method is urgently needed to detect and monitor metastatic patients in order to offer an intensified or personalized therapy. By analyzing glycoproteins in OS cells, we may identify biomarkers that would help detect early metastasis and shed light on how this cancer develops. Thus, we used a tumor-targeted glycoproteomic approach in order to identify metastasis-associated glycoproteins in OS cells, and test if these can be used as blood biomarkers to indicate metastasis or spread of disease at diagnosis. In this pilot study, we have analyzed a previously established human OS metastatic cell line (LM7). We captured a specific subset of N-linked glycoproteins from LM7 cells by Wheat Germ Agglutinin (WGA) affinity chromatography. Subsequently, we identified the captured glycoproteins by a multidimensional protein identification technology (MudPIT). In addition, two samples with or without PNGase treatment were tested to estimate the impact of protein glycosylation on the MS/MS analysis. A total of 121 and 368 unique peptides were identified in the non-treated and PNGase treated samples, respectively. All of the proteins identified with two or more distinct peptides were predicted to have potential N-linked glycosylation sites, and 57% of these were found to have one or more typical Asn-X-Ser/Thr N-glycosylation sites. Many of the identified glycoproteins are known to be involved in tumorigenesis. To this end, we have optimized the affinity lectin chromatography and mass spectrometry to capture and identify N-linked glycoproteins in metastatic osteosarcoma cells. Differentially expressed glycoproteins in metastatic OS will be further identified by comparing LM7 with a non-metastatic control. The candidate biomarkers will be validated in the plasma and tumor samples collected from OS patients. 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 5570.