BACKGROUND. The cell surface landscape of primary and relapsed neuroblastoma (NB) is currently unknown. An unbiased survey of these proteins and their isoforms would greatly facilitate the identification of candidate immunotherapeutic targets for preclinical validation.
METHODS. We are performing plasma membrane protein extraction utilizing an optimized sucrose density gradient methodology followed by nano-liquid chromatography coupled to mass spectrometry (nLC-MS/MS) to identify proteins on the cell surface of 10 NB cells lines and 10 primary/relapse tumor pairs. In addition, we are developing an analysis program in R optimized to take as input the output table from MaxQuant, a software tool for database searching and quantification commonly used in proteomics. Proteomic data will be integrated with RNA-sequencing data to assess the correlation between these data types, determine differential expression between NB (n=2242) and normal tissues (n=1641), and investigate NB-specific isoforms.
RESULTS. To date, we have optimized our method on the NB cell line IMR5. This methodology has yielded a 66% membrane protein enrichment with high reproducibility between biological replicates. This has allowed us to confirm known cell surface proteins that are currently being developed as immunotherapeutic targets (GPC2 and NCAM1). We have also identified other cell surface proteins that are being further evaluated in additional NB cell lines and tumors. We have created an efficient pipeline for data analysis and visualization. After normalization, our program outputs 27 plots and figure legends that assess data quality, regulated or abundant proteins, and Gene Ontology or motif enrichments.
CONCLUSION: We have developed a software package and robust methodology for cell surface protein isolation and quantification. Parallel proteomic and transcriptomic studies in additional NB cell lines and patient tumors are ongoing to define the cell surface landscape in primary and relapsed NB.