Confirmation of SOMAmer® Enrichment from Biological Matrices using Mass Spectrometry

In an effort to confirm binding of SOMAmer®s to their respective targets in an endogenous matrix, we have conducted a series of experiments using SOMAmer®s to enrich target proteins followed by measurement using two mass spectrometry techniques: data dependent analysis (DDA) and multiple reaction monitoring (MRM). For data dependent analysis, the library of 4783 SOMAmer®s were multiplexed in sets of 8 and used for enrichment of target proteins from cell lysate and conditioned media, as well as human plasma and serum. A subset of the SOMAmer®s were screened in urine by this global profiling technique as well. Cell lines were selected from Cancer Cell Line Encyclopedia for screening by comparing gene expression of the target proteins measured by RNA sequencing across the CCLE.1 Using the criterion of FPKM value greater than 5 for the target protein, the minimum number of cell lines was grown to maximize protein coverage across the SOMAmer® library. For a given cell line, the presence of at least 8 target proteins with FPKM values greater than 5was required for inclusion. Approximately 400 target proteins were not covered by this strategy were screened in serum and plasma only. For enrichment from biological matrices, SOMAmer®s were combined into sets of 8 such that the potential interaction with similar proteins or binding partners was minimized. Non-specific binding of the SOMAmer®s was assessed in each matrix by using a SOMAmer® generated against the bacterial protein CysH. Target protein spectral counts in the SOMAmer® enriched samples were compared to the respective CysH control. A positive hit is defined as target protein detection with a minimum of 2 spectral counts and signal over CysH background greater than 10X (if applicable).