Surfaceome Capture by Multiplex Biotinylation Enables Enhanced Identification of Cell Surface Proteins by Mass Spectrometry.

A key goal of surfaceomic discovery technologies is to comprehensively interrogate the cell surface proteome to identify targets for immunotherapy development. Considerable progress has made in the application of surfaceomics to profile cancer cells. However, challenges still exist in the sensitivity of current surfaceomic approaches, which consequently, are restricted to the analysis of cell lines or primary tumour material that contain a relatively large number of cells. In addition, since current approaches are based on labelling a single functional group on polypeptides, it is not clear if these recover the full spectrum of proteins present on cell surfaces. To circumvent these limitations, we developed a biotinylation-based combinatory approach for isolating a more diverse group of proteins facing the cell periphery. Our proposed approach, named Surfaceome Capture by Multiplex (SUCAM) biotinylation consists of "multiplexing" biotin reagents to enable for multiple-functional group derivatisation of the surface proteome. LC-MS/MS is then used to identify and characterize proteins pulled down by streptavidin magnetic beads. We found that SUCAM identified more plasma membrane and cell surface proteins than methods based on labelling with single reagents, leading to enhanced identification of cell surface proteins from just 1.2 million cells. Replicate experiments revealed that surfaceomic proteins could be quantified with good precision across repeats (coefficient of variation of 1.7% on average). Application of the approach to a panel of leukaemia cell lines identified well-known leukemic cell surface antigens, as well as proteins with hitherto uncharacterised roles in this disease. SUCAM is a complementary biotinylating strategy that will facilitate surfaceomic profiling for discovery of therapeutic targets in haematological and solid tumours.