A new photolabeling probe for efficient enrichment and deep profiling of cell surface membrane proteome by mass spectrometry

The cell surface membrane proteome is a class of proteins encoded by ∼ 25% of all protein-coding genes in living organisms and plays a key role in mediating communication between the cells and their surrounding environment. However, most cell surface membrane proteins (CSMPs) are naturally expressed at very low levels compared with intracellular proteins. The difficulties in their purification with high specificity further hinder the understanding of their structure and function. In this study, we developed a new photolabeling probe to achieve efficient tagging and facile enrichment of the CSMPs. The probe is composed of a lipid tail for cell surface localization, a polyethylene glycol (PEG) spacer for increased water solubility, two 4-(N-maleimido)benzophenone (MBP) groups for UV-active tagging of the CSMPs, and a biotin tag for subsequent isolation. Application of this photolabeling probe resulted in the successful enrichment and identification of 3098 annotated CSMPs in HT22 cells with close to 70% selectivity. The proposed photolabeling probe and enrichment strategy were demonstrated to be a powerful method for deep cell surface proteome profiling, representing one of the largest groups of current drug targets.