In the paper, the authors introduce an approach referred to as functional single-cell proteomic profiling (FUNpro). The FUNpro approach allows for screening a population containing a large quantity of cancer cells (>103) with high spatiotemporal resolution by a custom-built, ultrawide field-of-view optical microscope. This enabled the authors to perform real-time identification of cells with different (intra)cellular dynamics by an integrated automatic cell-tracking algorithm. This allowed the authors to separate different phenotypes of cells with selective photolabeling of desired cells, subsequent cell sorting and mass spectrometry based single-cell proteomic profiling. The FUNpro approach allows the authors to pre-select cells ahead of time before single-cell proteomics profiling based on any microscopically observable cellular and/or intracellular behaviors.
The SCP workflow used here is SCOPE-MS on an Orbitrap Eclipse. The authors applied FUNpro to profile and investigate a subpopulation of U2OS cells displaying abnormal DDR induced by ionizing radiation. Approximately 2129 unique proteins (10,575 unique peptides) across different cells.