IBT-16plex-Based Quantitative Proteomics at the Single-Cell Level: Enabling Protein Profiles toward the Single Cells...

Isobaric tag-based mass spectrometry has been successfully employed in single-cell proteomics. As a novel and powerful isobaric reagent, 16plex isobaric tags (IBT-16plex) have not yet been applied to this area. For the first time, IBT-16plex was examined to determine whether it could be worked upon in proteomic quantification at the single-cell level. Optimal reaction conditions for IBT-16plex labeling single-cell proteome were systematically evaluated, resulting in an appropriate mass ratio of IBT-16plex versus peptides over 10,000 for complete labeling. For identifying more proteins in single cells, the strategy of carrier proteins was manipulated with multiple ratios of single-cell proteins versus carrier proteins, leading to the conclusion that the carrier proteins with 200 times more than single-cell proteins reached an acceptable peptide identification. Two isobaric reagents, TMTpro-16plex and IBT-16plex, were used to label the single-cell peptides in parallel. Comparison of the single-cell proteomics revealed that the relative quantification made by IBT-16plex was comparable with that produced from TMTpro-16plex. In contrast to traditional methods with isobaric reagents to label single-cell proteins in cultured cells, the IBT-16plex was used to profile and quantify the single-cell proteomes that were extracted from mouse spleen tissues. The single cells of the mouse spleen were isolated by CellenONE, followed by completely labeling the peptides with the IBT-16plex reagent. Based on the single-cell proteomics, those single cells were clustered into four cell types, which were annotated to monocytes, dendritic cells, macrophages, and T cells. Hence, IBT-16plex was convincingly demonstrated as a potential reagent in single-cell proteomics, also feasible for proteomic analysis of single cells isolated from animal tissues.