IBT16plex-based Quantitative Proteomics: Enabling Protein Profiles of Mouse Spleen Tissues at the Single Cell Level

ABSTRACT: Isobaric tag-based mass spectrometry has been successfully employed in single-cell proteomics. As a novel and powerful isobaric reagent, 16-plex isobaric tags (IBT-16plex) have not been applied to this area yet. For the first time, IBT-16plex was examined to determine if it was workable 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, leading to the conclusion that the carrier proteins with 200 times more than single-cell proteins reached an acceptable identification of peptides. Two isobaric reagents, TMTpro-16plex and IBT-16plex, were used to label the single-cell peptides in parallel. Comparison of the single-cell proteomics unveiled 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 cell suspension, IBT-16plex was used to profile and quantify the single-cell proteomes that were extracted from solid tissues, the mouse spleen. The single cells of the mouse spleen were isolated by CellenONE and completely reacted with IBT-16plex reagent. Based on the single-cell proteomics, those single cells isolated from the mouse spleen were clustered into 4 cell types, which were successfully annotated to 4 spleen cells, monocytes and dendritic cells, macrophage, and T cells. Hence, IBT-16plex was convincingly demonstrated as a potential reagent in single-cell proteomics, also feasible for proteomic analysis towards single cells isolated from tissues.

ORGANISM(S): Homo Sapiens

SUBMITTER: Siqi Liu  

PROVIDER: PXD068645 | iProX | Fri Sep 19 00:00:00 BST 2025

REPOSITORIES: iProX