Cell Map of Mouse Peripheral Blood Mononuclear Cells with a Label-free Single-cell Proteomics Method

Fluorescently labeled antibodies were used to isolate T cells, B cells, natural killer cells, and dendritic cells. Single cells were then sorted using the CellenONE single-cell sorting system. During the sorting process, an approach of mass-adaptive coating-assisted single-cell proteomics(Mad-CASP) was pre-added to the wells of the sorting plate to reduce subsequent protein adsorption to the plate and chromatography column. The single-cell proteins were extracted and digested, and the resulting peptides were analyzed using a liquid chromatography-tandem mass spectrometry(LC-MS/MS). Protein identification was conducted using the Maxquant software's spectral library function in combination with the "match-between-runs" feature. Venn diagrams and UMAP(Uniform manifold approximation and projection) were employed to analyze the protein expression differences among the four cell types. The specific proteins of each cell type were subjected to molecular signature database mouse immune pathway enrichment, with the top two pathways selected for further analysis. Simultaneously, fuzzy C-means clustering and KEGG(Kyoto encyclopedia of genes and genomes) pathway enrichment were used to analyze the quantitative changes of shared proteins among immune cells. Finally, a single-cell proteomics map specifically for mouse peripheral blood mononuclear cells was generated, providing significant insights into understanding the functional characteristics of immune cells and identifying key protein markers related to diseases.