Fig. 1
Workflow for the proteomic analysis of five immune cell lines and major primary immune cells and generation of the immune cell spectral library. In the process of generating the spectral library, proteins were extracted from both cell lines and primary cells of immune cells. Specifically, 200 μg of cell line proteins and 100 μg of primary cell proteins underwent tryptic digestion using the FASP method. To alleviate sample complexity, the peptide pre-fractionation step was implemented, involving high-pH fractionation of over 100 μg of proteins from cell lines and primary cells, resulting in 12 and 24 fractions, respectively. The fractionated tryptic peptides from both cell lines and primary cells were then subjected to analysis using the DDA method with Q-Exactive plus and Exploris 240, respectively. For trypsin digestion of proteins from a limited number of primary immune cells (10,000 cells per primary cell type), the SP3 method was employed, and the DIA method with Exploris 240 was applied to obtain the proteome profiles. The raw files acquired from DDA and DIA methods were searched and employed to create the combined immune cell library via Spectronaut (version 18). This combined library was subsequently applied to identify the proteome of the small number of primary immune cells (10,000 cells per primary cell type). The resulting proteome profiles were visualized using SIMCA-P (version 17). Statistical analysis, aiming to identify significant proteins among the four types of primary immune cells, was conducted using the Kruskal-Wallis test through SPSS (version 27). Violin plots, generated using the ggplot2 package in R, visually represented some statistically evaluated proteins.
