Comprehensive spectral library generation for primary human immune cells using data-dependent acquisition

Abstract

Immune cells play crucial regulatory roles and exhibit remarkable cellular diversity in human physiology. Previous studies have extensively analyzed T-cell proteomes in order to clarify the comprehensive profiles according to the status of cells. However, generating cell-specific proteome libraries for small immune cell components like NK or B cells has not yet been undertaken due to the difficulties of sample collection. In this work, we report novel cell-specific libraries for four primary human immune cell types obtained through sorting with high-purity using fluorescence-activated cell sorting (FACS) and data-dependent spectrum acquisition. As a result, we generated 10,300 spectral libraries with 6,125 common spectra among helper T cells, cytotoxic T cells, natural killer (NK) cells, and B cells. Each primary cell library included cell type-specific protein markers and comprised 7,879, 7,347, 7,516, and 7,070 spectra for helper T cells, cytotoxic T cells, NK cells, and B cells, respectively. With these spectral libraries, we further analyzed a limited number of immune cells and identified proteins using the integrated spectral library. Obtaining sufficient protein quantities from primary immune cells, which are present in absolutely limited quantities in whole blood, poses challenges for reproducibility in sample analysis. Therefore, providing a comprehensive candidate list of proteins in small quantity of samples can enhance quantitative reproducibility among technical replicates. In this study, we observed that the identified proteins were proportionally increased with the number of cells, and functional analysis of the identified proteins revealed multiple signaling pathways characteristic of each immune cell type. In conclusion, our established immune cell-specific libraries, ensuring high cellular purity, can be readily applied to biomarker discovery, immune status assessment, and drug response monitoring, particularly in diseases associated with immune system dysfunction. Moreover, these libraries enhanced quantitative reproducibility among the technical replicates even with a limited sample amount.