A reference database enabling in-depth proteome and PTM analysis of mouse immune cells

Abstract

Spectral libraries fulfill multiple functions in biological and analytical applications. For biologists, these libraries provide a valuable resource to verify the presence and abundance of proteins or pathways within a selected cell type thus determine the feasibility of further experiments. Despite advances, existing libraries are incomplete and provide researchers only a limited amount of information. To address this, we introduce the reference database - Spectral Library of Immune Cells (SpLICe), a resource covering B-cells, CD4 and CD8 T-cells, macrophages and dendritic cells containing nearly 9,000 protein groups and 110,346 proteotypic peptides. Additionally, the database provides data on > 20,000 post-translationally modified proteotypic peptides (oxidation, phosphorylation, methylation, acetylation, deamidation and N-glycosylation) across the selected immune cell populations. SpLICe supports the quantification of more than half of total murine proteins annotated by UniProtKB/Swiss-Prot, enabling monitoring of selected proteins or pathways from Reactome pathways and Gene Ontology databases. The platform provides relative protein abundances and supports the generation of targeted mass spectrometry assays by identifying and scoring proteotypic peptides.

Fig. 1

The workflow to generate the primary immunological spectral libraries. (a) Key procedures encompassed the isolation of mouse splenocytes followed by purification of CD8 T-cells, CD4 T-cells, and B cells, along with the isolation of bone marrow and subsequent differentiation into BMDMs and BMDCs; (b) Proteomic sample preparation, peptide fractionation, and LC-MS/MS data acquisition; (c) Individual spectral library generation for distinct immune cells integrated in data analysis platform facilitating efficient extraction of information about pathways, associated proteins and corresponding peptides from a HTML file. Partially created with BioRender.com.

Fig. 2

Coverage and characteristics of the Spectral Libraries of Immune Cells (SpLICe). (a) Stacked histogram of precursor charges for all cell type libraries. (b) Peptide length distribution and (c) number of peptides with oxidation (Ox), deamidation (Da), phosphorylation (P), methylation (Me), acetylation (Ac), or glycosilation (Gly) PTMs. (d) Molecular mass distribution of identified proteins, (e) the distribution of proteotypic peptides per protein, and (f) their subcellular distribution. (g) Relative abundance of each protein as indicated by the normalized spectral abundance factor (NSAF) across respective cell types. Characteristic cluster differentiation (CD) markers are specified for each proteome. (h) UpsetPlot displaying the number of proteins unique to each cell type and all possible combinations of intersections. The core proteome shared among all cell types comprises N=4598 proteins. (i) Clustermap indicating selected cluster of differentiation (CD) markers.