Single Cell Proteomics Reveals Novel Cell Phenotypes in Marfan Mouse Aneurysm

Abstract Background Single-cell omics technology is a powerful tool in biomedical research. However, single cell proteomics has lagged due to an inability to amplify peptides in a similar fashion to nucleotide strings. Single cell proteomics is important because proteins are the main functional unit in cells, and they often poorly correlate with mRNA quantities. In this paper we describe the first single cell proteomic analysis of complex tissue, comparing aneurysmal and normal mouse aorta from males and females. We also compare and integrate our single cell proteomic profiles with a matching single cell transcriptomics dataset. Methods We compared single cell proteomes between male and female, wild-type and Fbn1 C1041G/+ Marfan mice (N=3 per group). Individual cells from mouse aortic root single cell suspensions were deposited in 384 well plates and subjected to ultra-sensitive nanoflow liquid chromatography-ion mobility-time of flight-mass spectrometry. The data were analyzed with leiden clustering to identify cell types. Statistical analyses were performed to detect differential proteins within cell types and multi-omics analysis integrated single cell proteomics with published single cell RNA-seq. Results We identified all major aortic cell types including 7 distinct smooth muscle cell subtypes. The proportion of these cells varied based on sex and the Fbn1 C1041G/+ genotype. Differentially expressed proteins between male and female in addition to wild-type and Marfan samples uncovered enhanced endothelial to mesenchymal transition patterns in endothelial cells from male Marfan mice. Comparisons between single cell RNA and single cell proteomic profiles showed similarities in major subtypes but not smooth muscle cell subtypes. Multi-omics analysis of these two single cell platforms demonstrated a potential novel role for smooth muscle cell derived angiotensin signaling in the Marfan phenotype. Conclusions Single cell proteomics identified new subpopulations of vascular smooth muscles cells and novel cell type specific protein signatures related to sex differences and aneurysm formation. Abbreviations Next generation sequencing (NGS), Mass spectrometer (MS), Single cell proteomics by Mass Spectrometry (ScOPE-MS), Marfan’s syndrome (MFS), Fibrillin 1 (FBN1), Transforming growth factor β (TGFβ), Smooth muscle cell (SMC), Single cell proteomic (scProteomic), Differentially expressed proteins (DEPs), Wild-type (WT), Hanks’ balanced salt solution (HBSS), Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), Data-independent acquisition parallel accumulation-serial fragmentation (DIA-PASEF), Magnetic assisted cell sorted (MACS), Single Cell Analysis in Python (Scanpy), Kyoto Encyclopedia of Genes and Genomes (KEGG), Principal component analysis (PCA), Uniform manifold projection (UMAP), Single cell transcriptomic (scTranscriptomic), Smoothelin (Smtn), Transgelin (Tagln), Myosin heavy chain 11 (Myh11), Platelet endothelial cell adhesion molecule 1 (Pecam1), Dipeptidase 1 (Dpep1), Uncoupling protein 1 (Ucp1), Low-density lipoprotein receptor-related protein (Lrp1), DNA ligase 3 (Lig3), Capsaicin channel transient receptor potential vanilloid 1 (Trpv1), Endothelial to mesenchymal transition (endMT), Intercellular adhesion molecule 1 (Icam1), Intercellular adhesion molecule 2 (Icam2), Endothelial cell-selective adhesion molecule (Esam), Calponin 1 (Cnn1), Vimentin (Vim), Zinc finger E-box-binding homeobox 1 (Zeb1), Snail family transcriptional repressor 1 (Snai1), Tropomyosin alpha-4 chain (Tpm4), Angiotensin converting enzyme (Ace)