Overcoming the challenges in sample multiplexing for next generation single cell proteomics

Single-cell proteomics has emerged in the past five years as a powerful approach to investigate the heterogeneity of proteomes within cell populations. Understanding how protein expression and modification vary from cell to cell is essential, as population averages can mask critical regulatory mechanisms and rare cell states. This question is particularly relevant in the context of aging, where tissues progressively lose cellular identity and individual cells accumulate distinct DNA mutations and proteomic profiles, potentially contributing to functional decline. Among the molecular mechanisms that shape proteome regulation, chromatin plays a central role by modulating gene accessibility and expression through histone post-translational modifications (hPTMs). Therefore, studying chromatin-associated proteins at single-cell resolution can provide key insights into how epigenetic diversity influences cellular function and identity. Despite recent advances, single-cell proteomics still faces important challenges in sensitivity and throughput. Unlike sequencing-based methods, proteins and peptides cannot be amplified or easily barcoded, making efficient sample processing and precise quantification. During my PhD, I addressed these limitations through three complementary projects. First, I developed and optimized a workflow for single-cell analysis of histone post-translational modifications, enabling the study of chromatin regulation at unprecedented depth. Second, I refined a series of chemical labeling strategies to tag peptides site-specifically and multiplex samples, increasing the throughput of single-cell proteomics. Finally, I applied the optimized workflow to a mouse model of extended healthspan, using single-cell proteomics to quantify how the liver proteome and its heterogeneity are regulated during aging by metabolic intervention. Together, these studies contribute methodological and conceptual advances toward a more comprehensive understanding of proteome regulation at the single-cell level and open new directions for applying proteomics to complex biological questions such as aging and epigenetic control.