Refining Liquid Chromatography Conditions across Platforms To Democratize Single-Cell Proteomics

Single-cell proteomics has emerged as a powerful approach for characterizing cellular heterogeneity. Here, we present an optimized scProteomic workflow that enhances proteome coverage and quantification by refining liquid chromatography (LC) conditions across platforms (both Evosep and nanoElute2). Importantly, our results show that, using our optimized LC conditions, even with Bruker timsTOF HT, a machine not designed for scProteomics applications, we achieved solid performance with single-cell samples that allows meaningful biological discoveries. First, we compared the power to detect differentially expressed genes/proteins between scRNA-seq and scProteomics at the single-cell level and demonstrated that scProteomics showed excellent performance at small sample sizes. To further validate this finding, we applied our optimized workflow to study the proteomic response to oxidative stress by using a limited number of single cells. Our scProteomics results successfully detected 3 distinct cell populations, correctly reflecting the 3 cell lines used, and captured the dysregulation of antioxidant enzymes, molecular chaperones, and the ubiquitin-proteasome system, reflecting a multifaceted response to oxidative stress that is uniform across distinct cell lines. Our results highlighted the potential of scProteomics to resolve subtle perturbations and provide a readout of cellular states for the broader community, including users operating nonscProteomics-dedicated machines.