Quantification Quality Control Emerges as a Crucial Factor to Enhance Single-Cell Proteomics Data Analysis

Mass spectrometry (MS)-based single-cell proteomics (SCP) provides us the opportunity to unbiasedly explore biological variability within cells without the limitation of antibody availability. This field is rapidly developed with the main focuses on instrument advancement, sample preparation refinement, and signal boosting methods; however, the optimal data processing and analysis are rarely investigated which holds an arduous challenge because of the high proportion of missing values and batch effect. Here, we introduced a quantification quality control to intensify the identification of differentially expressed proteins (DEPs) by considering both within and across SCP data. Combining quantification quality control with isobaric matching between runs (IMBR) and PSM-level normalization, an additional 12% and 19% of proteins and peptides, with more than 90% of proteins/peptides containing valid values, were quantified. Clearly, quantification quality control was able to reduce quantification variations and q-values with the more apparent cell type separations. In addition, we found that PSM-level normalization performed similar to other protein-level normalizations but kept the original data profiles without the additional requirement of data manipulation. In proof of concept of our refined pipeline, six uniquely identified DEPs exhibiting varied fold-changes and playing critical roles for melanoma and monocyte functionalities were selected for validation using immunoblotting. Five out of six validated DEPs showed an identical trend with the SCP dataset, emphasizing the feasibility of combining the IMBR, cell quality control, and PSM-level normalization in SCP analysis, which is beneficial for future SCP studies. [Display omitted] •IMBR effectively expands the protein pools for DE analysis in multiplexed SCP.•Removing cells and proteins with massive missing values improves cell separation.•PSM normalization preserves the original data profile with efficient cell separation.•Consistent trends in immunoblotting affirm this pipeline’s feasibility in SCP. Single-cell proteomics (SCP) via mass spectrometry offers unbiased insight into cellular variability, overcoming antibody limitations. Despite instrument and sample prep advances, optimal data processing remains underexplored. Our approach, incorporating isobaric matching between runs, quantification quality control, PSMlevel normalization, and adjusted imputation, significantly improved SCP analysis. A 12 to