An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

Through evaluating and optimizing boosting ratio and MS acquisition conditions (automatic gain control and ion injection time), the improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) strategy allows for precise and robust quantitative single-cell proteomics. A total of 2,622 proteins were identified and 1,452 proteins (58%) were quantified in more than 70% of the sample channels in the analysis of 104 FACS-isolated AML single cells, which recapitulates the key biological differences amongst three AML cell lines. [Display omitted] Highlights •Higher AGC significantly improves quantitation quality in single-cell analysis.•The boosting-to-sample ratio should be carefully evaluated and optimized.•iBASIL allows for precise quantitation of 1,500 proteins from 104 AML single cells.•iBASIL recapitulates major biological differences in different AML single cells. Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general