Abstract
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 <100×) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ∼2500 proteins and precise quantification of ∼1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.
Highlights
An Improved Boosting to Amplify Signal with Isobaric Labeling Strategy for Precise Quantitative Single-cell Proteomics*□S
To evaluate the effects of the B/S ratios on protein identification and quantitation, a fixed amount of tryptic peptides from MCF7 cells labeled with TMT126, TMT127N and TMT127C were mixed with varying amounts of TMT131N-labeled boosting peptides to generate the B/S ratios of 10ϫ, 50ϫ, 100ϫ and 200ϫ (Fig. 1A)
Taking advantage of the multiplexed detection enabled by isobaric labeling, the boosting/carrier strategies are increasingly being used in nanoproteomics or single-cell proteomics analysis for enhanced sensitivity
Summary
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. We found that excessively high boosting ratios degrade both signal stabilities and signal-to-noise ratios (S/N) of lower abundance proteins, due mainly to the limited charge capacity of the Orbitrap; significantly increased automatic gain control (AGC) and ion injection time (IT) settings that those used in typical bulk analysis, on the other hand, help improve the signal in the sample channels, resulting in precise protein quantification in the single cells while achieving improved proteome coverage This improved BASIL (iBASIL) strategy was demonstrated by the reliable precise quantification of ϳ1500 proteins in 104 FACS-isolated single cells, which led to robust separation and clustering of these cells from 3 different acute myeloid leukemia (AML) cell lines
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