Quantitative accuracy and precision in multiplexed single-cell proteomics

Single-cell proteomics workflows have considerably improved in sensitivity and reproducibility to characterize yet unknown biological phenomena. With the emergence of multiplexed single-cell proteomics, studies increasingly present single-cell measurements in conjunction with an abundant congruent carrier to improve precursor selection and enhance identifications. While these extreme carrier spikes are often >100-times more abundant than the investigated samples, undoubtedly the total ion current increases, but quantitative accuracy possibly is affected. We here focus on narrowly titrated carrier spikes (i.e., <20x) and assess their elimination for comparable sensitivity at superior accuracy. We find that subtle changes in the carrier ratio can severely impact measurement variability and describe alternative multiplexing strategies to evaluate data quality. Lastly, we demonstrate elevated replicate overlap while preserving acquisition throughput at improved quantitative accuracy with DIA-TMT and discuss optimized experimental designs for multiplexed proteomics of trace samples. This comprehensive benchmarking gives an overview of currently available techniques and guides conceptualizing the optimal single-cell proteomics experiment.