Quantitative accuracy and precision in multiplexed single-cell proteomics

Abstract

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.

An automated workflow for multiplexed single-cell proteomics sample preparation at unprecedented sensitivity

“…2b-c). 6,7 Next, we aimed at determining if precursor selection is influenced by the carrier ratio or preparation. For this, we spiked bulk digested equivalents of 20 or 100 cells, sorted 20 cells and no carrier.…”

“…1e). 6,7 Although the carrier undoubtedly boosts identifications in SCP, the reduced RI S/N and precursor selection influence suggests to keep the carrier ratio to a minimum (Fig. 1d-e; Supplemental Fig.…”

“…1e). 7 This is presumably due to the different fragmentation behavior of the longer TMTpro tag. 4 Nevertheless, single cell RI S/N is reduced by 74% or 8% in the 20x versus the no-carrier samples, for TMT10 and TMTpro, respectively (Fig.…”

“…2a-e). 6,7,10 For quantitative reproducibility and data quality evaluation, we compared missing quantitative signal per PSM and cumulative missing data across multiple analytical runs. For both labeling reagents, with or without a carrier, the RI S/N of two individual sample channels correlates well (i.e., r = >0.85) and outperforms previous reports of multiplexed SCP (Fig.…”

“…5 However, such extreme carrier ratios were demonstrated to impair quantitative accuracy and possibly affect biological conclusions. 6,7…”