posted on 2021-12-30, 14:09authored byClaudia Ctortecka, Karel Stejskal, Gabriela Krššáková, Sasha Mendjan, Karl Mechtler
Single-cell proteomics
workflows have considerably improved in
sensitivity and reproducibility to characterize as-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 the precursor selection
and enhance identifications. While these extreme carrier spikes are
often >100× more abundant than the investigated samples, the
total ion current undoubtably increases but the quantitative accuracy
possibly is affected. We here focus on narrowly titrated carrier spikes
(i.e., <20×) and assess their elimination for a comparable
sensitivity with superior accuracy. We find that subtle changes in
the carrier ratio can severely impact the measurement variability
and describe alternative multiplexing strategies to evaluate data
quality. Lastly, we demonstrate elevated replicate overlap while preserving
acquisition throughput at an 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 the conceptualization
of the optimal single-cell proteomics experiment.