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Quantitative Accuracy and Precision in Multiplexed Single-Cell Proteomics

[Image: see text] 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...

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Detalles Bibliográficos
Autores principales: Ctortecka, Claudia, Stejskal, Karel, Krššáková, Gabriela, Mendjan, Sasha, Mechtler, Karl
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2021
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8829824/
https://www.ncbi.nlm.nih.gov/pubmed/34967612
http://dx.doi.org/10.1021/acs.analchem.1c04174
Descripción
Sumario:[Image: see text] 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.