Optimizing accuracy and depth of protein quantification in experiments using isobaric carriers

Abstract

The isobaric carrier approach, which combines small isobarically-labeled samples with a larger isobarically-labeled carrier sample, is finding diverse applications in ultrasensitive mass-spectrometry analysis of very small samples, such as single cells. To inform the growing use of isobaric carriers, we characterized the trade-offs of using isobaric carriers in controlled experiments with complex human proteomes. The data indicate that isobaric carriers directly enhances peptide sequence identification without simultaneously increasing the number of protein copies sampled from small samples. The results also indicate strategies for optimizing the amount of isobaric carrier and analytical parameters, such as ion accumulation time, for different priorities such as improved quantification or increased number of identified proteins. Balancing these trade-offs enables adapting isobaric carrier experiments to different applications, such as quantifying proteins from limited biopses or organoids, building single-cell atlases, or modeling protein networks in single cells. In all cases, the reliability of protein quantification should be estimated and incorporated in all subsequent analysis. We expect that these guidelines will aid explicit incorporation of the characterized trade-offs in experimental designs and transparent error propagation in data analysis.