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As the use of isobaric carriers expands, we are often asked for guidelines on experimental designs. So, @harrisonspecht did controlled experiments, and we used the data to explore the trade-offs of different experimental regimes.…
A refresher on the isobaric carrier concept.
The theory.
A major bottleneck for ultrasensitive MS analysis of individual cells is peptide sequence identification.
The isobaric carrier overcomes this bottleneck by enhancing peptide sequence identification.
Sampling fewer ion copies from single cells is not simply a function of a large isobaric carrier and hardware limitations; rather, it is a reflection of analytical parameters that favor speed of analysis and number of identified peptides over sampling many ion copies.
A single experiment contains both well quantified and poorly quantified proteins. Therefore, we see great benefit in estimating the reliability of quantification for each protein and using this reliability for further quantitative analysis of the data.

1. The size of the isobaric carrier should reflect the project priorities.

2. Evaluate whether the isobaric carrier affects peptide sampling, e.g., by using DO-MS &…
3. Estimate the reliability of quantification for each protein, e.g., based on the consistency peptides originating from the same protein…

4. Incorporate estimates of reliability in all subsequent analysis
Explore the data for yourself!…
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