Figure 3 ∣. Fractionation prior to counting molecules improves the dynamic range in proteomics.

a, The dynamic range problem of the human proteome is far more extreme than that of transcriptomics. The enormous dynamic range of peptide abundances requires massive oversampling of the most abundant peptide (solid blue) to obtain counts for the least abundant peptide (vertical stripe red) b, LC-MS separates peptides biochemically, ionizes them, and samples the peptides at different times and with different spectra. While a mass spectrometer works in the gas phase, it is analogous to separating peptides/proteins prior to counting and then applying normalization to make the quantities comparable between spectra or flow cells. This strategy significantly improves the counting statistics of low abundance molecules in the presence of high abundance molecules. In ion trap mass spectrometry, the normalization approach to optimize ions in each spectrum and adjust the signal by the variable fill time is known as automatic gain control (AGC).