Differential Precipitation of Proteins: A Simple Protein Fractionation Strategy to Gain Biological Insights with Proteomics

Differential precipitation of proteins (DiffPOP) is asimple techniquefor fractionating complex protein mixtures. Using stepwise additionof acidified methanol, ten distinct subsets of proteins can be selectivelyprecipitated by centrifugation and identified by mass spectrometry-basedproteomics. We have previously shown that the ability of a proteinto resist precipitation can be altered by drug binding, which enabledus to identify a novel drug-target interaction. Here, we showthat the addition of DiffPOP to a standard LC-MS proteomicsworkflow results in a three-dimensional separation of peptides thatincreases protein coverage and peptide identifications. Importantly,DiffPOP reveals solubility differences between proteoforms, potentiallyproviding valuable insights that are typically lost in bottom-up proteomics.

Automated summary

Differential precipitation of proteins (DiffPOP) is a simple technique for fractionating complex protein mixtures. It enables the selective precipitation of ten distinct subsets of proteins, which can be identified using mass spectrometry-based proteomics. Addition of DiffPOP to a standard LC-MS proteomics workflow allows for a three-dimensional separation of peptides, resulting in increased protein coverage and peptide identifications. Importantly, DiffPOP reveals solubility differences between proteoforms, providing valuable insights that are often missed in bottom-up proteomics.