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

Abstract

Differential precipitation of proteins (DiffPOP) is a simple technique for fractionating complex protein mixtures. Using stepwise addition of acidified methanol, ten distinct subsets of proteins can be selectively precipitated by centrifugation and identified by mass spectrometry-based proteomics. We have previously shown that the ability of a protein to resist precipitation can be altered by drug binding, which enabled us to identify a novel drug−target interaction. Here, we show that the addition of DiffPOP to a standard LC−MS proteomics workflow results in a three-dimensional separation of peptides that increases protein coverage and peptide identifications. Importantly, DiffPOP reveals solubility differences between proteoforms, potentially providing valuable insights that are typically lost in bottom-up proteomics.

Essential role of MFSD1-GLMP-GIMAP5 in lymphocyte survival and liver homeostasis

“…We hypothesized that if Mfsd1 −/− or Gimap5 −/− mice have a deficiency or excess of the substrate molecule transported by the MGG complex, they might also display a deficiency or excess of a substrate carrier protein and proteomic analysis of serum might detect a difference between WT and mutant animals. We employed a protein fractionation strategy to facilitate detection of low-abundance serum proteins while minimizing interference by abundant proteins in serum such as albumin ( 38 ).…”

“…Serum protein fractionation was performed by DiffPOP [Differential Precipitation of Proteins; ( 38 )]. Briefly, 10 μL of serum was mixed with 100 μL of Phosphoprotein Buffer A (Takara) and 140 μL water.…”

TMT-Based Proteomics Analysis of Senescent Nucleus Pulposus from Patients with Intervertebral Disc Degeneration

“…Quantitative proteomics is a high-throughput screening technique commonly used to quantify proteins and metabolites in complex samples. It can be used to identify novel targets for disease treatment and elucidate the changes in target proteins involved in disease pathogenesis and drug interventions [14][15][16][17][18][19]. Tandem mass tag (TMT)-based proteomics technology has the advantages of high throughput, high sensitivity, good reproducibility, low systematic bias, and low noise.…”