Depletion of abundant plasma proteins by poly(N-isopropylacrylamide-acrylic acid) hydrogel particles.

Protein and proteome analysis of human blood plasma presents a challenge to current analytical platforms such as mass spectrometry (MS). High abundance plasma proteins interfere with detection of potential protein biomarkers that are often 3-10 orders of magnitude lower in concentration. We report the application of pH-sensitive poly(N-isopropylacrylamide-acrylic acid) hydrogel particles for removal of abundant plasma proteins, prior to proteome analysis by MS. Protein depletion occurs by two separate mechanisms: (1) hydrogel particles incubated with low concentrations of plasma capture abundant proteins at higher efficiency than low abundance proteins, which are enriched in the supernatants, whereas (2) hydrogel particles incubated with high concentrations of plasma capture and irreversibly trap abundant proteins. During the elution step, irreversibly trapped proteins remain captured while low abundance proteins are released and recovered in the eluate. We developed a series of distinct depletion protocols that proved useful for sample depletion and fractionation and facilitated targeted analysis of putative biomarkers such as IGF1-2, IBP2-7, ALS, KLK6-7, ISK5, and PLF4 by selected reaction monitoring (SRM) liquid chromatography (LC)-MS/MS. This novel use of hydrogel particles opens new perspectives for biomarker analysis based on mass spectrometry.