High-throughput protein modification quantitation analysis using intact protein MRM and its application on hENGase inhibitor screening.

Proteins are widely used as drug targets, enzyme substrates, and biomarkers for numerous diseases. The emerging demand for proteins quantitation has been increasing in multiple fields. Currently, there is still a big gap for high-throughput protein quantitation at intact protein level using label-free method. Here we choose ribonuclease B (RNB) as a model, which is the substrate for human endo-β-N-acetylglucosaminidase (hENGase), a promising drug target for the treatment of N-Glycanase deficiency. Intact proteinlevel multiple reaction monitoring (MRM) methods were initally developed and optimized to quantify RNB and deglycosylated RNB (RNB-deg), with the S/N ratio improved by nearly 20-fold compared to the traditional full MS scan methods. To further increase the throughput making it possible for hENGase inhibitors screen, the protein MRM methods were introduced to the RapidFire-MS/MS system, achieving at least 12-fold throughput improvement. This assay was further optimized into 384-well plate format for compound screening with S/B ratio >37-fold and Z' factor >0.7 that is suitable for high-throughput screening of compound collections with a speed of 2 h per 384-well plate and an ability to screen over 3000 compounds per day at a single concentration dose. This 384-well plate based automated SPE-MS/MS assay is efficient and robust for compound screening and the assay format has a wide applicability to protein targets for other disease models.