A robust new strategy for high-molecular-weight proteome research: A 2-hydroxyethyl agarose/polyacrylamide gel enhanced separation and ZnO–PMMA nanobeads assisted identification

A new mass spectrometry based analysis strategy has been established here for high-molecular-weight (HMW) proteome research. First, a 2-hydroxyethyl agarose/polyacrylamide (HEAG/PAM) electrophoresis gel was designed for the first time to realize an easy-handling separation method with high spatial resolution for HMW proteins, good reproducibility and mass spectrometry-compatible sliver staining. Second, ZnO–polymethyl methacrylate (ZnO–PMMA) nanobeads were applied here for enriching and desalting the peptides from the HMW proteins. Third, the peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) with the presence of the ZnO–PMMA nanobeads, and their MS signals were enhanced markedly. The success rate of identification for HMW proteins was significantly increased due to high enriching efficiency and salt tolerance capability as well as signal enhancing capability of the ZnO–PMMA nanobeads. We believe that this analysis strategy will inspire and accelerate the HMW proteome studies.