A robust new strategy for high-molecular-weight proteome research: A 2-hydroxyethyl agarose/polyacrylamide gel enhanced separation and ZnO-PMMA nanobeads assisted identification |
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| Authors: | Wenwen Shen Chengpin Shen Huanming Xiong Pengyuan Yang |
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| Institution: | a Department of Chemistry, Fudan University, Shanghai, China
b Institutes of Biomedical Sciences, Fudan University, Shanghai, China |
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| Abstract: | 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. |
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| Keywords: | HEAG 2-hydroxyethyl agarose gel PAM polyacrylamide gel HMW high-molecular-weight IEF isoelectric focusing MS mass spectrometry MALDI matrix-assisted laser desorption/ionization CHCA α-cyano-4-hydroxycinnamic acid ZnO-PMMA ZnO-polymethyl methacrylate APS ammonium persulfate SD Sprague-Dawley GST glutathione-S-transferase 1-DE one dimensional electrophoresis 2-DE two dimensional electrophoresis TOF time-of-flight MYO horse heart myoglobin BSA bovine serum albumin GRAVY the grand average of hydropathy |
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