Online Proteolysis and Glycopeptide Enrichment with Thermoresponsive Porous Polymer Membrane Reactors for Nanoflow Liquid Chromatography-Tandem Mass Spectrometry

In this Article, we have reported a fully automated online method to carry out proteolysis and glycopeptide enrichment in sequence for nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) analysis. By implementing two serial thermoresponsive porous polymer membrane reactors (TPPMRs), in which the TPPM could be immobilized either with trypsin for proteolysis or with lectins for glycopeptide enrichment, the entire pretreatment procedure can be performed online in about an hour. The TPPM was fabricated by coating polystyrene-maleic anhydride-N-isopropylacrylamide (PS-MAn-PNIPAm), which was synthesized by reversible addition–fragmentation chain transfer polymerization, on a Nylon sheet. Because of the thermoresponsive nature of PNIPAm, it formed micelle cavities and changed its morphology at elevated temperatures, resulting in enhanced interactions between the enzyme or lectins and the proteins/peptides flowing through the membrane. The performances of the TPPMs were evaluated by varying th...