title: A hydrophilic immobilized trypsin reactor with N-vinyl-2-pyrrolidinone modified polymer microparticles as matrix for highly efficient protein digestion with low peptide residue.
abstract: In this work, a novel kind of N-vinyl-2-pyrrolidinone (NVP) modified poly acrylic ester microspheres was prepared, followed by trypsin immobilization to prepare a hydrophilic immobilized enzyme reactor (IMER), to achieve highly efficient protein digestion with low peptide residue. The nonspecific adsorption of peptides on such an IMER was evaluated by the in sequence digestion of bovine serum albumin (BSA) and myoglobin. Without NVP modification, both proteins could be identified after digestion by a 5 cm-length IMER, but 18 peptides of BSA were found in the digests of myoglobin caused by the nonspecific adsorption of the matrix. With NVP modification, the hydrophilicity of IMER was greatly improved, resulting in not only the sequence coverage of myoglobin increased from 63% to 73%, but also no residual peptides from BSA observed in myoglobin digests. Although the sequence coverages of proteins obtained by the IMER were comparable to those obtained by in-solution digestion, the digestion time was shortened from 24h to 1 min. By such an IMER, a protein mixture, containing BSA, myoglobin, and cytochrome c (100, 1 and 0.01 μg/mL, respectively), was digested, and all proteins were unambiguously identified with improved sequence coverages than that achieved by in-solution digestion. Furthermore, the hydrophilic IMER was also off-line coupled to nano-RPLC-ESI-MS/MS for the analysis of proteins extracted from yeast. After 1.5 min digestion, 271 protein groups with at least 2 distinct peptides were identified, much more than those obtained by 24h in-solution digestion (192 protein groups), indicating the great potential of such an IMER for proteome analysis.
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, 457 Zhongshan Road, Dalian 116023, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Jiang
authors.forename: Huiming
authors.initials: H
authors.lastname: Yuan
authors.forename: Yu
authors.initials: Y
authors.lastname: Liang
authors.forename: Simin
authors.initials: S
authors.lastname: Xia
authors.forename: Qun
authors.initials: Q
authors.lastname: Zhao
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000327:Adsorption; D000818:Animals; D019149:Bioreactors; D002417:Cattle; D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D006736:Horses; D057927:Hydrophobic and Hydrophilic Interactions; D008863:Microspheres; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011760:Pyrrolidinones / Q000737:chemistry*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011506:Proteins; D011760:Pyrrolidinones; C042670:N-vinyl-2-pyrrolidinone; D014357:Trypsin