title: Porous silica enhanced proteolysis during Off-Gel separation for efficient protein identification.
abstract: An advanced approach is developed in this work for simultaneous on-line separation and digestion of proteins by combining the Off-Gel isoelectric focusing (IEF) and enzymatic nanoreactor enhanced proteolysis. The nanoreactor was prepared by preloading trypsin in amino-functionalized macroporous silica, and then directly added into Off-Gel wells. With the nanoreactor loaded Off-Gel device, effective digestion of proteins happened during IEF electrophoresis to generate directly fractionated tryptic peptides, which not only accelerated the experimental flow but also avoided sample loss, leading to a more comprehensive protein identification from complex biological samples. A successful identification of 3592 proteins was achieved from Hela cell line by using the approach followed with LC-MS/MS analysis, while only 1877 proteins were identified from the same sample when using standard in-solution proteolysis followed with Off-Gel electrophoresis and then LC-MS/MS analysis. Therefore, we have demonstrated that the approach can greatly simplify high-throughput proteomics and significantly improve protein identification.
authors.affiliation: Department of Chemistry, Institute of Biomedical Sciences and State Key Lab of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China.
authors.forename: Jinrui
authors.initials: J
authors.lastname: Gan
authors.affiliation: Department of Chemistry, Institute of Biomedical Sciences and State Key Lab of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China.
authors.forename: Yixin
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Chemistry, Institute of Biomedical Sciences and State Key Lab of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China; Laboratoire d'Electrochimie Physique et Analytique, Ecole Polytechnique Fédérale de Lausanne, CH-1950 Sion, Switzerland.
authors.forename: Liang
authors.initials: L
authors.lastname: Qiao
authors.affiliation: Laboratoire d'Electrochimie Physique et Analytique, Ecole Polytechnique Fédérale de Lausanne, CH-1950 Sion, Switzerland.
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Pourhaghighi
authors.affiliation: Department of Chemistry, Institute of Biomedical Sciences and State Key Lab of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China.
authors.forename: Guoquan
authors.initials: G
authors.lastname: Yan
authors.affiliation: Department of Chemistry, Institute of Biomedical Sciences and State Key Lab of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China.
authors.forename: Huizhi
authors.initials: H
authors.lastname: Fan
authors.affiliation: Laboratoire d'Electrochimie Physique et Analytique, Ecole Polytechnique Fédérale de Lausanne, CH-1950 Sion, Switzerland. Electronic address: hubert.girault@epfl.ch.
authors.forename: Hubert H
authors.initials: HH
authors.lastname: Girault
authors.affiliation: Department of Chemistry, Institute of Biomedical Sciences and State Key Lab of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China. Electronic address: bhliu@fudan.edu.cn.
authors.forename: Baohong
authors.initials: B
authors.lastname: Liu
mesh_terms: D006367:HeLa Cells; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D016062:Porosity; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D059748:Proteolysis; D012822:Silicon Dioxide / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
chemical_list: D011506:Proteins; D012822:Silicon Dioxide; D014357:Trypsin
keywords: Macroporous silica; Mass spectrometry; Off-Gel separation; Proteolysis