title: On-plate digestion of proteins using novel trypsin-immobilized magnetic nanospheres for MALDI-TOF-MS analysis.
abstract: In this study, a novel method of on-plate digestion using trypsin-immobilized magnetic nanospheres was developed followed by MALDI-TOF-MS for rapid and effective analysis and identification of proteins. We utilized a facile one-pot method for the direct preparation of amine-functionalized magnetic nanospheres with highly magnetic properties and the amino groups on the outer surface. Through the reaction of the aldehyde groups with amine groups, trypsin was simply and stably immobilized onto the magnetic nanospheres. The obtained trypsin-linked magnetic nanospheres were then applied for on-plate digestion of sample proteins (myoglobin and Cytochrome c). Moreover, after digestion, the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS. The effects of the temperature and incubation time on the digestion efficiency were characterized. Within only 5 min, proteins could be efficiently digested with the peptide sequence coverage higher than or equal to that of the traditional in-solution digestion for 12 h. Furthermore, RPLC fractions of rat liver extract were also successfully processed using this novel method. These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins.
authors.affiliation: Department of Chemistry, Institute of Biomedical Sciences, Fudan University, Shanghai, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Bo
authors.initials: B
authors.lastname: Yan
authors.forename: Chunhui
authors.initials: C
authors.lastname: Deng
authors.forename: Jia
authors.initials: J
authors.lastname: Tang
authors.forename: Junyan
authors.initials: J
authors.lastname: Liu
authors.forename: Xiangmin
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004800:Enzymes, Immobilized; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D008280:Magnetics; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D054118:Nanospheres; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D014357:Trypsin
chemical_list: D004800:Enzymes, Immobilized; D009211:Myoglobin; D011506:Proteins; D014108:Tosylphenylalanyl Chloromethyl Ketone; D014357:Trypsin