Rapid and efficient proteolysis through laser-assisted immobilized enzyme reactors.
In this report, laser radiation (808nm) for the first time was employed to enhance the efficiency of proteolysis through immobilized enzyme reactor (IMER). IMER based monolithic support was prepared in the fused-silica capillary via a simple two-step procedure including acryloylation on trypsin surface and in situ aqueous polymerization/immobilization. The feasibility and high efficiency of the laser-assisted IMER were demonstrated by the digestion of bovine serum albumin (BSA), cytochrome c (Cyt-c) and β-casein. The digestion process was achieved in 60s. The peptides were identified by MALDI-TOF-MS, yielding the sequence coverage of 33% for BSA, 73% for Cyt-c and 22% for β-casein. The comparisons between the in-solution digestion and on IMER reaction with/without laser assistance were made. To further confirm its efficiency in proteome analysis, the laser-assisted IMER was also applied to the analysis of one fraction of human serum sample through two-dimensional (2-D) separation of strong anion exchange/reversed-phase liquid chromatography (SAX/RPLC). After a database search, 49 unique peptides corresponding to 5 proteins were identified. The results showed that the laser-assisted IMER provides a promising platform for the high-throughput protein identification.
Department of Chemistry and Institute of Biomedical Sciences, Fudan University, Shanghai 200433, China.
Peng
P
Zhang
Mingxia
M
Gao
Shaochun
S
Zhu
Jie
J
Lei
Xiangmin
X
Zhang
D000818:Animals; D019149:Bioreactors; D001798:Blood Proteins / Q000032:analysis / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D056148:Chromatography, Reverse-Phase; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007834:Lasers; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D001798:Blood Proteins; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin