Integrated protein analysis platform based on column switch recycling size exclusion chromatography, microenzymatic reactor and microRPLC-ESI-MS/MS.
An integrated platform with the combination of proteins and peptides separation was established via the unit of on-line proteins digestion, by which proteins were in sequence separated by column switch recycling size exclusion chromatography (csrSEC), on-line digested by an immobilized trypsin microreactor, trapped and desalted by two parallel C8 precolumns, separated by microRPLC with the linear gradient of organic modifier concentration, and identified by ESI-MS/MS. A 6-protein mixture, with Mr ranging from 10 kDa to 80 kDa, was used to evaluate the performance of the integrated platform, and all proteins were identified with sequence coverage over 5.67%. Our experimental results demonstrate that such an integrated platform is of advantages such as good time compatibility, high peak capacity, and facile automation, which might be a promising approach for proteome study.
Yuan
Huiming
H
Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R.&A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China.
Zhou
Yuan
Y
Zhang
Lihua
L
Liang
Zhen
Z
Zhang
Yukui
Y
D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods*; D007782:Lactoglobulins / Q000032:analysis; D009211:Myoglobin / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012260:Ribonucleases / Q000032:analysis; D012710:Serum Albumin, Bovine / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014168:Transferrin / Q000032:analysis; D014357:Trypsin / Q000378:metabolism*
D007782:Lactoglobulins; D009211:Myoglobin; D011506:Proteins; D014168:Transferrin; D012710:Serum Albumin, Bovine; D012260:Ribonucleases; D014357:Trypsin; D002256:Carbonic Anhydrases