Online multidimensional separation with biphasic monolithic capillary column for shotgun proteome analysis.
A biphasic monolithic capillary column with 10 cm segment of strong-cation exchange monolith and 65 cm segment of reversed-phase monolith was prepared within a single 100 microm i.d. capillary. Separation performance of this column was evaluated by a five-cycle online multidimensional separation of 10 microg tryptic digest of yeast proteins using nanoflow liquid chromatography coupled with tandem mass spectrometry, and it took 12 h for whole separation under the operating pressure only approximately 900 psi. Totally, 780 distinct proteins were positively identified through assignment of 2953 unique peptides at false-positive rate less than 1%. The good separation performance of this biphasic column was largely attributed to the good orthogonality of the strong-cation exchange monolith and reversed-phase monolith for multidimensional separation.
Wang
Fangjun
F
National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Dong
Jing
J
Ye
Mingliang
M
Jiang
Xiaogang
X
Wu
Ren'an
R
Zou
Hanfa
H
D053801:Capillary Electrochromatography / Q000295:instrumentation* / Q000592:standards; D002411:Cation Exchange Resins; D002853:Chromatography, Liquid; D005656:Fungal Proteins / Q000302:isolation & purification*; D009862:Online Systems; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation*; D053719:Tandem Mass Spectrometry
D002411:Cation Exchange Resins; D005656:Fungal Proteins; D020543:Proteome