Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column.
An approach was developed to automate sample introduction for nanoflow LC-MS/MS (microLC-MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 microm id x 2 cm SCX trap column and a 75 microm id x 12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current microLC-MS/MS system. This system represented a promising platform for routine proteome analysis.
Jiang
Xiaogang
X
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.|School of Medicine, Suzhou University, Suzhou, Jiangsu, China.
Feng
Shun
S
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
Tian
Ruijun
R
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
Han
Guanghui
G
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
Jiang
Xinning
X
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
Ye
Mingliang
M
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
Zou
Hanfa
H
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
D000595:Amino Acid Sequence; D001331:Automation; D002412:Cations / Q000737:chemistry; D045744:Cell Line, Tumor; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D005656:Fungal Proteins / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002412:Cations; D005656:Fungal Proteins; D020543:Proteome; D012710:Serum Albumin, Bovine