3D-SISPROT: A simple and integrated spintip-based protein digestion and three-dimensional peptide fractionation technology for deep proteome profiling

Chen, Wendong1,3; Adhikari, Subash1; Chen, Lan1; Lin, Lin1; Li, Hua1; Luo, Shusheng1; Yang, Pengyuan3; Tian, Ruijun1,2

2017-05-19

10.1016/j.chroma.2017.01.033

JOURNAL OF CHROMATOGRAPHY A   影响因子和分区

0021-9673

1873-3778

Multidimensional peptide fractionation strategies have been approved as the efficient approaches to significantly improve the depth of proteome coverage. In this study, a simple and integrated spintip-based protein digestion and three-dimensional peptide fractionation technology (3D-SISPROT) was developed for the deep proteome profiling from low microgram of proteins as starting materials. All the sample preparation steps, including protein digestion, strong anion exchange (SAX)-based fractionation, and high-pH reversed phase (RP) fractionation were integrated into one pipette tip packed with SAX and C-18 membranes for the first time. The SAX plus C-18 membranes design minimizes the sample loss and ensures high efficient SAX-based digestion. 4275 proteins were identified with 1.4h of MS time when 6 mu g cell lysates was processed. More importantly, the SAX-based digestion procedure did not influence the SAX-based peptide fractionation efficiency which was done in the same SAX membrane. The 3D-SISPROT was exemplified by the analysis of 30 mu g of HEK 293T cell lysates with 20.4 h of MS time, which resulted in the identification of 8222 proteins including 3215 annotated membrane proteins. Gene Ontology annotations indicated that the 3D-SISPROT was unbiased for the proteins from major cellular components. Taking advantages of the efficient SAX-based and high-pH RP-based fractionation strategies, we expect that the 3D-SISPROT can be applied for the deep proteome profiling with limited starting material. (C) 2017 Elsevier B.V. All rights reserved.

Three-dimensional separation Protein digestion Deep proteome profiling Integrated sample preparation Liquid chromatography-tandem mass spectrometry

SCI ; EI

英语

第一 ; 通讯

Shenzhen Innovation of Science and Technology Commission[JCYJ20150901153557178] ; Shenzhen Innovation of Science and Technology Commission[JSGG20160301103415523]

Biochemistry & Molecular Biology ; Chemistry

Biochemical Research Methods ; Chemistry, Analytical

WOS:000401043000024

ELSEVIER SCIENCE BV

20170403279383

Biological membranes ; Liquid chromatography ; Mass spectrometry

Biological Materials and Tissue Engineering:461.2 ; Biology:461.9 ; Chemistry:801

CHEMISTRY

Web of Science

1.Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China
2.Southern Univ Sci & Technol, Shenzhen Key Lab Cell Microenvironm, Shenzhen 518055, Peoples R China
3.Fudan Univ, Dept Chem, Shanghai 200433, Peoples R China

Chen, Wendong,Adhikari, Subash,Chen, Lan,et al. 3D-SISPROT: A simple and integrated spintip-based protein digestion and three-dimensional peptide fractionation technology for deep proteome profiling[J]. JOURNAL OF CHROMATOGRAPHY A,2017,1498:207-214.

Chen, Wendong.,Adhikari, Subash.,Chen, Lan.,Lin, Lin.,Li, Hua.,...&Tian, Ruijun.(2017).3D-SISPROT: A simple and integrated spintip-based protein digestion and three-dimensional peptide fractionation technology for deep proteome profiling.JOURNAL OF CHROMATOGRAPHY A,1498,207-214.

Chen, Wendong,et al."3D-SISPROT: A simple and integrated spintip-based protein digestion and three-dimensional peptide fractionation technology for deep proteome profiling".JOURNAL OF CHROMATOGRAPHY A 1498(2017):207-214.