title: 3D-SISPROT: A simple and integrated spintip-based protein digestion and three-dimensional peptide fractionation technology for deep proteome profiling.
abstract: 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 C18membranes for the first time. The SAX plus C18membranes design minimizes the sample loss and ensures high efficient SAX-based digestion. 4275 proteins were identified with 1.4h of MS time when 6μg 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 30μg of HEK 293T cell lysates with 20.4h 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.
authors.affiliation: Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China; Department of Chemistry, Fudan University, Shanghai 200433, China.
authors.forename: Wendong
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China.
authors.forename: Subash
authors.initials: S
authors.lastname: Adhikari
authors.affiliation: Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.affiliation: Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China.
authors.forename: Shusheng
authors.initials: S
authors.lastname: Luo
authors.affiliation: Department of Chemistry, Fudan University, Shanghai 200433, China.
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
authors.affiliation: Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China; Shenzhen Key Laboratory of Cell Microenvironment, Southern University of Science and Technology, Shenzhen 518055, China. Electronic address: tianrj@sustc.edu.cn.
authors.forename: Ruijun
authors.initials: R
authors.lastname: Tian
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D056148:Chromatography, Reverse-Phase; D057809:HEK293 Cells; D006801:Humans; D036103:Nanotechnology; D010455:Peptides / Q000032:analysis* / Q000302:isolation & purification; D011506:Proteins / Q000378:metabolism*; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D011506:Proteins; D020543:Proteome
keywords: Deep proteome profiling; Integrated sample preparation; Liquid chromatography–tandem mass spectrometry; Protein digestion; Three-dimensional separation