title: A tunable isoelectric focusing via moving reaction boundary for two-dimensional gel electrophoresis and proteomics.
abstract: Routine native immobilized pH gradient isoelectric focusing (IPG-IEF) and two-dimensional gel electrophoresis (2DE) are still suffering from unfortunate reproducibility, poor resolution (caused by protein precipitation) and instability in characterization of intact protein isoforms and posttranslational modifications. Based on the concept of moving reaction boundary (MRB), we firstly proposed a tunable non-IPG-IEF system to address these issues. By choosing proper pairs of catholyte and anolyte, we could achieve desired cathodic and anodic migrating pH gradients in non-IPG-IEF system, effectively eliminating protein precipitation and uncertainty of quantitation existing in routine IEF and 2DE, and enhancing the resolution and sensitivity of IEF. Then, an adjustable 2DE system was developed by combining non-IPG-IEF with polyacrylamide gel electrophoresis (PAGE). The improved 2DE was evaluated by testing model proteins and colon cancer cell lysates. The experiments revealed that (i) a tunable pH gradient could be designed via MRB; (ii) up to 1.65 fold improvement of resolution was achieved via non-IPG-IEF; (iii) the sensitivity of developed techniques was increased up to 2.7 folds; and (iv) up to about 16.4% more protein spots could be observed via the adjustable 2DE as compared with routine one. The developed techniques might contribute to complex proteome research, especially for screening of biological marker and analysis of extreme acidic/alkaline proteins.
authors.affiliation: Laboratory of Bioseparation and Analytical Biochemistry, State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Chen-Gang
authors.initials: CG
authors.lastname: Guo
authors.affiliation: Laboratory of Bioseparation and Analytical Biochemistry, State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Shang
authors.affiliation: Laboratory of Bioseparation and Analytical Biochemistry, State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Yan
authors.affiliation: Laboratory of Bioseparation and Analytical Biochemistry, State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Si
authors.initials: S
authors.lastname: Li
authors.affiliation: Laboratory of Bioseparation and Analytical Biochemistry, State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Guo-Qing
authors.initials: GQ
authors.lastname: Li
authors.affiliation: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Rong-Zhong
authors.initials: RZ
authors.lastname: Liu
authors.affiliation: Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Qing
authors.affiliation: Laboratory of Bioseparation and Analytical Biochemistry, State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Liu-Yin
authors.initials: LY
authors.lastname: Fan
authors.affiliation: Laboratory of Bioseparation and Analytical Biochemistry, State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: huaxiao@sjtu.edu.cn.
authors.forename: Hua
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Laboratory of Bioseparation and Analytical Biochemistry, State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: cxcao@sjtu.edu.cn.
authors.forename: Cheng-Xi
authors.initials: CX
authors.lastname: Cao
mesh_terms: D011232:Chemical Precipitation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D045325:HCT116 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D040901:Proteomics / Q000379:methods*
keywords: Isoelectric focusing; Moving reaction boundary; Proteomics; Tunable pH gradient; Two-dimensional gel electrophoresis