title: Two-dimensional liquid chromatography separation of peptides using reversed-phase/weak cation-exchange mixed-mode column in first dimension.
abstract: Selection of a first-dimensional separation method is very important in proteomic studies performed with two-dimensional liquid chromatography (2D-LC), since reversed-phase chromatography (RPLC) is usually chosen as the second-dimensional separation method in most studies. In this paper, we assess the potential use of mixed-mode reversed-phase/weak cation-exchange (RP/WCX) chromatography in 2D-LC proteomic studies. First, a new RP/WCX mixed-mode stationary phase (named C18WCX) was synthesized based on the polar-copolymerized approach, and a C18WCX column with separation efficiency comparative to conventional C18 columns was developed. This new mixed-mode column primarily provides hydrophobic interactions under acidic condition, but can offer hydrophobic and cation-exchange interactions under neutral and weak basic conditions. An off-line 2D-RP/WCX-RPLC system was established using the C18WCX column with a mobile phase of pH 6.5 in the first dimension and a C18 column with a mobile phase of pH 3.0 in the second dimension. The orthogonality of this two-dimensional system, evaluated through the separation of 123 tryptic peptides, was shown to be higher than that of the conventional 2D-RP-RPLC approach. Applying this 2D-LC method to rat brain samples, we identified 1031 proteins and 4397 unique peptides. In addition, this new two-dimensional method improved the identification of basic peptides. Therefore, we propose that this novel 2D-RP/WCX-RPLC system can be used as an alternative approach for the two-dimensional separation of peptides.
authors: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors: Xiaoming
authors: X
authors: Cai
authors: Zhimou
authors: Z
authors: Guo
authors: Xingya
authors: X
authors: Xue
authors: Junyan
authors: J
authors: Xu
authors: Xiuli
authors: X
authors: Zhang
authors: Xinmiao
authors: X
authors: Liang
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D051381:Rats; D012822:Silicon Dioxide
chemical_list: D010446:Peptide Fragments; D020543:Proteome; D012822:Silicon Dioxide