title: Fully automatable two-dimensional reversed-phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics.
abstract: Herein, we describe the development of a fully automatable technology that features online coupling of high-pH RP separation with conventional low-pH RP separation in a two-dimensional capillary liquid chromatography (2-D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first-dimension, high-pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second-dimension, low-pH RP separation, each under identical gradient-elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non-redundant proteins, of which 50% were observed in all three replicates. A comparison of RP-RP 2-D LC and strong cation exchange-RP 2-D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong, P R China.
authors.forename: S O
authors.initials: SO
authors.lastname: Siu
authors.forename: Maggie P Y
authors.initials: MP
authors.lastname: Lam
authors.forename: Edward
authors.initials: E
authors.lastname: Lau
authors.forename: Ricky P W
authors.initials: RP
authors.lastname: Kong
authors.forename: Simon M Y
authors.initials: SM
authors.lastname: Lee
authors.forename: Ivan K
authors.initials: IK
authors.lastname: Chu
mesh_terms: D000818:Animals; D057205:Automation, Laboratory; D002460:Cell Line; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004625:Embryo, Nonmammalian; D004867:Equipment Design; D005347:Fibroblasts; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D029961:Zebrafish Proteins / Q000032:analysis / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D011506:Proteins; D029961:Zebrafish Proteins