title: Integrated strong cation-exchange hybrid monolith coupled with capillary zone electrophoresis and simultaneous dynamic pH junction for large-volume proteomic analysis by mass spectrometry.
abstract: A sulfonate-silica hybrid strong cation-exchange (SCX) monolith was synthesized at the proximal end of a capillary zone electrophoresis column and used for on-line solid-phase extraction (SPE) sample preconcentration. Sample was prepared in an acidic buffer and deposited onto the SCX-SPE monolith and eluted using a basic buffer. Electrophoresis was performed in an acidic buffer. This combination of buffers results in formation of a dynamic pH junction, which allows use of relatively large elution buffer volume while maintaining peak efficiency and resolution. All experiments were performed with a 50 µm ID capillary, a 1cm long SCX-SPE monolith, a 60cm long separation capillary, and a electrokinetically pumped nanospray interface. The volume of the capillary is 1.1 µL. By loading 21 µL of a 1×10(-7) M angiotensin II solution, an enrichment factor of 3000 compared to standard electrokinetic injection was achieved on this platform while retaining efficient electrophoretic performance (N=44,000 plates). The loading capacity of the sulfonate SCX hybrid monolith was determined to be ~15 pmol by frontal analysis with 10(-5) M angiotensin II. The system was also applied to the analysis of a 10(-4) mg/mL bovine serum albumin tryptic digest; the protein coverage was 12% and 11 peptides were identified. Finally, by loading 5.5 µL of a 10(-3) mg/mL E. coli digest, 109 proteins and 271 peptides were identified in a 20 min separation; the median separation efficiency generated by these peptides was 25,000 theoretical plates.
authors: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 USA.
authors: Zhenbin
authors: Z
authors: Zhang
authors: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 USA.
authors: Liangliang
authors: L
authors: Sun
authors: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 USA.
authors: Guijie
authors: G
authors: Zhu
authors: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 USA.
authors: Xiaojing
authors: X
authors: Yan
authors: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 USA. Electronic address: ndovichi@nd.edu.
authors: Norman J
authors: NJ
authors: Dovichi
mesh_terms: D000818:Animals; D002411:Cation Exchange Resins / Q000737:chemistry; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D052616:Solid Phase Extraction / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002411:Cation Exchange Resins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide
keywords: Bottom-up proteomics; Capillary zone electrophoresis; Large volume injection; Solid phase extraction; Strong cation exchange