title: Third-generation electrokinetically pumped sheath-flow nanospray interface with improved stability and sensitivity for automated capillary zone electrophoresis-mass spectrometry analysis of complex proteome digests.
abstract: We have reported a set of electrokinetically pumped sheath flow nanoelectrospray interfaces to couple capillary zone electrophoresis with mass spectrometry. A separation capillary is threaded through a cross into a glass emitter. A side arm provides fluidic contact with a sheath buffer reservoir that is connected to a power supply. The potential applied to the sheath buffer drives electro-osmosis in the emitter to pump the sheath fluid at nanoliter per minute rates. Our first-generation interface placed a flat-tipped capillary in the emitter. Sensitivity was inversely related to orifice size and to the distance from the capillary tip to the emitter orifice. A second-generation interface used a capillary with an etched tip that allowed the capillary exit to approach within a few hundred micrometers of the emitter orifice, resulting in a significant increase in sensitivity. In both the first- and second-generation interfaces, the emitter diameter was typically 8 μm; these narrow orifices were susceptible to plugging and tended to have limited lifetime. We now report a third-generation interface that employs a larger diameter emitter orifice with very short distance between the capillary tip and the emitter orifice. This modified interface is much more robust and produces much longer lifetime than our previous designs with no loss in sensitivity. We evaluated the third-generation interface for a 5000 min (127 runs, 3.5 days) repetitive analysis of bovine serum albumin digest using an uncoated capillary. We observed a 10% relative standard deviation in peak area, an average of 160,000 theoretical plates, and very low carry-over (much less than 1%). We employed a linear-polyacrylamide (LPA)-coated capillary for single-shot, bottom-up proteomic analysis of 300 ng of Xenopus laevis fertilized egg proteome digest and identified 1249 protein groups and 4038 peptides in a 110 min separation using an LTQ-Orbitrap Velos mass spectrometer; peak capacity was ∼330. The proteome data set using this third-generation interface-based CZE-MS/MS is similar in size to that generated using a commercial ultraperformance liquid chromatographic analysis of the same sample with the same mass spectrometer and similar analysis time.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Guijie
authors.initials: G
authors.lastname: Zhu
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Zhenbin
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Si
authors.initials: S
authors.lastname: Mou
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D000818:Animals; D057205:Automation, Laboratory; D002417:Cattle; D045424:Complex Mixtures / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D010455:Peptides / Q000302:isolation & purification*; D059748:Proteolysis; D020543:Proteome / Q000302:isolation & purification*; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000295:instrumentation*; D014982:Xenopus laevis; D015053:Zygote / Q000737:chemistry
chemical_list: D045424:Complex Mixtures; D010455:Peptides; D020543:Proteome; D012710:Serum Albumin, Bovine
keywords: Xenopus laevis; bottom-up proteomics; capillary electrophoresis; developmental proteomics