title: Improving the comprehensiveness and sensitivity of sheathless capillary electrophoresis-tandem mass spectrometry for proteomic analysis.
abstract: We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) procedure which employs a high sensitivity porous electrospray ionization (ESI) sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency, we primarily employed electrokinetic methods for elution and separation after loading the sample by application of pressure. Conditions were developed for optimum simultaneous electrokinetic elution and sample stacking using a tryptic digest of 16 proteins to maximize peptide identifications and minimize band broadening. We performed comparative proteomic analysis of a dilution series using CE and nanoflow liquid chromatography (nLC). We found complementary peptide and protein identifications with larger quantities (100 ng) of a Pyrococcus furiosus tryptic digest, but with mass-limited amounts (5 ng) CE was 3 times more effective at identifying proteins. We attribute these gains in sensitivity to lower noise levels with the porous CE sprayer, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides when compared directly to nLC. From comparative analysis of SPME-tITP-CE with direct injection CE, the SPME-tITP process improved comprehensiveness and sensitivity.
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Yueju
authors.initials: Y
authors.lastname: Wang
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Fonslow
authors.forename: Catherine C L
authors.initials: CC
authors.lastname: Wong
authors.forename: Aleksey
authors.initials: A
authors.lastname: Nakorchevsky
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
mesh_terms: D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis / Q000302:isolation & purification; D057625:Isotachophoresis / Q000379:methods; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D020027:Pyrococcus furiosus / Q000737:chemistry; D012680:Sensitivity and Specificity; D052617:Solid Phase Microextraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D019843:Archaeal Proteins; D029968:Escherichia coli Proteins; D011506:Proteins