title: In-capillary enrichment, proteolysis and separation using capillary electrophoresis with discontinuous buffers: application on proteins with moderately acidic and basic isoelectric points.
abstract: Advances in mass spectrometry and capillary-format separation continue to improve the sensitivity of protein analysis. Of equal importance is the miniaturization of sample pretreatment such as enrichment and proteolysis. In a previous report (Nesbitt et al., Electrophoresis, 2008, 29, 466-474), nanoliter-volume protein enrichment, tryptic digestion, and partial separation was demonstrated in capillary electrophoresis followed by MALDI mass spectral analysis. A discontinuous buffer system, consisting of ammonium (pH 10) and acetate (pH 4), was used to create a pH junction inside the capillary, trapping a protein with a neutral isoelectric point, myoglobin (pI 7.2). Moreover, co-enrichment of myoglobin with trypsin led to an in-capillary digestion. In this paper, the ability of this discontinuous buffer system to perform similar in-capillary sample pretreatment on proteins with moderately acidic and basic pI was studied and reported. Lentil lectin (pI 8.6) and a multi-phosphorylated protein, beta-casein (pI 5.1), were selected as model proteins. In addition to the previously shown tryptic digestion, proteolysis with endoproteinase Asp-N was also performed. Digestion of these acidic and basic pI proteins produced a few peptides with extreme pI values lying outside the trapping range of the discontinuous buffer. An alteration in the peptide trapping procedure was made to accommodate these analytes. Offline MALDI mass spectral analysis confirmed the presence of the expected peptides. The presented miniaturized sample pretreatment methodology was proven to be applicable on proteins with a moderately wide range of pI. Flexibility in the choice of protease was also evident.
authors.affiliation: Department of Chemistry, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Chandra A
authors.initials: CA
authors.lastname: Nesbitt
authors.forename: Ken K-C
authors.initials: KK
authors.lastname: Yeung
mesh_terms: D000818:Animals; D002021:Buffers; D002364:Caseins / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D037102:Lectins / Q000032:analysis; D008666:Metalloendopeptidases; D008904:Miniaturization; D011506:Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin
chemical_list: D002021:Buffers; D002364:Caseins; D037102:Lectins; D011506:Proteins; D014357:Trypsin; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N