title: Peptide mapping using capillary electrophoresis offline coupled to matrix-assisted laser desorption ionization time of flight mass spectrometry.
abstract: This article reports the results of a study carried out to evaluate the offline hyphenation of capillary zone electrophoresis with matrix-assisted lased desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for the analysis of low-abundant complex samples, represented by the tryptic phosphorylated peptides of phosphoproteins, such as α-casein, β-casein, and fetuin. The proposed method employs a latex-coated capillary and consists in the online preconcentration of the tryptic peptides by a pH-mediated stacking method, their separation by capillary zone electrophoresis, and subsequent deposition of the separated analytes onto a MALDI target for their MS analysis. The online preconcentration method allows loading a large sample volume (∼150 nL), which is introduced into the capillary after the hydrodynamic injection of a short plug of 1.0 M ammonium hydroxide solution and is sandwiched between two plugs of the acidic background electrolyte solution (BGE) filling the capillary. The sample spotting of the separated analytes onto the MALDI target is performed either during or postseparation using an automatic spotting device connected to the exit of the separation capillary. The proposed method allows the separation and identification of multiphosphorylated peptides from other peptides and enables their identification at femtomole level with improved efficiency compared with LC approaches hyphenated to MS.
authors.affiliation: Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Bachmann
authors.forename: Rania
authors.initials: R
authors.lastname: Bakry
authors.forename: Christian W
authors.initials: CW
authors.lastname: Huck
authors.forename: Fabio
authors.initials: F
authors.lastname: Polato
authors.forename: Danilo
authors.initials: D
authors.lastname: Corradini
authors.forename: Günther K
authors.initials: GK
authors.lastname: Bonn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000032:analysis / Q000737:chemistry; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D060748:Fetuins / Q000032:analysis / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D002364:Caseins; D060748:Fetuins; D010446:Peptide Fragments; D010750:Phosphoproteins