title: On-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry for the analysis of large biomolecules: a preliminary report.
abstract: The analysis of large biomolecules by on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) remains unexplored because of the complex issues that need to be addressed. In this preliminary study, we used the human glycoprotein transferrin (Tf) as a model of a large biomolecule. First, we established by CE-UV a novel method compatible with IA-SPE-CE-MS, based on the use of a fused silica capillary coated with an anionic derivative of polyacrylamide (UltraTrol(TM) Dynamic Pre-Coat High Normal, HN) to prevent protein adsorption. The methodology allowed the detection of the most abundant Tf sialoforms. Repeatability studies demonstrated high stability of the coated capillaries, which was required for on-line immunoextraction and MS detection. IA-SPE-CE-UV and IA-SPE-CE-MS methods were optimized for the analysis of Tf standards and human serum samples using a laboratory-made IA sorbent. Three peaks corresponding to Tf were detected with UV detection when on-line immunoextraction was applied to the standards. The use of MS detection, however, reduced the resolution of the electrophoretic separation. Finally, we demonstrated that it was possible to detect Tf in human serum samples, after off-line serum sample de-salting by centrifugal filtration.
authors.affiliation: Department of Analytical Chemistry, University of Barcelona, Barcelona, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Medina-Casanellas
authors.forename: Fernando
authors.initials: F
authors.lastname: Benavente
authors.forename: Estela
authors.initials: E
authors.lastname: Giménez
authors.forename: José
authors.initials: J
authors.lastname: Barbosa
authors.forename: Victoria
authors.initials: V
authors.lastname: Sanz-Nebot
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D013058:Mass Spectrometry; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D014168:Transferrin / Q000032:analysis / Q000302:isolation & purification
chemical_list: D014168:Transferrin
keywords: Capillary electrophoresis; Immunoaffinity; Mass spectrometry; On-line preconcentration; Transferrin