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On‐line immunoaffinity solid‐phase extraction capillary electrophoresis mass spectrometry for the analysis of large biomolecules: A preliminary report
Author(s) -
MedinaCasanellas Silvia,
Benavente Fernando,
Giménez Estela,
Barbosa José,
SanzNebot Victoria
Publication year - 2014
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201400119
Subject(s) - chromatography , capillary electrophoresis , chemistry , solid phase extraction , mass spectrometry , biomolecule , detection limit , repeatability , sample preparation , extraction (chemistry) , capillary electrophoresis–mass spectrometry , analytical chemistry (journal) , electrospray ionization , biochemistry
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.