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Advanced online mass spectrometry detection of proteins separated by capillary isoelectric focusing after sequential injection
Author(s) -
Páger Csilla,
Biherczová Nikoleta,
Ligetvári Roland,
Berkics Balázs Viktor,
Pongrácz Tamás,
Sándor Viktor,
Bufa Anita,
Poór Viktória,
Vojs Staňová Andrea,
Kilár Ferenc
Publication year - 2017
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201700695
Subject(s) - chemistry , isoelectric focusing , chromatography , mass spectrometry , analyte , capillary action , immobilized ph gradient , capillary electrophoresis , capillary electrophoresis–mass spectrometry , analytical chemistry (journal) , polyvinyl alcohol , isoelectric point , electrospray ionization , biochemistry , materials science , organic chemistry , composite material , enzyme
Capillary isoelectric focusing hyphenated with mass spectrometry detection, following the sequential injection of the carrier ampholytes and the sample zone, is highly efficient for the characterization of proteins. The main advantage of the sequential injection protocol is that ampholytes, with pH ranges, which are not supposed to cover the isoelectric points of the sample components, can be used for separation. The method then allows online mass spectrometry detection of separated analytes either in the absence (substances that have left the pH gradient) or in the presence of low‐level ampholytes (substances that are migrating within the pH gradient). The appearance of the substances within, or outside the pH gradient depends on, e.g., the composition of the ampholytes (broad or narrow pH range) or on the composition of electrolyte solutions. The experiments performed in coated capillaries (with polyvinyl alcohol or with polyacrylamide) show that the amount and the injection length of the ampholytes influence the length of the pH gradient formed in the capillary.