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A miniaturized multichamber solution isoelectric focusing device for separation of protein digests
Author(s) -
Tan Aimin,
Pashkova Anna,
Zang Li,
Foret Frantisek,
Karger Barry L.
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200210)23:20<3599::aid-elps3599>3.0.co;2-m
Subject(s) - chromatography , chemistry , isoelectric focusing , mass spectrometry , electrophoresis , analytical chemistry (journal) , peptide , peptide mass fingerprinting , isoelectric point , capillary electrophoresis , proteomics , biochemistry , gene , enzyme
A miniaturized multichamber device was constructed for solution isoelectric focusing (IEF) separation of complex peptide mixtures. The system, based on immobilized pH gels, consisted of 96 minichambers (∼75 νL each) arranged in eight rows. Neighboring chambers in a given row were separated by short glass tubes (4 mm inner diameter, 3 mm long), within which Immobiline gels of specific pH values were polymerized. During focusing, the device was sandwiched between two supporting blocks incorporating the reservoirs for anolyte and catholyte. In principle, multiple samples could be simultaneously fractionated, each separated into 12 fractions of various p I ranges. A variety of standard peptide mixtures and tryptic digests of proteins were separated by IEF using this device, and the fractions were characterized by mass spectrometry. For a codigested nine‐protein mixture, both the total number of peptides identified and the average sequence coverage were similar to the results of ion‐exchange chromatography (IEC), according to matrix assisted laser/desorption/ionization – time of flight (MALDI‐TOF) data. The IEF separation provided concentrated and desalted fractions, suitable for an additional separation liquid chromatography, capillary electrophoresis (LC, CE) or mass spectrometry (MS) detection without additional sample cleanup. High loading capacity was achieved for the miniaturized multichamber IEF device. Importantly, a linear correlation was found between the experimentally determined and calculated p I values of peptides.