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Multidimensional high performance liquid chromatography – capillary electrophoresis separation of a protein digest: An update
Author(s) -
Issaq Haleem J.,
Chan King C.,
Liu ChangSheng,
Li Qingbo
Publication year - 2001
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()22:6<1133::aid-elps1133>3.0.co;2-s
Subject(s) - chromatography , electropherogram , capillary electrophoresis , chemistry , electrophoresis , high performance liquid chromatography , capillary action , micellar electrokinetic chromatography , myoglobin , analytical chemistry (journal) , capillary electrochromatography , materials science , biochemistry , composite material
Abstract The trypsin digest of a mixture of two proteins, namely cytochrome c and myoglobin, was first separated in the first dimension by high‐performance liquid chromatography (HPLC). Fractions from the HPLC were collected every 30s with the aid of a fraction collector into a 96‐well microtiter plate. After concentration, all the collected fractions were analyzed simultaneaosly in the second dimension by a 96‐array capillary electrophoresis system. The labeled peptides were detected by laser‐induced fluorescence. An internal standard, allura red, was added to all the fractions, prior to capillary electrophoretic analysis. The internal standard serves two functions, migration time correction and signal intensity correction. The data are presented in two different formats, as an electropherogram of all the fractions and in a two‐dimensional (2‐D) format. The 2‐D plot of the data shows the density of each spot, which corresponds to the concentration of the migrating peptides. The total experimental time for the HPLC and capillary electrophoretic analyses ist less than 1 h, which ist much faster than using 2‐D slab‐gel electrophoresis or single‐capillary capillary electrophoresis.