Informace o publikaci

Divergent-flow isoelectric focusing for separation and preparative analysis of peptides

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DUŠA Filip KŘENKOVÁ Jana MORAVCOVÁ Dana KAHLE Vladislav ŠLAIS Karel

Rok publikování 2012
Druh Článek v odborném periodiku
Časopis / Zdroj Electrophoresis
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
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Doi http://dx.doi.org/10.1002/elps.201100587
Obor Biochemie
Klíčová slova Autofocusing; Caseinomacropeptide; Isoelectric focusing; Mass spectrometry; Peptides
Popis A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.
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