Pre-Separation of High Volume Biological Samplesusing Divergent Flow Isoelectric Focusing

DUŠA, Filip, Jana KŘENKOVÁ, Dana MORAVCOVÁ, Vladislav KAHLE and Karel ŠLAIS. Pre-Separation of High Volume Biological Samplesusing Divergent Flow Isoelectric Focusing. In HPLC 2011 Budapest Symposium. 2011. ISBN 978-963-89335-0-8.

Basic information

Original name

Pre-Separation of High Volume Biological Samplesusing Divergent Flow Isoelectric Focusing

Authors

DUŠA, Filip, Jana KŘENKOVÁ, Dana MORAVCOVÁ, Vladislav KAHLE and Karel ŠLAIS

Edition

HPLC 2011 Budapest Symposium, 2011

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Other information

Language

English

Type of outcome

Konferenční abstrakt

Field of Study

10600 1.6 Biological sciences

Country of publisher

Czech Republic

Confidentiality degree

není předmětem státního či obchodního tajemství

Organization unit

Faculty of Science

ISBN

978-963-89335-0-8

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Abstract

V originále

Biological samples are very complex mixtures differing in species of substances as well as in their concentration range. Due to difficulties in their direct analyses, efficient pre-separation steps are very often required in order to simplify the complexity of samples and enhance detection and identification of individual components. We present an application of newly developed divergent flow isoelectric focusing (DFIEF) instrument for fractionation of protein digests and its comparison with currently used instrumentation. Model protein mixture consisting of bovine serum albumin, myoglobin, and cytochrome c was digested with trypsin and resulting peptide fragments were preseparated by DF-IEF in autofocusing mode in which the sample was desalted and separated without addition of carriers forming pH gradient. The obtained fractions were analyzed using u-LC coupled with UV/Vis or ESI/MS detector. Obtained chromatograms and MS spectra show well separated and focused peptides in IEF fractions covering their isoelectric points. Only some peptides were focused poorly due to the lack of charged residues in their sequences. The DF-IEF instrument proved very good pre-separation efficiency and ability to process a high volume throughput of low concentrated samples.