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

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DUŠA Filip KŘENKOVÁ Jana MORAVCOVÁ Dana KAHLE Vladislav ŠLAIS Karel

Rok publikování 2011
Druh Konferenční abstrakty
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
Popis 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.