Univerzita PardubiceFakulta chemicko- technologická

Univerzita Pardubice

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Publikace detail

MICROCHIP ISOLATION COMBINED WITH MS-IDENTIFICATION OF PHOSPHOPROTEINS FROM FRANCISELLA TULARENSIS

Autoři: Kupčík Rudolf | Řehulka Pavel | Jankovičová Barbora | Svobodová Zuzana | Klimentová Jana | Hernychová Lenka | Stulík Jiří | Bílková Zuzana

Rok: 2012

Druh publikace: ostatní - přednáška nebo poster

Strana od-do: nestránkováno

Tituly:

Jazyk	Název	Abstrakt	Klíčová slova
eng	MICROCHIP ISOLATION COMBINED WITH MS-IDENTIFICATION OF PHOSPHOPROTEINS FROM FRANCISELLA TULARENSIS	Posttranslational modifications of proteins, such as phosphorylation, play a key role in many biological processes. Due to low abundances phosphoproteins digested into peptides typically require isolation and preconcentration before MS analysis. For these purposes we developed new methodical approach based on phosphopeptides enrichment by magnetic TiO2 particles in microfluidic chip. Enriched phosphopeptides were subsequently fractionated by microcolumn RP-LC using gradient elution and offline application to MALDI plate. For confirmation of phosphorylation MS and MS/MS analysis was followed by on-plate dephosphorylation by alkaline phosphatase. The obtained spectra were compared before and after dephosphorylation, which enhanced the probability of a correct identification. This procedure was applied firstly to tryptic digest of alpha-casein and then to digest of Francisella tularensis subsp. holarctica strain FSC200 (FTH FSC200) lysate. Finally bacterial phosphoproteins were identified using database searching and manual evaluation.	microfluidics; phosphoproteins; Francisella tularensis