University of PardubiceFaculty of Chemical Technology

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

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

Year: 2012

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
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