Lipid transfer proteins of barley
J. Žídková1, M. Matejková2, I. Petry-Podgórska1, L. Žídek2, K. Sikorová2, M. Nálezková2, J.Chmelík1, V. Sklenář2
1Institute of Analytical Chemistry of the Academy of Sciences of the Czech Republic, Veveří 97, 60200 Brno, Czech Republic
2Masaryk University, Faculty of Science, NCBR, Kotlářská 2, 61137 Brno, Czech Republic
Nonspecific lipid transfer protein 1 (LTP1) was isolated from barley seeds and malt. MALDI-TOF mass spectrometry in combination with in-solution tryptic digestion revealed that Asp 7 of LTP1 was modified by cis-14-hydroxy-10,13-dioxo-7-heptadecenoic ester in both samples. However, no modification was observed when the protein was digested by the standard proteomic protocol using SDS-PAGE. It was found that the ester was hydrolyzed by the harsh conditions of protein denaturation. In addition to the lipid modification, glycations of the protein were identified in the intact LTP1 samples.
A large-scale purification protocol was optimized for isolation of LTP1 from barley flour, yielding approximately 20 mg of pure LTP1. The sample was studied by proton NMR spectroscopy at 600 MHz. In addition to NOESY and TOCSY spectra recorded at 310 K, a series of NOESY spectra was measured up to 350 K. Inspection of the spectra showed that the protein was not denatured in the temperature range tested. The obtained spectra differed from the results reported for the non-modified LTP1 in literature. Assignment of the resonance frequencies is in progress.
Nonspecific lipid transfer protein 2 (LTP2) was identified in barley by MALDI-TOF/TOF MS-MS. No modification was observed. Therefore, a synthetic gene was obtained, in order to clone it into a overexpression vector. This approach should provide an access to large quantities of isotope-labeled LTP1 for NMR studies.