10 research outputs found
Complete resonance assignment of a natural abundance solid peptide by through-bond heteronuclear correlation solid-state NMR
The assignment of the NMR spectra of natural abundance medium-sized solid-state organic molecules still represents a challenging problem. In this paper, we show that a complete assignment of all the carbon-13, nitrogen-15, and proton NMR lines of a natural abundance solid peptide can be performed by combining various one-bond and multiple-bond correlation techniques for rotating solids. The assignment of the MAS spectra is shown to be unambiguous and relatively straightforward
Proteomic analysis of β-1,3-glucanase in grape berry tissues
Grape berries are considered recalcitrant
materials in proteomic analysis, because berry tissues
contain large amounts of secondary metabolites, especially
phenolic compounds, which severely interfere with protein
extraction and electrophoresis separation. We report hereby
a PVPP/TCA-based protein extraction protocol for grape
berries. Phenolic compounds in berry extracts were
removed with repeated PVPP cleanups, and proteins were
recovered with TCA precipitation. Protein resolution in
2-D gels was gradually improved with the increase of
PVPP cleanup steps. By the protocol, about 760 protein
spots of berry tissues were clearly resolved in 2-D gels with
CBB staining. This protocol was also used to analyze
b-1,3-glucanase (EC 3.2.1.39) in berry tissues. An antisynthetic
peptide antibody was prepared against 15 amino
acid sequence residing on the surface of b-1,3-glucanase
molecule. It detected two major spots in 2-D blots of berry
extracts. The spots were identified by MALDI-TOF analysis
as b-1,3-glucanase. The present study validates that
b-1,3-glucanase is present in higher abundance in berry
skins than in pulps, and in red berries than in white berries.
Therefore, b-1,3-glucanase displays a tissue-specific
expression. The preferential accumulation of b-1,3-glucanase
in skins may be relevant to berry ripening