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Title: Identification of thermolabile pectin methylesterases isolated from sweet orange fruit by mass spectrometry peptide profiles

item SAVARY, BRETT - Arkansas State University
item VASU, PRASANNA - Arkansas State University
item NUNEZ, ALBERTO - Arkansas State University
item Cameron, Randall - Randy

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/20/2010
Publication Date: 11/5/2010
Citation: Savary, B.J., Vasu, P., Nunez, A., Cameron, R.G. 2010. Identification of thermolabile pectin methylesterases from sweet orange fruit by peptide mass fingerprinting. Journal of Agricultural and Food Chemistry. 58:12462-12468.

Interpretive Summary: We demonstrated how biochemical and molecular foundations developed previously for Citrus pectin methylesterases (PMEs) can be combined with current proteomics tools (mass spectrometry, bioinformatics, and sequence databases) to define diagnostic peptides and use them to obtain highly specific structural identification of PMEs. These tools are now commonly available to laboratories with minimal biochemical capabilities (chromatography and electrophoresis) to generate samples from SDS-PAGE gels that can be submitted for MALDI-TOF MS analysis, which is now widely available at service and core facilities. This will enable a better understanding of functional properties of PMEs in their action on pectin substrates.

Technical Abstract: The multiple forms of the enzyme pectin methylesterase (PME) present in citrus fruit tissues vary in activity towards juice cloud-associated pectin substrates and subsequently their impact on juice cloud stability and product quality. Proteins responsible for PME activities are rarely isolated and identified by their structural properties or correlated to specific PME genes. In this report we applied matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) to match peptide mass fingerprints generated from the two major thermolabile (TL-) forms of PME isolated from Valencia orange [Citrus sinensis (L.) Osbeck] fruit tissue to theoretical peptide sets generated from existing PME nucleic acid sequences. Affinity-purified PME preparations were used for intact protein analysis and for separation by SDS-PAGE prior to trypsin digestion and MS analysis of tryptic peptides. This approach is direct and highly sensitive for detecting both conserved common and isoenzyme-specific peptide ion signatures, providing unequivocal identification between structurally similar PMEs.