Identification of thermolabile pectin methylesterases from sweet orange fruit by peptide mass fingerprinting.
The multiple forms of the enzyme pectin methylesterase (PME) present in citrus fruit tissues vary in activity toward juice cloud-associated pectin substrates and, thus, in their impact on juice cloud stability and product quality. Because the proteins responsible for individual PME activities are rarely identified by structural properties or correlated to specific PME genes, matrix-assisted laser desorption-ionization with tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was investigated as a direct means to unequivocally identify the thermolabile (TL-) PME isoforms isolated from sweet orange [ Citrus sinensis (L.) Osbeck] fruit tissue. Affinity-purified TL-PME preparations were separated by SDS-PAGE prior to trypsin digestion and analyzed by MS for peptide mass fingerprinting. The two major PME isoforms accumulated in citrus fruit matched existing accessions in the SwissProt database. Although similar in size by SDS-PAGE, isoform-specific peptide ion signatures easily distinguished the two PMEs.
Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 Mermaid Lane, Wyndmoor, Pennsylvania 19038, United States.
Brett J
BJ
Savary
Prasanna
P
Vasu
Alberto
A
Nunez
Randall G
RG
Cameron
D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D032084:Citrus sinensis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005638:Fruit / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D010940:Plant Proteins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase