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.