Abstract
In two-dimensional polyacrylamide gel electrophoresis (2-DE), peptides with the same primary structure are often separated as several spots because of the post-translational modifications. Immunoblotting and MALDI-TOF mass spectrometry analysis revealed that the RuBisCO large subunit gave two spots in 2-DE gel of proteins in the crude extracts of plant leaves; one was at pI 6.0, similar to that predicted from its sequence, and the other was unfocused around pI 4.3-4.5. The post-translational modifications reported for RuBisCO could not explain the appearance of the low-pI spot. Thus further analysis of the low-pI spot was carried out here. The low-pI spot was not detected in 2-DE with purified RuBisCO. When the low-pI spot extracted from the isoelectric focusing gel was subjected to 2-DE, the peptide in the low-pI spot moved to pI 6.0. We are investigating the cause of the appearance of the low-pI spot with crude extracts.
