A common polymorphism at
codon 72 of human TP53 gene determines a
proline to
arginine aminoacidic substitution within the
proline-rich domain of p53
protein. The two resulting
isoforms (p53P(72) and p53R(72)) are different from a biochemical and
biological point of view and many reports suggest that they can modulate individual
cancer susceptibility and overall survival. In the attempt to explain the observed
biological differences, we characterized the two
isoforms by mass spectrometry and circular dichroism (CD) to evaluate the possible alteration in the secondary structure of p53 introduced by this polymorphism. Recombinant human p53R(72) and p53P(72) were produced by using E. coli expression system then purified by chromatography (affinity chromatography and RP-HPLC), and the whole
proteins identified by HPLC-ESI-IT and MALDI-TOF analysis. A bottom-up approach, using both MALDI-TOF and HPLC-ESI-QTOF analysis, was then adopted to obtain the sequence information on the two p53
isoforms. To this purpose,
peptide maps were obtained by
trypsin proteolysis on the two p53
isoforms. The two
isoforms proteolytic digests were separated by LC and subsequent mass spectrometry analysis of both entire and fragmented
peptides was performed. In particular, precursor
peptide ions obtained by ESI were subjected to collision by the triple quadrupole and TOF separation, allowing us to determine the
isoforms aminoacidic
peptide sequence by
peptide ladder sequencing. Because of the presence of
arginine, a selective
trypsin proteolytic cleavage at R(72), giving rise to two selective shorter
peptides, occurred in p53R(72), but was missing in the case of p53P(72)
trypsin digest, in which an uncleaved longer
peptide was instead identified. Upon primary structure confirmation, the two p53
isoforms were studied by CD in order to investigate the experimental variables, which affect ordered secondary structure adoption. CD analysis indicated that the two
isoforms are not structurally different, thus allowing us to exclude that the observed
biological differences can be due to a different conformation of the two
isoforms introduced by this polymorphism. Furthermore, these studies establish a mass spectrometry method to identify the two
isoforms that can be useful for future interactome studies and
cancer drug discovery.