Structural characterization of p53 isoforms due to the polymorphism at codon 72 by mass spectrometry and circular dichroism

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• 作者：Marina Naldi ; Marco Pistolozzi ; Carlo Bertucci ; Angela De Simone ; Serena Altilia ; Michela Pierini ; Claudio Franceschi ; Stefano Salvioli ; Vincenza Andrisano
• 关键词：p53 Isoforms p53P⁷² and p53R⁷² ; Primary and secondary structure characterizations ; LC– ; ESI-QTOF ; LC– ; ESI-IT ; MALDI-TOF ; Tryptic digest ; Circular dichroism
• 刊名：Journal of Pharmaceutical and Biomedical Analysis
• 出版年：2010
• 出版时间：10 October 2010
• 年：2010
• 卷：53
• 期：2
• 页码：200-206
• 全文大小：701 K

文摘

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 (p53P⁷² and p53R⁷²) 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 p53R⁷² and p53P⁷² 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 R⁷², giving rise to two selective shorter peptides, occurred in p53R⁷², but was missing in the case of p53P⁷² 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.