Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Targeted mass spectrometry quantification of IFN-γ induced ADP/ATP translocase 2 suppression related to cancer cell metabolism (#893)

Marie Vajrychova 1 , Barbora Salovska 2 , Ivo Fabrik 1 , Miroslav Pribyl 2 , Blanka Mrazkova 2 , Vojtech Tambor 1 , Zdenek Hodny 2
  1. Biomedical Research Centre, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic
  2. Department of Genome Integrity, Institute of Molecular Genetics of the ASCR, Prague, Czech Republic

Background: Cellular senescence represents a primary tumorigenesis barrier. On the other hand, senescent cells may participate on aging as well as cancer process development due to secretion of inflammatory cytokines like interferon (IFN) type I and II and TGF-β. We have recently shown that IFN-γ suppresses ADP/ATP translocase 2 (ANT2), which may result in elevation of reactive oxygen species, DNA damage and DNA damage response, persistent activation of cell cycle checkpoints and induction of senescent-like phenotype in cancer cell lines (1). Nevertheless, the mechanism of ANT2 growth-promoting role as well as detailed mechanism of its transcriptional suppression during exit from cell growth is unclear. Moreover, there is a lack of methods to accurately measure cellular concentration of different proteins from ADP/ATP translocases family. For illustration, it is not possible to distinguish between ANT1, ANT2, and ANT3 isoforms by Western blot. Thus, the aim of this work was to develop a method enabling us to quantitatively estimate levels of individual ATP/ADP translocators in mitochondria of normal and cancer cells from tissue samples and cell cultures undergoing various treatments.

Methodology: Firstly, unique ANT2 peptides to quantify protein level were carefully chosen and used for targeted quantification of ANT isoforms. The level of ANT2 peptide was determined using parallel reaction monitoring method upon normalization to synthetic internal ANT2 peptide standards.

Principal findings: Based on unique ANT2 peptides and synthetic peptide standards, we developed and optimized method for targeted quantification of ANT2. We applied this approach to assess ANT2 suppression in cellular senescence induced by IFN-γ.          

Conclusions: Given the high similarity of amino acid composition of all human ANTs, we optimized a MS-based targeted method for quantification of ANT2 as an alternative to immunoblotting. Subsequently, we assessed the ratio of ADP/ATP translocators in normal and cancer cells on relative level.   

  1. Hubackova, S., et al. (2015) Oncogene 35, 1236-1249