Original Paper

Analytical and Bioanalytical Chemistry

, Volume 398, Issue 5, pp 2099-2107

Arsenic-induced protein phosphorylation changes in HeLa cells

  • Orkun AlpAffiliated withAnalytical Chemistry Department, Faculty of Pharmacy, Gazi UniversityUniversity of Cincinnati/Agilent Technologies Metallomics Center of the Americas, Department of Chemistry, University of Cincinnati
  • , Edward J. MerinoAffiliated withDepartment of Chemistry, University of Cincinnati
  • , Joseph A. CarusoAffiliated withUniversity of Cincinnati/Agilent Technologies Metallomics Center of the Americas, Department of Chemistry, University of CincinnatiDepartment of Chemistry, University of Cincinnati Email author 

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Abstract

Arsenic is well documented as a chemotherapeutic agent capable of inducing cell death while at the same time is considered a human carcinogen and an environmental contaminant. Although arsenic toxicity is well known and has formed an impressive literature over the time, little is known about how its effects are exerted at the proteome level. Protein phosphorylation is an important post-translational modification involved in the regulation of cell signaling and likely is altered by arsenic treatment. Despite the importance of phosphorylation for many regulatory processes in cells, the identification and characterization of phosphorylation, as effected by arsenic through mass spectrometric detection, are not fully studied. Here, we identify phosphorylated proteins, which are related to post-translational modifications after phenylarsine oxide (PAO) inoculation to HeLa cells. PAO was chosen because of its high cytotoxicity, measured earlier in these labs. In this study, size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS) is used to establish several molecular weight fractions with phosphorylated proteins by monitoring 31P signal vs. time via ICP-MS. SEC-ICP-MS fractions are collected and then separated by the nano-LC-CHIP/ITMS system for peptide determination. Spectrum Mill and MASCOT protein database search engines are used for protein identification. Several phosphorylation sites and proteins related to post-translational modifications are also identified.

Keywords

Bioanalytical methods Cell systems/single-cell analysis Mass spectrometry/ICP-MS Speciation HPLC Genomics/proteomics