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.
Similar content being viewed by others
References
Hirano S, Cui X, Li S, Kanno S, Kobayashi Y, Hayakawa T, Shraim A (2003) Arch Toxicol 77:305–312
Leermakers M, Baeyens W, De Gieter M, Smedts B, Meert C, De Bisschop HC, Morabito R, Quevauviller P (2006) TrAC, Trends Anal Chem 25:1–10
Tornes JA, Opstad AM, Johnsen BA (2006) Sci Total Environ 356:235–246
Stock T (1996) Sea-dumped chemical weapons and the chemical weapons convention. In: Kafka AV (ed) Sea-dumped chemical weapons: aspects, problems and solutions. Kluwer Academic, Dordrecht
Kroening KK, Solivio MJV, Garcia-Lopez G, Puga A, Caruso JA (2008) Metallomics 1:59–66
Pitten FA, Muller G, Konig P, Schmidt D, Thurow K, Kramer A (1999) Sci Total Environ 226:237–245
Charoensuk V, Gati WP, Weinfeld M, Le XC (2009) Toxicol Appl Pharmacol 239:64–70
Hirano S, Kobayashi Y, Hayakawa T, Cui X, Yamamoto M, Kanno S, Shraim A (2005) Arch Toxicol 79:54–61
Sahara N, Takeshita A, Kobayashi M, Shigeno K, Nakamura S, Shinjo K, Naito K, Maekawa M, Horii T, Ohnishi K, Kitamura K, Naoe T, Hayashi H, Ohno R (2004) Leuk Lymphoma 45:987–995
Cline DJ, Thorpe C, Schneider JP (2003) J Am Chem Soc 125:2923–2929
Wang Z, Zhang H, Li XF, Le XC (2007) Rapid Commun Mass Spectrom 21:3658–3666
Kitchin KT, Wallace K (2005) Toxicol Appl Pharmacol 206:66–72
Guo Y, Ling Y, Thomson BA, Siu KWM (2005) J Am Soc Mass Spectrom 16:1787–1794
Ngu TT, Stillman MJ (2006) J Am Chem Soc 128:12473–12483
Pinkse MWH, Uitto PM, Hilhorst MJ, Ooms B, Heck AJR (2004) Anal Chem 76:3935–3943
Wolschin F, Wienkoop S, Weckwerth W (2005) Proteomics 5:4389–4397
Loyet KM, Stults JT, Arnott D (2005) Mol Cell Proteomics 4:235–245
Schlosser A, Vanselow JT, Kramer A (2005) Anal Chem 77:5243–5250
Chalmers MJ, Kolch W, Emmett MR, Marshall AG, Mischak H (2004) J Chromatogr B Anal Technol Biomed Life Sci 803:111–120
Zeller M, Konig S (2004) Anal Bioanal Chem 378:898–909
Wind M, Edler M, Jakubowski N, Linscheid M, Wesch H, Lehmann WD (2001) Anal Chem 73:29–35
Marshall P, Heudi O, Bains S, Freeman HN, Abou-Shakra F, Reardon K (2002) Analyst 127:459–461
Wind M, Feldmann I, Jakubowski N, Lehmann WD (2003) Electrophoresis 24:1276–1280
Wind M, Wesch H, Lehmann WD (2001) Anal Chem 73:3006–3010
Wind M, Gosenca D, Kubler D, Lehmann WD (2003) Anal Biochem 317:26–33
Wind M, Kelm O, Nigg EA, Lehmann WD (2002) Proteomics 2:1516–1523
Abdullaev FI, Rivera-Luna R, García-Carrancá A, Ayala-Fierro F, Espinosa-Aguirre JJ (2001) Mutat Res Genet Toxicol Environ Mutagen 493:31–38
Chen XW, Zou AM, Chen ML, Wang JH, Dasgupta PK (2009) Anal Chem 81:1291–1296
Gerlach VL, Feaver WJ, Fischhaber PL, Friedberg EC (2001) J Biol Chem 276:92–98
Briggs SD, Bryant SS, Jove R, Sanderson SD, Smithgall TE (1995) J Biol Chem 270:14718–14724
Tourrière H, Chebli K, Zekri L, Courselaud B, Blanchard JM, Bertrand E, Tazi J (2003) J Cell Biol 160:823–831
Moon SY, Zheng Y (2003) Trends Cell Biol 13:13–22
Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC, Gygi SP (2004) Proc Natl Acad Sci U S A 101:12130–12135
Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M (2006) Cell 127:635–648
Shen X, Valencia CA, Szostak J, Dong B, Liu R (2005) Proc Natl Acad Sci U S A 102:5969–5974
Cosen-Binker LI, Kapus A (2006) Physiology 21:352–361
Liu H, Sugiura M, Nava VE, Edsall LC, Kono K, Poulton S, Milstien S, Kohama T, Spiegel S (2000) J Biol Chem 275:19513–19520
Conus NM, Hannan KM, Cristiano BE, Hemmings BA, Pearson RB (2002) J Biol Chem 277:38021–38028
Ostergaard E (2008) J Inherit Metab Dis 31:226–229
Cvetkovic A, Angeli Lal Menon A, Thorgersen M, Scott J, Poole F II, Jenney F Jr, Lancaster W, Praissman J, Shanmukh S, Vaccaro B, Trauger S, Kalisiak E, Apon J, Siuzdak G, Yannone S, Tainer J, Adams M (2010) Nature. doi:10.1038/nature09265
Acknowledgements
The authors are grateful to Agilent Technologies for their continuing support via chromatographic and mass spectrometric instrumentation. OA is particularly thankful to the Scientific and Technical Research Council of Turkey (TUBITAK) for post-doctoral fellowship support.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Alp, O., Merino, E.J. & Caruso, J.A. Arsenic-induced protein phosphorylation changes in HeLa cells. Anal Bioanal Chem 398, 2099–2107 (2010). https://doi.org/10.1007/s00216-010-4128-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-010-4128-3