Absolute Identification and Quantification of DNA Repair Proteins: An Emerging Measurement Trend in Nanotoxicology
Erdem Coskun, Pawel Jaruga, Bryant C. Nelson, Prasad T. Reddy, M Miral Dizdar, Shareen H. Doak, Neenu Singh
Oxidatively induced DNA damage is repaired in vivo by various mechanisms involving numerous DNA repair proteins. If not repaired, DNA damage may lead to mutagenesis, which is a fundamental part of the molecular basis of all cancers. Due to the unique surface-active properties (i.e. enhanced catalysis, etc.) of nanomaterials, it is important to understand and characterize their molecular-scale interactions with key biomolecules inside the cell. Characterizing and quantifying the effects of engineered nanomaterials on endogenous DNA repair protein expression levels is an emerging area of nanosafety research. Here, we present a novel methodology to positively identify and accurately measure key DNA repair proteins in cell cultures and human tissues using LC-MS/MS with isotope-dilution. We measured hAPE1 and hMTH1 levels in human normal and malignant breast tissues, and in human cultured cell lines, some of which were exposed to superparamagnetic iron oxide nanoparticles (USPIONs). We also developed the methodology for other DNA repair proteins such as NEIL1, NTH1, OGG1, PARP1 and Pol β and identified and measured these proteins in various cell lines and human tissues. The use of 15N-labeled analogs of these proteins as internal standards is critical for their accurate measurement. We, therefore, overexpressed and purified 15N-labeled versions of these proteins, added a known amount of the 15N-labeled protein analog to nuclear or cytoplasmic protein extracts from cells, hydrolyzed both labeled and unlabeled proteins with trypsin and used LC-MS/MS to separate and identify resulting tryptic peptides by their full-scan and product-ion mass spectra. Finally, selected-reaction monitoring of the tryptic peptides was utilized for positive identification and absolute quantification of protein levels. This novel method, developed in the NIST laboratories, may help to improve our understanding of protein-nanoparticle interactions and specifically, this method may eventually s
June 1-4, 2016
NanoTOX 2016 (8th International Congress of Nanotoxicology)
LC-MS/MS with isotope-dilution, iron oxide nanoparticles, NEIL1, NTH1, OGG1, PARP1 and Pol ß
, Jaruga, P.
, Nelson, B.
, Reddy, P.
, , M.
, Doak, S.
and Singh, N.
Absolute Identification and Quantification of DNA Repair Proteins: An Emerging Measurement Trend in Nanotoxicology, NanoTOX 2016 (8th International Congress of Nanotoxicology), Boston, MA
(Accessed November 30, 2023)