Production, Purification, and Characterization of ¹⁵N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements

Abstract

Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA-protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length (15)N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of (15)N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented.

Keywords: (15)N-labeled proteins; DNA damage; DNA repair; DNA repair proteins; Mass spectrometry; Protein measurements.

Figure 1

SDS (13%)-PAGE analysis of the purification steps of E. coli¹⁵N-Fpg: lane 1, 21 µg protein of the extract of BL21(DE3) cells harboring pET11a control plasmid; lane 2,31 µg protein of the extract of BL21(DE3) cells harboring Fpg/pET11a plasmid induced with IPTG; lane 3,26 µg of 10000 × g supernatant; lane 4,8.6 µg flow through from DEAE cellulose column, 29 µg; lane 5, 8.5 µg ¹⁵N-Fpg eluted from CM cellulose column, 26 µg; and lane 6, molecular mass markers. Data from Reddy et al. (2011).

Figure 2

SDS (10%)-PAGE analysis of the purification steps of ¹⁵N-hOGG1: lane 1, 20 µg protein of the extract of BL21(DE3) cells harboring pET11a control plasmid; lane 2, 21 µg protein of the extract of BL21(DE3) cells harboring hOGG1/pET15b plasmid induced with IPTG; lane 3, 17 µg of 48000 × g supernatant; lane 4, 15.5 µg from Ni-agarose flow through; lane 5, 8.3 µg ¹⁵N-hOGG1 eluted from Ni-agarose column; and lane 6, molecular mass markers. Data from Reddy et al. (2011).

Figure 3

SDS (10%)-PAGE analysis of the purification steps of ¹⁵N-hNEIL1: lane 1, uninduced cell extract, 24 µg; lane 2, induced cell extract, 28 µg; lane 3,20000 × g supernatant, 22 µg; lane 4, flow through from nickel resin, 17 µg; lane 5, pure ¹⁵N-NEIL1 eluted from nickel resin, 15 µg; and lane 6, molecular mass markers. Data from Reddy et al. (2013).

Figure 4

SDS (11%)-PAGE analysis of the purification steps of ¹⁵N-hAPE1: lane 1, uninduced cell extract, 24 µg; lane 2, induced cell extract, 28 µg; lane 3, 70000 × g supernatant, 24 µg; lane 4, flow through from DEAE cellulose column, 17 µg; lane 5, flow through from CM cellulose column, 16µg; lane 6, pure ¹⁵N-APE1 eluted from CM cellulose column, 21 µg; and lane 7, molecular mass markers. Data from Kirkali et al. (2013).

Figure 5

SDS–PAGE analysis of purified hMTH1 and ¹⁵N-hMTH1. Lane 1, hMTH1; lane 2, ¹⁵N-hMTH1; and lane 3, molecular mass markers. Data from Coskun et al. (2015).

Figure 6

Full-scan mass spectra of the tryptic peptides GAVAEDGDELR (A) and ¹⁵N-GAVAEDGDELR (B). Data from Kirkali et al. (2013).

Figure 7

Product ion spectra of GAVAEDGDELR (A) and ¹⁵N-GAVAEDGDELR (B). The (M+2H)²⁺ ions m/z 566.3 (A) and m/z 573.3 (B) were used as the precursor ions. The insert shows the fragmentation pathways leading to the b- and y-ions. Data from Kirkali et al. (2013).

Figure 8

Ion-current profiles of mass transitions of five tryptic peptides of hAPE1 and ¹⁵N-hAPE1 obtained using the tryptic digest of a nuclear extract of MCF-7 cells. The extract was spiked with an aliquot of ¹⁵N-hAPE1 prior to separation by HPLC. Data from Kirkali et al. (2013).