Nuclease digestion and mass spectrometric characterization of oligodeoxyribonucleotides containing 1,2-GpG, 1,2-ApG, and 1,3-GpXpG cisplatin intrastrand cross-links

Abstract

Background: The primary mode of action for cis-diamminedichloroplatinum (II), referred to as cisplatin, toward the treatment of solid malignancies is through formation of cross-links with DNA at purine sites, especially guanines.

Methods: We prepared oligodeoxyribonucleotides (ODNs) containing a 1,2-GpG, 1,2-ApG, or 1,3-GpXpG cisplatin intrastrand cross-link and the corresponding ODNs modified with (15)N2-labeled cisplatin, and characterized these ODNs with electrospray ionization mass spectrometry (ESI-MS) and tandem MS (MS/MS). We also employed LC-MS/MS to characterize the digestion products of these ODNs after treatment with a cocktail of 4 enzymes (nuclease P1, phosphodiesterases I and II, and alkaline phosphatase).

Results: 1,2-GpG was released from the ODNs as a dinucleoside monophosphate or a dinucleotide. Analyses of the digestion products of ODNs containing a 1,2-GpG cross-link on the 5' or 3' terminus revealed that the dinucleotide carries a terminal 5' phosphate. On the other hand, digestion of the 1,3-GpXpG intrastrand cross-link yielded 3 dinucleoside products with 0, 1, or 2 phosphate groups.

Conclusion: The availability of the ODNs carrying the stable isotope-labeled lesions, MS/MS analyses of the cisplatin-modified ODNs, and the characterization of the enzymatic digestion products of these ODNs set the stage for the future LC-MS/MS quantification of the 1,2-GpG, 1,2-ApG, and 1,3-GpXpG lesions in cellular DNA.

Figure 1

Negative-ion ESI-MS (a,c) and high-resolution “zoom-scan” analysis showing the experimental isotopic distribution of the [M-3H]³⁻ ion with an inset depicting the corresponding theoretical isotopic distribution (b,d) of unlabeled (a,b) and ¹⁵N₂-labeled (c,d) d(ATCCG*G*CCTA), where * represents light [Pt(NH₃)₂]²⁺ or heavy [Pt(¹⁵NH₃)₂]²⁺ cisplatin coordination, respectively.

Figure 2

Negative-ion ESI-MS/MS of the unlabeled [M-3H]³⁻ ion with an inset showing a list of the observed fragment ions (a) and the peak assignments (with relative ion abundance in parenthesis) for the product-ion spectrum (b) of d(ATCCG*G*CCTA) where * (a) and bold m/z values (b) represent [Pt(NH₃)₂] ²⁺ coordination.

Figure 3

Unique fragment ion pairs and the corresponding m/z values {[Pt(NH₃)₂]²⁺/ [Pt(¹⁵NH₃)₂]²⁺} generated during the CID of ODN sequence 1, which harbors a 1,2-GpG cisplatin (*) intrastrand cross-link. Numbers in parenthesis represents relative ion abundance.

Figure 4

Negative-ion ESI-MS/MS of the unlabeled [M-3H]³⁻ ion with an inset showing a list of the observed fragment ions (a) and the peak assignments (with relative ion abundance in parenthesis) for the product-ion spectrum (b) of d(ATCCA*G*CCTA) where * (a) and bold m/z values (b) represent [Pt(NH₃)₂] ²⁺ coordination.

Figure 5

Unique fragment ion pairs and the corresponding m/z values {[Pt(NH₃)₂]²⁺/ [Pt(¹⁵NH₃)₂]²⁺} generated during the CID of ODN sequence 2, which harbors a 1,2-ApG cisplatin (*) intrastrand cross-link. Numbers in parenthesis represents relative ion abundance.

Figure 6

Negative-ion ESI-MS/MS of the unlabeled [M-3H]³⁻ ion with an inset showing a list of the observed fragment ions (a) and the peak assignments (with relative ion abundance in parenthesis) for the product-ion spectrum (b) of d(ATCG*CG*CCTA) where * (a) and bold m/z values (b) represent [Pt(NH₃)₂] ²⁺ coordination.

Figure 7

Unique fragment ion pairs and the corresponding m/z values {[Pt(NH₃)₂]²⁺/ [Pt(¹⁵NH₃)₂]²⁺} generated during the CID of ODN sequence 3, which harbors a 1,2-GpXpG cisplatin (*) intrastrand cross-link. Numbers in parenthesis represents relative ion abundance.

Figure 8

Structures and m/z values for the [M+H]⁺ ions for light [Pt(NH₃)] ²⁺ / heavy [Pt(¹⁵NH₃)] ²⁺ cisplatin adducts of the enzymatic digestion products.

Figure 9

HPLC traces for the separation of the enzymatic digestion mixture of synthetic sequences 4 (a) and 5 (b) upon cisplatin treatment; and the corresponding positive-ion ESI-MS of the hydrolysis products d(pG*pG*) (c) and d(G*pG*) (d), where * indicates the coordination of a cisplatin adduct.