Jettison-MS of Nucleic Acid Species

Abstract

While MALDI-MS of intact genomic DNA is unheard of, actually many DNA adducts can be detected in this way under certain MALDI conditions: relatively high molar ratio of DNA nucleobases to matrix (0.01 to 0.3), hot matrix (CCA), and high laser fluence. This is because many DNA adducts create "bubbles" on dsDNA (disruption of base pairing), making it easier for these adducts as modified nucleobases to be jettisoned by the laser-derived energy of MALDI (jettison mass spectrometry or JeMS). The method also works for other nucleic acid species, namely nucleobases, nucleosides, nucleotides, and RNA. Examples of what we have detected in this way are as follows: methyladenine in E. coli DNA, 5-hydroxymethylcytosine in human brain DNA, melphalan-adenine in leukocyte DNA from patients on corresponding chemotherapy, wybutosine in tRNA, benzyl DNA adducts in E. coli cell culture treated with benzyl bromide, and various DNA adducts formed in test tube exposure experiments with calf thymus DNA. Noteworthy, in the chemotherapy study, principle component analysis of the data encourages the hypothesis that patient DNA undergoes much more damage than just melphalan adducts. Overall, our work leads to the preliminary generalization that about 5 fmol of a nucleobase deficient in base pairing, and present in a MALDI spot, will be detected by JeMS (on the equipment that we used), irrespective of the type of nucleic acid species which houses it, as long as the nucleobase is relatively basic such as A, C, or G.

Figure 1.

Concept of Jettison-MS (JeMS).

Figure 2.

(A) JeMS background spectrum from CCA matrix; (B) JeMS spectrum of calf thymus DNA: nucleobase peaks for C (peak 1 at m/z 112.044), mC (peak 2 at m/z 126.064), A (peak 3 at m/z 136.057), G (peak 4 at m/z 152.054), and possibly 8-oxo-G (peak 6 at m/z 168.049). Inset I shows mA at m/z 150.077 (peak 5) from E. coli DNA. Inset II shows calf thymus DNA that has been treated with H₂O₂, displaying an enhanced peak for 8-oxo-G at m/z 168.054 (peak 6).

Figure 3.

JeMS2 (TOF/TOF mode of JeMS) of human brain DNA. (A) Detection of dehydroxylated hydroxymethylC at m/z 124. (B) Relative abundance of hmC in DNA samples from 24 human brains (six Cockayne syndrome, three xeroderma pigmentosum, and 15 controls (normals).

Figure 4.

JeMS2 of calf thymus DNA modified with melphalan. (A) Detection of second product ions of monochloro-melphalan-adenine at m/z 359 and 269. (B) Similar detection at m/z 341 and 251 from the corresponding monohydroxy adduct. (C) JeMS2 of blood DNA samples from a subject administered melphalan at time zero, 2.5, 8, and 24 h, where the level of the monochloro melphalan adduct of adenine (m/z 269 in A) was monitored.