Understanding gas phase modifier interactions in rapid analysis by differential mobility-tandem mass spectrometry

Abstract

A systematic study involving the use and optimization of gas-phase modifiers in quantitative differential mobility-mass spectrometry (DMS-MS) analysis is presented using nucleoside-adduct biomarkers of DNA damage as an important reference point for analysis in complex matrices. Commonly used polar protic and polar aprotic modifiers have been screened for use against two deoxyguanosine adducts of DNA: N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP) and N-(deoxyguanosin-8-y1)-2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP). Particular attention was paid to compensation voltage (CoV) shifts, peak shapes, and product ion signal intensities while optimizing the DMS-MS conditions. The optimized parameters were then applied to rapid quantitation of the DNA adducts in calf thymus DNA. After a protein precipitation step, adduct levels corresponding to less than one modification in 10(6) normal DNA bases were detected using the DMS-MS platform. Based on DMS fundamentals and ab initio thermochemical results, we interpret the complexity of DMS modifier responses in terms of thermal activation and the development of solvent shells. At very high bulk gas temperature, modifier dipole moment may be the most important factor in cluster formation and cluster geometry, but at lower temperatures, multi-neutral clusters are important and less predictable. This work provides a useful protocol for targeted DNA adduct quantitation and a basis for future work on DMS modifier effects.

Figure 1

(A): Effect on compensation voltage of varying concentrations of modifiers (i) Isopropanol and (ii) Ethyl acetate on dG-C8-4-ABP CoV shifts. (B) Effect of varying concentrations of modifiers (i) Isopropanol, (ii) Ethyl acetate, (iii)1-butanol on dG-C8-PhIP CoV shifts

Figure 2

Comparison of peak shapes of dG-C8-4-ABP with and without modifier. Improvements in peak shape by the introduction of modifiers can be realized by comparing the FWHM (full width at half maximum) at a constant SV of 3500 volts

Figure 3

(A) Separation of a mixture of dC, dA, dG and dG-C8-4-ABP in the presence of ethyl acetate modifier at a fixed concentration done on the DMS- ion trap. (B) Separation of dG and dG-C8-4-ABP (i) without modifier, and using IPA modifier at two different modifier concentration done on DMS-triple quadrupole (ii) 0.60% and (iii) 1.10%

Figure 4

Effect of separation voltage and modifier isopropanol (IPA) concentration on the MS/MS signal intensity of the DNA adduct dG-C8-PhIP (A) at 0.6% (B) 1.1% (C) 2.2% (D) Trends of AB SCIEX DMS API 3000 signal intensities at separation voltages of 3500, 4000, 4500 volts with increasing modifier percentages. Effect of modifiers on the product ion intensity at a fixed separation voltage of 3500 volts: (E) Modifiers Isopropanol (IPA) and 1-butanol on the DNA adduct dG-C8-PhIP & (F) Modifiers Isopropanol (IPA) and Ethyl acetate (EtoAc) on the DNA adduct dG-C8-4-ABP. Calibration curves: (G) dG-C8-4-ABP with ethyl acetate (0.6%), SV(3500V) and (H)dG-C8-PhIP with IPA(0.6%), SV 3500V. Selection of optimum conditions of SV and modifier allow accurate calibration of low adduct concentrations as indicated by calibration curves.

Figure 5

Comparison of LC-MS and DMS-MS speed of analysis for DNA adducts. For SPE-LC-MS, a second lyophilization step is required.

Figure 6

(A) Flyback and two-harmonic waveforms. The two-harmonic shape is used by AB SCIEX in the DMS API 3000, and the flyback shape on the ion trap. (B) Using ab-initio thermochemical values, the mean number of bound neutrals is shown as a function of DMS field and transport gas temperature at 1 atm. The maximum neutral count is limited to 3 because shell thermochemistry has not been calculated. These results for R-alphamethylhistamine, which has a polar core similar to dG, with 1.5% isopropanol show that bulk gas temperature is an essential controlling parameter in modifier DMS selectivity and that multiple clustering is common even at relatively high bulk gas temperatures.