Reactive desorption electrospray ionization mass spectrometry (DESI-MS) of natural products of a marine alga

Abstract

Presented here is the optimization and development of a desorption electrospray ionization mass spectrometry (DESI-MS) method for detecting natural products on tissue surfaces. Bromophycolides are algal diterpene-benzoate macrolide natural products that have been shown to inhibit growth of the marine fungal pathogen Lindra thalassiae. As such, they have been implicated in antimicrobial chemical defense. However, the defense mechanisms are not yet completely understood. Precise detection of these compounds on algal tissue surfaces under ambient conditions without any disruptive sample processing could shed more light onto the processes involved in chemical defense of marine organisms. Conventional DESI-MS directly on algal tissue showed relatively low sensitivity for bromophycolide detection. Sensitivity was greatly improved by the addition of various anions including Cl(-), Br(-), and CF(3)COO(-) into the DESI spray solvent. Chloride adduction gave the highest sensitivity for all assayed anions. Density functional optimization of the bromophycolide anionic complexes produced during DESI supported this observation by showing that the chloride complex has the most favorable binding energy. Optimized DESI protocols allowed the direct and unambiguous detection of bromophycolides, including A, B, and E, from the surface of untreated algal tissue.

Fig. 1

Conventional DESI-MS, MS², and MS³ spectra of pure bromophycolides (10 μL, 1 mg/mL) deposited on PTFE substrates and interrogated with a neat MeOH spray solution: a mass spectrum of bromophycolide A, b corresponding MS² spectrum of the precursor ion at m/z 665 (the insert in b shows the MS³ spectrum of the precursor ion at m/z 583); c mass spectrum of bromophycolide B, d corresponding MS² spectrum of the precursor ion at m/z 665 (the insert in c shows the MS³ spectrum of the precursor ion at m/z 665); e mass spectrum of bromophycolide E, and f corresponding MS² spectrum of the precursor ion at m/z 583

Fig. 2

Geometries for a deprotonated bromophycolide A and b [2M–H]⁻ complex of neutral and deprotonated bromophycolide A optimized at the B3LYP/6-31+G* level of theory

Fig. 3

Reactive DESI-MS and MS² spectra of bromophycolide A standards (10 μL, 1 mg/mL), deposited on PTFE surfaces and interrogated with spray solutions of various anions dissolved in MeOH; a 100 μM NH₄Cl with b corresponding MS² spectrum of the precursor ion at m/z 701; c 10 μM HBr, with d corresponding MS² spectrum of the precursor at m/z 745; and e 0.001% v/v (135 μM) CF₃COOH with f corresponding MS² spectrum of the ion at m/z 779

Fig. 4

Optimized geometries for: a bromophycolide A and its complexes with b Cl⁻, c Br⁻, and d CF₃COO⁻ optimized at the B3LYP-D/6-31+G* level of theory

Fig. 5

Effect of chloride ion concentration on the intensity of the [bromophycolide A+Cl]⁻ and [2 bromophycolide A–H]⁻ ions. The intensity values were normalized to that of the maximum value. For all experiments, 10 μL, 1 mg/mL bromophycolide A were deposited and allowed to air dry prior to analysis

Fig. 6

DESI spectrum obtained from directly interrogating the surface of the Fijian red alga C. serratus with a spray solution of 100 μM NH₄Cl in MeOH

Scheme 1

Chemical structure of bromophycolides identified from the Fijian red alga C. serratus