The photophysics of some UV-MALDI matrices studied by using spectroscopic, photoacoustic and luminescence techniques

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Abstract

The photophysical behaviour of classical UV-MALDI matrices 2,5-dihydroxybenzoic acid (gentisic acid; GA), 2,4,6-trihydroxyacetophenone (THAP), trans-3,5-dimethoxy-4-hydroxycinnamic acid (SA), trans-4-hydroxy-α-ciano-4-hydroxycinnamic acid (CHC), 9H-pirido[3,4-b]indole (nor-harmane; norHo) and 1-methyl-9H-pirido[3,4-b]indole (harmane; Ho) in acetonitrile was studied by using spectroscopic, luminescence and photoacoustic techniques.

Graphical abstract

The photophysical behaviour of UV-MALDI matrices 2,5-dihydroxybenzoic acid (gentisic acid; GA), 2,4,6-trihydroxyacetophenone (THAP), trans-3,5-dimethoxy-4-hydroxycinnamic acid (SA), trans-4-hydroxy-α-ciano-4-hydroxycinnamic acid (CHC), 9H-pirido[3,4-b]indole (nor-harmane; norHo) and 1-methyl-9H-pirido[3,4-b]indole (harmane; Ho) in acetonitrile was studied by using spectroscopic, luminescence and photoacoustic techniques.

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Introduction

Since its introduction, matrix-assisted UV laser desorption/ionization mass spectrometry (UV-MALDI MS) [1], [2] has rapidly become a vital tool in the study of macromolecules.

UV-MALDI is the method of dispersing a macromolecule (analyte) in a large excess of a matrix. The solid mixture is irradiated with a laser pulse and vaporizes. Analyte gas ions are formed and then detected by MS. The mechanism of ionization during MALDI is still poorly understood and no adequate quantitative model for the complete process exists. Whatever the mechanism may be, one can certainly say UV-MALDI has proven to be of great utility in the MS analysis of otherwise intractable bio- and synthetic polymers. The UV-MALDI technique involves both laser ablation and ionization of the matrix/analyte mixture after electronic excitation of the matrix. Spectra are dominated by matrix photoproducts, protonated molecules (analyte and matrix), matrix molecular radical cations, and adduct ions. Several groups have developed models for the ablation process [3], [4], [5], [6]. However, full models of UV-MALDI wait a more detailed knowledge of ionization mechanisms, kinetics and thermodynamics of the species involved [7], [8]. Evidences exist that analyte ionization occurs both in the surface [6], [9] and in the expanding plume by a collisional mechanism in the gas phase (matrix-analite collisional mechanism; matrix as [M]+radical dot, and/or [M + H]+, and/or [M + alkali metal cation]+) [7], [10]. Both processes prompt ionization and ionization in the plume, might involve the ground state and electronic excited states of the matrix.

Ehring and Sundqvist [11] studied the 2,5-dihydroxybenzoic acid (gentisic acid; GA), finding that its luminescence quantum yield was very low (<0.2). The authors suggested that most of the energy absorbed by the molecule relaxes via non-radiative pathways and is therefore available for the desorption/ionization process. In spite of this suggestion, and the models of ablation and ionization previously mentioned, the thermal deactivation process from the electronic excited state of the currently used UV-MALDI matrices has not been described yet.

A knowledge of the thermal deactivation process of common UV-MALDI matrices should play a critical role in understanding why some matrices are ‘hotter’ than others, leading to more abundant post-source decay as well as prompt decay of the analyte (i.e., fragmentation processes of carbohydrates [12], [13]). Furthermore, the knowledge of this matrix property might be helpful in providing an additional aspect to keep in mind in order to choose the proper matrix to perform better UV-MALDI-MS analysis. As it is well known, the type of fragmentation observed also depends on the type of analyte molecular ion formed, which to some extent depends on the matrix too [7], [12], [13].

On the other hand, in an early work was demonstrated that laser ablation generates an acoustic signal which is directly proportional to the amount of material removed [14], [15]. This principle has been used for quantification in both UV-MALDI [16] and IR-MALDI [17]. From a different point of view, Golovelev [18] described the basic principles and experimental results of laser-induced acoustic desorption (LIAD) of some salts and biomolecules. As part of a comparative study of the photochemistry of the most popular matrices used in UV-MALDI-MS, we decided to examine the photophysical processes occurring after the electronic excitation of acetonitrile solutions of the following compounds: 2,5-dihydroxybenzoic acid (gentisic acid; GA), 2,4,6-trihydroxyacetophenone (THAP), trans-3,5-dimethoxy-4-hydroxycinnamic acid (SA), trans-α-ciano-4-hydroxycinnamic acid (CHC) together with the recently described 9H-pirido[3,4-b]indole (nor-harmane; norHo) and 1-methyl-9H-pirido[3,4-b]indole (harmane; Ho) [19], [20], [21].

As it is known, they behave in general as efficient matrices in UV-MALDI-MS analysis of proteins and carbohydrates, among other analytes [3], [7], [8], [12], [13], [19], [20], [21].

In this work, we study the absorption and emission properties of those molecules in steady-state conditions; its photostability; its singlet oxygen production by means of time resolved phosphorescence detection, and its calorimetric behaviour and triplet state properties by using photoacoustics measurements.

Section snippets

Experimental

Spectrograde and HPLC grade acetonitrile was purchased from J. T. Baker and was used without further purification. 2-Hydroxybenzophenone (2-HBP), phenalenone (PH), GA, THAP, SA, CHC, norHo and Ho were purchased from Aldrich and were used without further purification.

The absorption measurements were performed with a UV–Visible spectrophotometer Shimadzu UV-1203. All measurements were made with 1 cm stopped quartz cells at 298 K. Steady-state spectra and quantum yields were determined with a

Results and discussion

Absorption and emission data obtained for the studied compounds are shown in Table 1.

It is important to note that in UV-MALDI experiments (N2 laser, 337 nm), matrices and analyte are prepared in neutral solutions (i.e. acetonitrile, acetonitrile–water or water for carbohydrates and glycoconjugate compounds) and in acid medium (i.e. H2O–trifluoroacetic acid 0.1% for protein analysis) [7], [8], [19], [20]. Thus, matrix molecules are protonated species in the latter experiments. Therefore, we

Conclusions

Experiments conducted showed a strong dependence on the matrix structure. The ortho-hydroxyphenyl-carboxylic and ortho-hydroxyphenyl-carbonylic compounds GA and THAP behave as the well known CR 2-HBP under N2, Air and O2, showing a very minor fluorescence and good photostability in solution. The piridoindoles norHo and Ho in neutral solutions show high fluorescence and good photostability with lower α values depending strongly on the atmosphere. The presence of the cinnamic moiety induces

Acknowledgements

This project was partially financially supported by UBA (X022), CONICET (PIP05/5443) ANPCyT (PICT02-12312), and UNLP (11/I083). R.E.-B. is a research member of CONICET and G.M.B. is a research member of CIC and UNLP.

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