Using fluorescence dyes as a tool for analyzing the MALDI process

Abstract

In a recent paper (Setz, P. D.; Knochenmuss, R. Phys. Chem. A 2005, 109, 4030 -4037) energy-transfer from excited matrix molecules to fluorescent traps was used to study the role of pooling reactions for the ionization processes in matrix-assisted laser desorption ionization (MALDI) using 2,5-dihydroxybenzoic acid as matrix. Exciton trapping was shown to interfere with matrix ionization. These investigations were extended to analyze the influence of fluorescent traps on both matrix and analyte ions for ␣-cyano-4-hydroxycinnamic acid and further matrices. A strong influence of the fluorescent traps on both matrix and analyte ionization was revealed, depending on the matrix:trap ratio, and manifested itself differently for low and high mass analytes. (MALDI) has become an enabling technology for the fields of protein mass spectrometry (MS) and proteomics. Despite its widespread use, e.g., for protein identification via peptide mass fingerprinting [4], a comprehensive model for the generation of free gasphase ions has not yet been developed [5,6]. All matrices in use today have been found empirically and stem from the early days of MALDI, such as ␣-cyano-4-hydroxycinnamic acid (CHCA) [7]. With regard to analyte ionization, the essential role of protonated matrix species was already discussed in the early MALDI papers proposing that the central reaction of analyte ionization in the positive ion mode is a proton transfer from protonated matrix species to analyte molecules. A so-called "photochemical" ionization model was proposed by Ehring et al. [8]; energy pooling processes and resonant two-step absorption were assumed to occur and to facilitate the generation of the primary radical cation matrix species for which more than one UV photon is needed. Karbach et al. experimentally determined the ionization potential of 2,5-DHB to be higher than the energy content of two 337 nm UV photons [9]. Resonant two-step absorption is only possible taking into consideration that the missing energy is contributed by the thermal energy of the matrix system. This photo/ thermal ionization model was analyzed more in detail by Allwood et al. [10]. Different authors proposed the possibility of energy pooling processes of two neighboring (localized) excited molecules (multicenter ionization model) [8,11,12] with higher ionization efficiency than resonant two-step absorption processes. This model was further extended by Setz et al. proposing mobile matrix excitations [13].Laser excitation also induces the MALDI ablation and evaporation process above a distinct threshold fluence. Theoretical investigations of the MALDI process on the basis of the breathing sphere model predict two desorption mechanisms: thermal vaporization of single molecules up to small clusters (four molecules or less) and ablation of large clusters at higher laser fluences [14 -16]. The mechanistic discussions and considerations have been carefully reviewed by Dreisewerd [17]. Moreover, it is included in the considerations that the diverging initial velocities as m...

Comparison between vacuum sublimed matrices and conventional dried droplet preparation in MALDI-TOF mass spectrometry

“…Device parameters were set to identical values as given in [22]. The delayed extraction (DE) time was varied from 10 ns (shortest selectable interval) to 1000 ns in 200 ns steps.…”

“…Theoretical investigations predict two desorption mechanisms that occur in MALDI: thermal vaporization of single molecules up to small clusters consisting of up to four molecules and phase explosions with ablation of large clusters at higher laser fluences [26 -28]. It was shown that the process of thermal vaporization of small molecules gets favored by increase of the effective temperature, which is accompanied by lower initial ion velocities [22]. Therefore, the question rose whether higher matrix crystal temperatures caused from smaller crystals accompanied by higher effective analyte temperatures in sublimed matrices cause more efficient thermal vaporization by heating and vaporization of the matrix in a larger volume or increased phase explosions by stronger ablation processes.…”

The new matrix 4-Chloro-α-cyanocinnamic acid allows the detection of phosphatidylethanolamine chloramines by MALDI-TOF mass spectrometry

“…Device parameters were set to identical values as given in [17]. For each DE time the average flight time of the protonated analyte ions was determined using data from six individual measurements accumulated of 50 shots each.…”

The analyses of fluorescently labeled biomolecules using ultraviolet-wavelength fluorophore-assisted laser desorption/ionization-mass spectrometry (UV FALDI-MS)

“…High fluorescence quantum yields of laser dyes in the solution and likely solid states may also play a role in efficient energy transfer and soft ionization of derivatized biomolecules since thermal relaxation and decomposition of fluorescent molecules are minimized. Nevertheless, although it likely shares many similarities with the mechanisms of LDI and MALDI [2,19,20,[37][38][39][40], detailed mechanism of FALDI remains to be determined by analyzing structurally diverse fluorescently labeled biomolecules using LDI-MS and fluorescence techniques.…”

“…For example, coumarin 510, coumarin 519, and rhodamine isothiocyanate (RITC) have been tested as visible-wavelength MALDI matrices [10,11]. Fluorescent dyes have also been used as dye co-matrices for the analysis of protein samples by visible-wavelength MALDI-MS and the investigation of the mechanism of UV MALDI [18][19][20]. The fluorescent dyes, such as coumarin, can covalently label theamine of lysine residues and the N-termini of peptides and proteins in order to investigate labeled biomolecules in fluorescence-based applications [21].…”