UV Matrix-assisted Laser Desorption/ionization Research Articles

Heptachlorepoxides: theoretical versus experimental study of the embedded samples in the matrixes of organic crystals

The analysis of heptachlorepoxides in gas- and condense phase of embedded in the organic matrixes analyte samples is highlighted. The electrospray ionization, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), nuclear magnetic resonance, Raman and CD spectroscopy as well as quantum chemical methods are used. The competitive formation of the variety of epoxides, strongly depending of the reaction conditions. Thus, the products of interaction of matrix-analyte matrixes under the UV–MALDI–MS experiment are comprehensively examinated. Nonetheless, that for heptachlor (HA) is known an epoxide, existing in two isomeric forms, i.e. A and B (exo- and endo-one), the study on the mechanism of the epoxidation of HA, showed formation of new epoxides C–F and an intermediate (I), characterizing with high stability, decreasing in the raw F > A > C > B > I > D > E. The epoxide D appeared enantiomer of the insecticide oxychlodran. The reported results on the pesticides, their metabolites and decomposition reactivity products contributed significantly to the field of analytical chemistry, providing possibility for elaboration of highly selective protocols for assessment of environmental contaminant issues.

Two-laser mid-infrared and ultraviolet matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization (MALDI) was performed using two-pulsed lasers with wavelengths in the infrared and ultraviolet regions. A 2.94 μm pulsed optical parametric oscillator laser system and a 337 nm pulsed nitrogen laser irradiated the same spot on the sample target. Sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CCA), and 4-nitroaniline (NA) were used as matrices, and bovine insulin and cytochrome C were used as analytes. The laser energy was adjusted so that one-laser MALDI and LDI was at a minimum and the matrix and analyte ion signal was enhanced when the two lasers were fired together. Two-laser LDI was observed with SA, DHB, and NA matrices and two-laser MALDI was observed with SA and DHB. Plots of ion signal as a function of delay between the IR and UV lasers show two-laser signal from 0 ns up to a delay of 500 ns when the IR laser is fired before the UV laser. The results are interpreted in terms of IR laser heating of the target that leads to an enhancement in UV LDI and MALDI.

Two-laser infrared and ultraviolet matrix-assisted laser desorption/ionization.

Matrix-assisted laser desorption/ionization (MALDI) was performed using two pulsed lasers with wavelengths in the IR and UV regions. A 10.6 micro m pulsed CO(2) laser was used to irradiate a MALDI target, followed after an adjustable delay by a 337 nm pulsed nitrogen laser. The sample consisted of a 2,5-dihydroxybenzoic acid matrix and bovine insulin guest molecule. The pulse energy for both of the lasers was adjusted so that the ion of interest, either the matrix or guest ion, was not produced by either of the lasers alone. The delay time for maximum ion yield occurs at 1 micro s for matrix and guest ions and the signal decayed to zero in approximately 400 micro s. A mechanism is presented for enhanced UV MALDI ion yield following the IR laser pulse based on transient heating.

Hyphenation of capillary high-performance liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for nano-scale screening of single-bead combinatorial libraries.

This paper focuses on the technical aspects of chemical screening from 384-well plate nano-scale single-bead combinatorial libraries. The analytical technique utilized is a combination of capillary liquid chromatography with ultraviolet detection and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The HPLC/MALDI-MS hyphenation is achieved by means of a micro-fraction collector with a peak detection system that automatically collects the peaks onto the MALDI targets for subsequent characterization. Several experimental parameters such as type of 384-well plate, well-plate sealing foils, and a column-switching procedure were investigated using a small test library of nine components. Additionally, the influence of different MALDI matrices, different MALDI targets and sample-spotting techniques on the MALDI detection sensitivity as well as the ruggedness and sample throughput capacity of this technique were studied. Optimum results for the analytes investigated were obtained with 2,5-dihydroxybenzoic acid using on-line mixing of HPLC effluent and matrix solution. To demonstrate the potential of this capillary HPLC/MALDI-TOFMS method, its application to several single-bead libraries was investigated. The instrumental method allowed for the rapid identification and purity assessment of combinatorial libraries with detection limits down to the higher femtomole level using both UV detection and MALDI mass spectrometry.

Cytochrome c6 from Cyanophora paradoxa

In the eukaryotic alga Cyanophora paradoxa, which does not contain plastocyanin, photosynthetic electron transport from the cytochrome b6/f complex to photosystem I is mediated by cytochrome c6. Cytochrome c6 was purified to homogeneity by column chromatography and FPLC. The relative molecular mass of the holoprotein was determined by two different mass spectrometric methods (californium-252 plasma desorption and UV matrix-assisted laser desorption ionization) giving 9251 +/- 3.3 Da. N-terminal Edman microsequencing yielded information on approx. 30 amino acid residues. Based on these data and on highly conserved regions of cytochromes c6, degenerate oligonucleotides were designed and used for PCR to amplify the genomic DNA of C. paradoxa. Screening of a C. paradoxa cDNA library yielded several clones coding for preapo-cytochrome c6. The deduced sequence of the mature protein was verified by plasma desorption mass spectrometric peptide mapping and shows high similarity to those of cytochromes c6 from cyanobacteria and algae. Cytochrome c6 appears to be encoded by a single nuclear gene (petJ) in C. paradoxa. As the mature protein is located in the lumen of the thylakoid membrane, it has to traverse three biological membranes as well as the unique peptidoglycan layer of the cyanelles before it reaches its final subcellular locale. Thus the transit sequence is composed of two different targeting signals: a stroma targeting peptide resembling those of higher plants with respect to hydropathy plots and amino acid composition and a hydrophobic signal peptide functioning as a thylakoid-traversing domain. There are indications for alternative sorting of part of the cyanelle cytochrome c6 pool to the periplasmic space. This is the first known bipartite transit sequence of a cyanelle precursor protein from C. paradoxa, a model organism concerning the endosymbiotic origin of plastids. Labeled precursor is efficiently imported into isolated cyanelles, then routed into thylakoids and processed to the mature protein. Hitherto, in vitro protein translocation was not reported for cyanobacterial-type thylakoids.

Polyfunctional Fullerene Derivatives as UV-MALDI Matrices to Detect Peptides and Proteins

Fullerene derivatives with polyfunctional groups were synthesized and used as matrices in UV matrix-assisted laser desorption ionization (UV-MALDI) to detect polar biomolecules such as peptides and proteins. The fullerene derivatives are N,N-dimethylformamide (DMF) or water soluble. The matrix solutions (in DMF or water) can mix well with the aqueous solution containing the analyte. Visual observation with the assistance of magnifying glasses indicated that after drying, a homogenous sample solution was produced on the sample probe. MALDI/time-of-flight (MALDI/TOF) analyses results demonstrate that strong signals from peptides and proteins can be obtained. The fact that a homogenous sample solution was produced on the sample probe accounts for why the analyte's signals were detected everywhere on the probe.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Identifying the Composition of Labeled Proteins

Labeled proteins are extensively used in molecular biology and environmental science. The determination of the composition and label ratio is very important for monitoring the efficiency of their separation and purification. In this paper a novel method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry was developed for this purpose. The results obtained for three commercial labeled proteins showed that they are mixtures of different conjugates. In some cases, the label ratio obtained by UV spectrometry and MALDI mass spectrometry was strikingly different. For fluorescent labels such as fluorescein isothiocyanate, MALDI mass spectrometry determines the number of covalently bound labels, whereas UV absorption yields both bound and adsorbed labels. For biotinylated proteins, label ratios obtained by the 4-hydroxyazabenzene-2′-carboxylic acid (HABA)–avidin method were found to be much smaller those determined by MALDI mass spectrometry. The HABA–avidin method may therefore not be suitable for the determination of biotin label ratios.

Do single matrix molecules generate primary ions in ultraviolet matrix-assisted laser desorption/ionization

The ionization mechanisms in matrix-assisted laser desorption/ionization (MALDI) remain poorly understood. We have begun a program of study aimed at determining the properties of matrix molecules which make them suitable for MALDI. Initial results are presented here for one of the most widely used matrix materials, 2,5-dihydroxybenzoic acid (DHB). Spectroscopy of free DHB molecules in a molecular beam shows that the photoionization energy is much lower than expected from semiempirical calculations, only 8.05 eV, yet still not accessible with two nitrogen laser photons (7.36 eV). No evidence is found for labile protons in the first excited state. This is in spite of structural similarity with salicylates where excited state proton transfer from the 2-hydroxy group occurs. Conventional excited state proton transfer from single DHB molecules to analytes is thus deemed unlikely. On the other hand, a two-step reaction taking place via decarboxylated DHB (hydroquinone) is shown to be a potential analyte protonation mechanism. The conclusion is reached that single matrix molecules are probably not the primary ion generators in UV MALDI with this matrix. This is consistent with proposed models which require dimers or larger aggregates for ion generation. The photo/thermal combined ionization model of Allwood, Dyer and Dreyfus (Rapid Commun. Mass Spectrom. 11, 499 (1997)) is updated with measured physical parameters for DHB, and extended to include 2-center energy pooling mechanisms. © 1998 John Wiley & Sons, Ltd.

The matrix suppression effect in matrix-assisted laser desorption/ionization: application to negative ions and further characteristics

As recently shown for positive ions (Rapid Commun. Mass Spectrom. 10, 871 (1996)), small to moderate-sized analyte ions (<20 000 Da) can fully suppress all matrix signals in matrix-assisted laser desorption/ionization (MALDI) mass spectra at appropriate matrix:analyte mixing ratios. The technique is useful for removing matrix interferences and led to a model for primary ion formation mechanisms. We here extend the method to negative ions, and investigate the effects of analyte type and size on the matrix suppression concentration range. For a given matrix/analyte pair, suppression was observed in either positive- or negative-ion mode, but not both. Some matrices do not show the effect at all, which is attributed to unfavourable thermodynamics. The analyte concentration where suppression appears is correlated with the analyte molecular weight and supports the proximity requirement of the model. The dependence of matrix signals on analyte concentration is very similar in both modes, and may have implications for the role of plume reactions at low analyte concentrations. Delayed extraction was found to extend matrix suppression to lower analyte concentrations. Lack of homogenous matrix/analyte co-crystallization at low analyte concentrations is shown to prevent the appearance of matrix suppression or to modify the shape of the concentration curves in some cases. Evidence is presented for the activity of electronic excitations in IR as well as in UV MALDI. © 1998 John Wiley & Sons, Ltd.

Positive ion formation in the ultraviolet matrix-assisted laser desorption / ionization analysis of oligonucleotides by using 2,5-dihydroxybenzoic acid

The development of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and its demonstrated performance with large proteins has generated substantial interest in utilizing this technique as an alternative to gel electrophoresis for DNA sequence analysis. However, a lack of understanding of the desorption and ionization processes has greatly hampered advances in this field. This article explores the formation of positively charged oligonucleotides in UV (355-nm) MALDI analysis by using the matrix 2,5-dihydroxybenzoic acid. Whereas substantial fragmentation is observed in the positive-ion mode by using the short oligomer d(TAGGT), no fragmentation is evident in the negative-ion mode under identical conditions. The fragmentation products are consistent with a previously published model in which base protonation initiates base loss, which leads to subsequent cleavage of the phosphodiester backbone. Several polydeoxythymidilic acids containing modified nucleosides were used to investigate positive-ion formation. The results support the hypothesis that positive ions are formed by protonation of the nucleobases. Because base protonation initiates base loss, fragmentation is intrinsic to positive-ion formation in the MALDI analysis of oligonucleotides. This result explains the dramatic difference in fragmentation observed in positive-ion compared to negative-ion UV-MALDI mass spectra of oligonucleotides.

Characterization of specific noncovalent protein complexes by UV matrix-assisted laser desorption ionization mass spectrometry.

While electrospray (ESI) mass spectrometry has already established its potential for the characterization of non-covalent protein complexes, matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) seemed not to be applicable hitherto because of limitations in matrix chemistry and sample preparation. In this work, a sample preparation method has been developed in which 6-aza-2-thiothymine (ATT) was used as a matrix without any addition of organic cosolvents, and proteins were dissolved in aqueous buffers such as ammonium hydrogencarbonate, ammonium citrate and ammonium acetate. Under these conditions, the intact non-covalent protein complexes, RNAse S, the non-covalent complex of S-protein and S-peptide and specific dimers of coiled-coil leucine zipper polypeptides were observed by UV-MALDI/MS. The specificity of complex formation was ascertained by admixture of non-specific peptides which did not yield detectable aggregate ions. In addition, on-target tryptic digestion of cytochrome c and leucine zipper peptides was carried out after MALDI/MS molecular mass determination in the presence of the ATT matrix. Mass spectrometric analyses of these tryptic digests yielded spectra that showed complete digestion of the proteins. These results indicate that proteins maintained intact tertiary structures necessary for the formation of specific non-covalent complexes, and that trypsin retained its functional enzymatic structure and full biological activity with the present sample preparation method.