Characterization of tetrathiofulvalene compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Tetrathiofulvalene compounds are important components of charge-transfer complexes, which may be applied in various fields of scientific research and practical applications. Some of these compounds cannot be characterized by mass spectrometry. Here, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for the characterization of tetrathiofulvalenes. The samples could be easily desorbed and ionized to form singly charged ions, and mass spectra with isotopic resolution readily obtained. The mass spectrometric results for 26 compounds have shown that MALDI-TOF is more effective and convenient than other mass spectrometry methods, and resolves the problem of mass spectrometric characterization of tetrathiofulvalene compounds.     

Molar mass determination of macromolecular compounds by matrix-assisted laser desorption ionization mass spectrometry

Matrix-assisted laser desorption ionization is a recently developed new ionization technique which enables macromolecular compounds to be investigated by mass spectrometry. The general features of the technique are described, they have so far mainly been worked out for biopolymers such as proteins. Fast and precise molecular weight determination is possible up to 400,000 Dalton. First examples described in this paper indicate that the technique also holds great promise for the investigation of synthetic polymers.     

On-line atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

A new technique is presented for the coupling of atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) mass spectrometry with liquid delivery systems. Mass measurements of polymers and peptides are demonstrated using a co-dissolved matrix, e.g. alpha-cyano-4-hydroxycinnamic acid (HCCA). Improvements in terms of sensitivity are achieved by optimizing the shape und control of the exit capillary and by using a laser (355 nm) at a 1 kHz repetition rate. Two calibration experiments promise a good applicability of the presented coupling method for quantitative measurements. The limit of detection achieved so far is 500 nM for peptides in methanol solution containing 25 mM HCCA.     

Differentiation of enantiomers using matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has successfully been used to differentiate pseudo-enantiomeric (isotopically labelled) amino acids by using cyclodextrin as complexing host. By using different pseudo-enantiomeric mixtures (i.e. R(Dn) + S; and R + S(Dn)), it has been demonstrated that the preference of cyclodextrin for S-enantiomers is not due to the size differences caused by the hydrogen/deuterium substitution. It is postulated that this method can be extended to differentiate enantiomers (and determine enantiomeric excess) by using a pair of enantiomeric hosts, as demonstrated previously using other ionization techniques, but with much higher sensitivity.     

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.     

Covariance mapping in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A novel method for acquisition and numerical analysis of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectral data is described. The digitized ion current transient from each consecutive laser shot is first acquired and stored independently. Subsequently, statistical correlation parameters between all stored transients are computed. We illustrate the uses of this event-by-event analysis method for studies of sample surface heterogeneity as well as for elucidating the mechanisms of ion formation in MALDI. Other potential applications of the method are also outlined.     

Miniaturizing sample spots for matrix-assisted laser desorption/ionization mass spectrometry

The trend of miniaturization in bioanalytical chemistry is shifting from technical development to practical application. In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), progress in miniaturizing sample spots has been driven by the needs to increase sensitivity and speed, to interface with other analytical microtechnologies, and to develop miniaturized instrumentation.We review recent developments in miniaturizing sample spots for MALDI-MS. We cover both target modification and microdispensing technologies, and we emphasize the benefits with respect to sensitivity, throughput and automation.We hope that this review will encourage further method development and application of miniaturized sample spots for MALDI-MS, so as to expand applications in analytical chemistry, protein science and molecular biology.     

Accurate mass measurement of low molecular weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We report the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the accurate measurement of mass of low molecular weight compounds (smaller than 1500 Da), a linear peptide, two types of cyclic depsipeptides, a polyhydroxy-macrocyclic lactone, and two prenylated flavonoids, with delayed extraction in the reflector mode. The performance of the MALDI-TOF instrument was less than those of fast atom bombardment and Fourier-transform ion cyclotron resonance mass spectrometry instruments and insufficient to give acceptable accuracy for literature reporting. Nevertheless, when combined with NMR spectrometry and/or amino acid analysis to give information on the numbers of carbon atoms and index of hydrogen deficiency, MALDI was useful for determination of the elemental composition of the low molecular weight compounds available in small quantities.     

Clustering of polycyclic aromatic hydrocarbons in matrix-assisted laser desorption/ionization and laser desorption mass spectrometry

The desorption/ionization behaviour of polycyclic aromatic hydrocarbons (PAHs) in matrix-assisted laser desorption/ionization (MALDI) and laser desorption (LD) mass spectrometry was studied by the solvent-free sample preparation method. As the understanding of the desorption/ionization mechanism in MALDI is normally hampered by the different ionization and desorption efficiencies of the analytes, this work was focused on the analyses of a homologous series of four hexabenzocoronenes (HBCs) possessing virtually the same ionization efficiency: HBC parent, hexamethyl-hexabenzocoronene (HBC-C1), hexapropyl-hexabenzocoronene (HBC-C3) and hexakis(dodecyl)-hexabenzocoronene (HBC-C12). The different signal intensities obtained in their mass spectra can be related to differences in their desorption efficiencies, which are attributed to the different strengths of the intermolecular interactions between unsubstituted and alkylated HBCs in the solid state. The influence of the aromatic structure of PAHs on their photoionization/desorption probability was investigated. As a model system, an equimolar mixture composed of HBC-C12 and hexakis(dodecyl)-hexaphenylbenzene (HPB-C12) was chosen. The aromatic structures of both molecules and thus their absorption coefficients at the laser wavelength differ substantially and have a huge influence on their photoionization efficiency. The combined effect of laser light absorption and intermolecular interactions on the desorption/ionization behaviour of giant PAHs was further studied by using an equimolar mixture composed of a larger PAH (C(222)H(42)) and its dendritic precursor (C(222)H(150)). This mixture shows the opposite behaviour to that of the former example, because the balance between desorption and ionization efficiency has changed significantly. The present investigation should be of interest for providing a better understanding of MALDI and LD spectra obtained from natural PAH-containing samples, such as heavy oils, asphaltenes or pitches, for which our artificial mixtures represent suitable model systems.     

Surfactant-mediated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of small molecules

A variety of surfactants have been tested as matrix-ion suppressors for the analysis of small molecules by matrix-assisted laser desorption/ionization time-of flight mass spectrometry. Their addition to the common matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) greatly reduces the presence of matrix-related ions when added at the appropriate mole ratio of CHCA/surfactant, while still allowing the analyte signal to be observed. A range of cationic quaternary ammonium surfactants, as well as a neutral and anionic surfactant, was tested for the analysis of phenolics, phenolic acids, peptides and caffeine. It was found that the cationic surfactants, particularly cetyltrimethylammonium bromide (CTAB), were suitable for the analysis of acidic analytes. The anionic surfactant, sodium dodecyl sulfate, showed promise for peptide analysis. For trialanine, the detection limit was observed to be in the 100 femtomole range. The final matrix/surfactant mole ratio was a critical parameter for matrix ion suppression and resulting intensity of analyte signal. It was also found that the mass resolution of analytes was improved by 25-75%. Depth profiling of sample spots, by varying the number of laser shots, revealed that the surfactants tend to migrate toward the top of the droplet during crystallization, and that it is likely that the analyte is also enriched in this surface region. Here, higher analyte/surfactant concentration would reduce matrix-matrix interactions (known to be a source of matrix-derived ions).     

Detection of immune complexes by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to detect an immune complex formed between beta-lactoglobulin and polyclonal anti-beta-lactoglobulin antibody in the gas phase. The most important experimental parameters to detect such a specific antibody-antigen complex by MALDI were the use of solutions at near-neutral pH and of sinapinic acid matrix prepared by the dried-droplet method. Under such conditions, predominantly one but also two molecules of antigen protein were complexed by the antibody. Specific formation of the antibody-antigen complex was confirmed by performing competitive reactions. Addition of antibody to a 1:1 mixture of beta-lactoglobulin and one control protein resulted not only in the appearance of the expected antibody-antigen complex, but also in a strong decrease in the free beta-lactoglobulin signal, while the abundance of the control protein was not influenced.     

Investigation of calmodulin-peptide interactions using matrix-assisted laser desorption/ionization mass spectrometry

In this report, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to study the binding interactions between calmodulin and two target peptides (melittin and substance P). Various matrix conditions were tested and the less acidic matrix DHAP and THAP were found to favor the survival of the intact calcium-calmodulin as well as the calmodulin-peptide complexes. However, the application of direct MALDI-MS to detect the intact complexes turned out to be very difficult due to the dissociation of the complexes and the formation of nonspecific aggregates. In contrast, the specific binding of the target peptides to calmodulin could be easily deduced using intensity-fading (IF) MALDI-MS. Compared with the nonbinding control, clear reduction in the ion abundances of the target peptides was observed with the addition of calmodulin. Relative binding affinities of different peptides towards the protein could also be estimated using IF-MALDI-MS. This study may extend the application of IF-MALDI-MS in the analysis of noncovalent complexes and offer a perspective into the utility of MALDI-MS as an alternative approach to study the peptides binding to calmodulin.     

Identification of isoenzymes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The identification of isoforms is one of the great challenges in proteomics due to the large number of identical amino acids preventing their separations by two-dimensional electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has become a rapid and sensitive tool in proteomics, notably with the new instrumental improvements. In this study, we used several acquisition modes of MALDI-TOFMS to identify isoforms of porcine glutathiones S-transferase. The use of multiple proteases coupled to the different acquisition modes of MALDI-TOFMS (linear, reflectron, post-source decay (PSD) and in-source decay, positive and negative modes) allowed the identification of two sequences. Moreover, a third sequence is pointed out from a PSD study of a tryptic ion revealing the modification of the amino acid tyrosine 146 to phenylalanine.     

Characterization of unknown compounds from stainless steel plates in matrix-assisted laser desorption/ionization mass spectrometry

Peaks originating from unknown compounds on stainless steel plates used in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers are observed around m/z 304.3, 332.3, 360.4, and 388.4 regardless of the matrix and/or solvent, and are even observed with bare plates. These peaks were characterized using three different types of MALDI-MS instrumentation: MALDI-TOF MS, MALDI-TOF/TOF MS, and MALDI-FTMS. The fragmentation data from MALDI-TOF/TOF MS and accurate mass determination by MALDI-FTMS enabled identification of the chemical formulae and structures. The unknown compounds are, in fact, likely benzylalkylmethylammonium salts, as confirmed by closely matching fragmentation patterns with a commercially available benzalkonium chloride.     

Characterization of metal-labelled peptides by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry

Metal labelling of peptides and proteins using high-affinity metal-chelating compounds has found widespread applications in the medical and bioanalytical fields. In the present study we investigated the analysis of peptides derivatized either with cysteine- or amino group-directed metal-bound DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The metal complexes of DOTA were shown to be stable under MALDI-MS conditions. The introduction of the metal label led in a number of cases to significantly increased signal-to-noise (S/N) values and thus improved sensitivity of the labelled peptides compared to their unlabelled counterparts, especially for multiply labelled peptides. The presence of the labels did alter the tandem mass spectrometric (MS/MS) behaviour, namely the formation of sequence specific a-, b- and y-ion series, in dependence of the position of the label within the peptide sequence. For cysteine-derivatized peptides several label-specific reporter ions and characteristic immonium ions could be identified. Amino-directed labelling led only to the formation of characteristic immonium ions in ε-amino groups of lysine, whereas N-terminal labelling in some cases led to the formation of a(1)- and b(1)-ions. The results clearly show that MALDI-MS is suitable for the analysis of metal-labelled peptides, which was also confirmed in liquid chromatography (LC)/MALDI-based identification of proteins in a model protein mixture labelled with Cys-reactive DOTA. Here, in comparison to a run with alkylated cysteines, more than 50% more cysteine-containing peptides were identified.     

Improved mass accuracy in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides

One problem of matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry is the moderate mass accuracy that typically can be obtained in routine applications, Here we report improved mass accuracy for peptides, even when low amounts and complex peptide mixtures are used. A new procedure for preparing matrix surfaces is used, and there is no need to mix the matrix with the sample or to add internal standards. Examples are shown with a mass accuracy better than 50 ppm in a peptide mixture. Peptide mapping as well as sequencing by creating “ragged ends” or “ladder sequencing” should benefit especially from the improved mass accuracy.     

Interfacing matrix-assisted laser desorption/ionization mass spectrometry with column and planar separations

This paper presents a critical review of off-line and on-line coupling of matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry to liquid column separations (e.g. HPLC, GPC and CE) and planar separations (e.g. PAGE and TLC). Off-line MALDI analysis of fractions collected from HPLC, GPC and CE or spots scraped and extracted from TLC and PAGE has already become a routine practice for many laboratories. MALDI has also been used to obtain mass spectra directly from TLC plates and PAGE. The direct analysis methods range from dot-blotted samples to two-dimensional scanning of the entire gels/plates. Various combinations of on-line coupling of MALDI with column separations are also reviewed. The review discusses the strengths and limitations associated with different off-line and on-line coupling approaches.     

Interfacing matrix-assisted laser desorption/ionization mass spectrometry with column and planar separations

This paper presents a critical review of off-line and on-line coupling of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to liquid column separations (e.g. HPLC, GPC and CE) and planar separations (e.g. PAGE and TLC). Off-line MALDI analysis of fractions collected from HPLC, GPC and CE or spots scraped and extracted from TLC and PAGE has already become a routine practice for many laboratories. MALDI has also been used to obtain mass spectra directly from TLC plates and PAGE. The direct analysis methods range from dot-blotted samples to two-dimensional scanning of the entire gels/plates. Various combinations of on-line coupling of MALDI with column separations are also reviewed. The review discusses the strengths and limitations associated with different off-line and on-line coupling approaches.     

Analysis of plant phosphatidylcholines by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) for the quantitative determination of phospholipid (PL) molecular species has been problematic, due primarily to the formation of multiple signals (corresponding to the molecular ion and other adducts) for some classes of PL. For example, analysis of phosphatidylcholine (PC) yielded signals that corresponded to protonated and sodiated molecules in the MALDI spectrum. The resulting spectral overlap among various molecular species (e.g. [PC(16:0/18:2) + Na] and [PC(18:2/18:3)]) made it impossible to ascertain their relative amounts using this technique. Other spectral ambiguities existed among different structural isomers, such as PC(18:1/18:1) and PC(18:0/18:2). We determined that molecular species could be resolved by MALDI-TOFMS by first removing the polar head (e.g. phosphocholine) from the phospholipid to effect production of only the sodiated molecules of the corresponding diacylglycerols (DAGs). Analysis of the resulting spectrum allowed unequivocal determination of the molecular species profile of PC from potato tuber and soybean. Estimation of fatty acid composition based on the molecular species determined by MALDI-TOFMS analysis agreed with that from GC-FID analysis. Post-source decay (PSD) was used to resolve standard isomers of PC (e.g. 18:1/18:1 vs. 18:0/18:2). Our results indicated that PSD is a useful approach for resolving structural isomers of PL molecular species.     

Analysis of solid-supported oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This study presents matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) as a powerful tool to analyze and characterize oligonucleotides covalently linked to a solid support during their synthesis. The analysis of the fragment ions generated either in negative or positive mode allows direct and easy access to the nucleotide sequence and identification of the internucleosidic linkage. The mechanisms of the fragmentation of the solid-supported oligonucleotides induced by MALDI-TOFMS are discussed. Copyright 2000 John Wiley & Sons, Ltd.