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.     

Characterization of silsesquioxanes by size-exclusion chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Liquid chromatography in combination with spectroscopic methods like matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) or nuclear magnetic resonance (NMR) spectroscopy is a powerful method to characterize silsesquioxanes and silsesquioxane mixtures. As new examples, the formation of silsesquioxyl-substituted silsesquioxanes [(n-octyl)(7)(SiO(1.5))(8)](2)O and [(n-octyl)(7)(SiO(1.5))(8)O](2)[(n-octyl)(6)(SiO(1.5))(8)] as well as the cage rearrangement of octa-[(n-heptyl)silsesquioxane] to larger structures [(n-heptyl)SiO(1.5))](n) up to n=28 are shown.     

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.     

Characterization of mesogen-jacketed liquid crystalline polymers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

For synthetic polymers, a proper sample preparation method is essential for successful characterization by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In this work, six synthetic mesogen-jacketed liquid crystalline polymers (MJLCPs) with different main-chain, spacer and mesogenic units were investigated by MALDI-TOF mass spectrometry. Several factors that affect the analysis of these polymers were examined. These factors include matrices used, cationization salts used, the concentration of polymers, and the ratio of sample to matrix. After testing different conditions, we found a suitable sample preparation method for these six polymers. The number average molecular weight (M(n)), weight average molecular weight (M(w)) and polydispersity (PD) were calculated using data obtained in the linear mode. The end groups of the polymers were proposed using data obtained in reflectron mode. Copyright 2000 John Wiley & Sons, Ltd.     

Characterization of phosphopeptides from protein digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray quadrupole time-of-flight mass spectrometry

A two-step mass spectrometric method for characterization of phosphopeptides from peptide mixtures is presented. In the first step, phosphopeptide candidates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) based on their higher relative intensities in negative ion MALDI spectra than in positive ion MALDI spectra. The detection limit for this step was found to be 18 femtomoles or lower in the case of unfractionated in-solution digests of a model phosphoprotein, beta-casein. In the second step, nanoelectrospray tandem mass (nES-MS/MS) spectra of doubly or triply charged precursor ions of these candidate phosphopeptides were obtained using a quadrupole time-of-flight (Q-TOF) mass spectrometer. This step provided information about the phosphorylated residues, and ruled out nonphosphorylated candidates, for these peptides. After [(32)P] labeling and reverse-phase high-performance liquid chromatography (RP-HPLC) to simplify the mixtures and to monitor the efficiency of phosphopeptide identification, we used this method to identify multiple autophosphorylation sites on the PKR-like endoplasmic reticulum kinase (PERK), a recently discovered mammalian stress-response protein.     

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 humic substances by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and laser desorption/ionization (LDI-)TOFMS have been used to characterize Suwannee River humic substances, obtained from the International Humic Substances Society (IHSS), and Armadale soil fulvic acid (ASFA). An array of MALDI matrices were tested for use with humic substances, including alpha-cyano-4-hydroxycinammic acid (CHCA), 2-(4-hydroxyphenylazo)benzoic acid (HABA), 2,5-dihydroxybenzoic acid (DHBA), sinapinic acid, dithranol and norharmane. DHBA yielded the best results, exhibiting superior ionization efficiency, low noise, broad applicability to the analytes of interest, and most importantly producing an abundance of high mass ions, the highest observed being m/z 1848. A number of sample preparation modes were investigated; the overlayer method improved sample/matrix homogeneity and hence shot-to-shot reproducibility. The choice of the matrix, mass ratio of analyte to matrix, and the sample preparation protocol, were found to be the most critical factors governing the quality of the mass spectra. Matrix suppression was greatly enhanced by ensuring good mixing of matrix and analyte in the solid phase, proper optimization of the matrix/analyte ratio, and optimizing delayed extraction to ensure complete matrix-analyte reaction in the plume before ions are moved to the flight tube. A number of common features, in particular specific ions which could not be attributed to the matrices or to contaminants, were present in the spectra of all the humic substances, regardless of origin or operational definition. Additionally, a prominent repeating pattern of peaks separated by 55, 114 and 169 Da was clearly observed in both LDI and MALDI, suggesting that the humic compounds studied here may have quasi-polymeric or oligomeric features.     

Characterization of Aspergillus spores by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The intact fungal spores of several strains of four Aspergillus species, Aspergillus flavus, A. oryzae, A. parasiticus, and A. sojae, were directly analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Very simple MALDI mass spectra are obtained by directly mixing spores with a matrix such as alpha-cyano-4-hydroxycinnamic acid or sinapinic acid. The mass spectra are obtained from the ablation of cell walls of spores owing to the acidity of the matrix solution. The MALDI results show that aflatoxigenic strains and non-aflatoxigenic strains have different mass peak profiles. Furthermore, the MALDI results of non-aflatoxigenic A. flavus and A. parasiticus spores resemble those of the closely related A. oryzae and A. sojae spores, respectively.     

In-source decay characteristics of peptides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In-source decay (ISD) of peptides, coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, has been examined to determine the influence of the matrix, the susceptibility of amino-acid residues to ISD, and the effect of extraction delay times. Out of nine di- and tri-hydroxybenzoic acids and three cinnamic derivatives tested, the most suitable matrix for ISD was 2,5-dihydroxybenzoic acid. The amine bond at Xxx-Gly and Xxx-Val residues was less susceptible than other amino-acid residues to ISD; however, the more sensitive residue(s) were not as clear. Using a peptide that gave the y(n)- and (z(n) + 2)-series product ions, it was confirmed that amide-bond cleavage (formation of the y(n)-series ions) accompanied metastable peaks, whereas metastable peaks were never observed with amine-bond cleavage [formation of the (z(n) + 2)-series ions]. Furthermore, abundant c(n)-series ions, which originate from amine-bond cleavage on the peptide backbone, were observed whenever a minimum delay time of 38 ns or continuous extraction was used to obtain spectra. These data indicate that amine-bond cleavage in ISD takes place on the ionization time scale before the energy randomization is completed.     

Characterization of recombinant human serum albumin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Chromatographically purified recombinant human serum albumin (rHSA), produced in genetically transformed yeast cells, was characterized using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS techniques. The molecular mass of the intact protein was determined to be 66671, in good agreement with that of purified HSA which was used as a standard. The identity of rHSA to its natural counterpart was established with high precision using peptide mass fingerprinting of tryptic peptides. Partial amino acid sequence data for rHSA were obtained using Ettan CAF MALDI Sequencing Kit and post-source decay on the tryptic peptides. The results achieved provide strong evidence that MALDI-TOF-MS is an important analytical technique for characterising gene products and for establishing the identity and bio-compatibility of recombinant proteins relative to their natural counterparts.     

Ganglioside analysis by thin-layer chromatography matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry

Metastable decomposition of ions generated in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers complicates analysis of biological samples that have labile bonds. Recently, several academic laboratories and manufacturers of commercial instruments have designed instruments that introduce a cooling gas into the ion source during the MALDI event and have shown that the resulting vibrational cooling stabilizes these labile bonds. In this study, we compared stabilization and detection of desorbed gangliosides on a commercial orthogonal time-of-flight (oTOF) instrument with results we reported previously that had been obtained on a home-built Fourier transform mass spectrometer. Decoupling of the desorption/ionization from the detection steps resulted in an opportunity for desorbing thin-layer chromatography (TLC)-separated gangliosides directly from a TLC plate without compromising mass spectral accuracy and resolution of the ganglioside analysis, thus coupling TLC and oTOF mass spectrometry. The application of a declustering potential allowed control of the matrix cluster and matrix adduct formation, and, thus, enhanced the detection of the gangliosides.     

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.     

Tyrosinase-induced cross-linking of tyrosine-containing peptides investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Tyrosinase-induced oxidation of tyrosine is known to lead to melanin by cross-linking of 5,6-dihydroxyindole (DHI) and indole-5,6-quinone intermediates. However, tyrosinase-induced cross-linking of tyrosine-containing peptides has not been reported. We observed tyrosinase-induced adducts of tyrosine-containing peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). MALDI-TOFMS was also used to observe tyrosine adducts at various levels of oxidation derived from acid hydrolysis of the peptide adducts. The rate of tyrosinase-induced browning of lys-tyr-lys was about half of that of tyrosine. These results indicate that tyrosinase-induced browning of tyrosine-containing peptides via direct oxidation and cross-linking of the benzene ring of the tyrosine residue occurs at a significant rate and needs to be considered in melanogenesis.     

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.     

Characterization of polyether mixtures using thin-layer chromatography and matrix-assisted laser desorption/ionization mass spectrometry

Thin-layer chromatography (TLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were combined to achieve characterization of polyether mixtures. Three polyethers, polyethylene glycol (PEG), polypropylene glycol (PPG) and polytetramethylene glycol (PTMG), or mixtures of these compounds, were studied. One shortcoming of mixture analysis of synthetic polymers using MALDI-MS is that individual polymers in the mixture may display different detection sensitivities. For example, the MALDI mass spectrum of an equimolar mixture of PEG, PPG and PTMG displayed a high intensity of PPG ions, while no PTMG ions were detectable; however, PTMG ions were detected after the mixture had been separated by TLC. This combined TLC and MALDI-MS analysis of a PPG polymer bearing reactive epoxy groups showed that the polymer contained byproducts with different end-groups. These byproducts were identified as chloro-substituted polymers formed during polymer synthesis. Our study shows TLC to be a rapid and low-cost separation technique, and that it can be combined with MALDI-MS to achieve effective analysis of synthetic polymers.     

Sequencing of a branched peptide using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Chemical degradation methods combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and post-source decay (PSD)-MALDI reflex TOF mass spectrometry (MS) were used to determine the sequence of a peptide branched on to a known peptide backbone. This study was applied to a branched peptide model (derivative of substance P). The branched peptide mimics a digest of a membrane receptor on to which a derivative of substance P was photochemically linked. Chemical degradation based on N-terminal ladder sequencing in combination with MALDI-TOF-MS gave only partial sequence information. Although single PSD mass spectra still remain difficult to interpret unambiguously, PSD-MALDI-TOF-MS was combined with on-target acetylation and H -- D exchange to give a better and successful approach to the unambiguous determination of the complete amino acid side-chain sequence. This study shows the capability of MALDI-TOF-MS to help in characterizing ligand-receptor interactions.     

Capillary liquid chromatography interfaced to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an on-line coupled piezoelectric flow-through microdispenser

A piezoelectric flow-through microdispenser interfacing capillary liquid chromatography (LC) with matrix-assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-TOF MS) was developed for the identification of biomolecules. The MALDI target plate was placed on a computer controlled high-resolution x-y stage, on to which the column effluent was deposited as discrete spots, which thereby facilitated tracing of the chromatographic separation. The entire target plate was sprayed with a homogeneous layer of alpha-cyano-4-cinnamic acid mixed with nitrocellulose by using an air-brush. Hence the tedious manual handling of a micropipetter applying matrix solution on top of each fraction collected spot was avoided. The pre-made target plates were stable for at least 3 weeks if kept in darkness at room temperature, which easily allowed re-analysis of dispensed sample spots. The integrated microsystem was characterized and optimized by means of fluidics, dispersion, operational stability and sensitivity parameters. The dispensing unit was developed specifically to match high-resolution capillary LC separations using a dispenser with an internal volume from inlet to the ejecting nozzle of 250 nl. Minimizing dead volumes was crucial in order to maintain the chromatographic resolution. The volume of the ejected droplets was of the order of 60 pl. Successful separations of seven immunoregulating peptides were made: ACTH 1-17, bradykinin, enkephalin, angiotensin III, angiotensin II, angiotensin I and ACTH 18-39. On-line sample dispensing on the target plate in combination with trace enrichment followed by automated MALDI-TOF MS identification is demonstrated, reaching a sensitivity of 100 amol.     

Small molecule matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a polymer matrix

We have employed a light-absorbing electrically conductive polymer as a matrix to determine the molecular mass of small organic molecules using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method, which is in contrast to the usual MALDI strategy for matrix selection in which a small molecule matrix is used with a high molecular mass analyte, addresses the problem of matrix interference which limits the usefulness of MALDI-TOF for small molecule analysis. Use of negative ion mode offers advantages for this application. Using this approach, we have obtained clean molecular ion mass spectra of small organic molecules in the mass range 100-300 Da.