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.     

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.     

Application of matrix-assisted laser desorption/ionization to on-line aerosol time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectra were obtained from single biological aerosol particles using an aerosol time-of-flight mass spectrometer (ATOFMS). The inlet to the ATOFMS was coupled with an evaporation/condensation flow cell that allowed the aerosol to be coated with matrix material as the sampled stream entered the spectrometer. Mass spectra were generated from aerosol composed either of gramicidin-S or erythromycin, two small biological molecules, or from aerosolised spores of Bacillus subtilis var niger. Three different matrices were used: 3-nitrobenzyl alcohol, picolinic acid and sinapinic acid. A spectrum of gramicidin-S was generated from approximately 250 attomoles of material using a molar ratio of 3-nitrobenzyl alcohol to analyte of approximately 20:1. A single peak, located at 1224 Da, was obtained from the bacterial spores. The washing liquid and extract solution from the spores were analyzed using electrospray mass spectrometry and subsequent MS/MS product ion experiments. This independent analysis suggests that the measured species represents part of the B. subtilis peptidoglycan. The on-line addition of matrix allows quasi-real-time chemical analysis of individual, aerodynamically sized particles, with an overall system residence time of less than 5 seconds. These results suggest that a MALDI-ATOFMS can provide nearly real-time identification of biological aerosols. Copyright 2000 John Wiley & Sons, Ltd.     

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).     

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.     

Accurate mass measurement of synthetic analogues of prazosine by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) has been investigated as a tool for accurate determination of the molecular mass of synthetic analogues of prazosine, a molecule used for the treatment of hypertension. Samples were dissolved in methanol, mixed with mass calibration standards, and crystallised on the target with alpha-cyano-4-hydroxycinnamic acid as matrix. Acquisition of spectra was rapidly completed in reflectron mode, allowing high resolution (6000-10000) and sensitive (about 1-10 pmol of sample on target) determination of the synthetic products. The results show the effectiveness of MALDI-TOFMS for accurate mass determination of these fairly large molecules, which are otherwise difficult to analyse by other high-resolution mass spectrometric techniques.     

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.     

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.     

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.     

Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to rapidly screen for albuminuria

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is used as an alternative method for the rapid diagnosis of albuminuria. This technique requires no further sample pretreatment than simply mixing the urine sample with a MALDI matrix and drying under ambient conditions. The resulting MALDI mass spectra reveal albumin ions having charges ranging from +1 to +5. The detection of albumin is possible using any of the three most common MALDI matrices - sinapinic acid (SA), 2,5-dihydroxybenzoic acid (2,5-DHB), or 4-hydroxy-alpha-cyanocinnamic acid (alpha-CHC). Using this analytical approach, the limit of detection for albumin in urine is 10(-6) M, approximately 5 to 10 times lower than that detectable through conventional chemical testing.     

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.     

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.     

Dehydrogenation of tertiary amines in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis of various compounds synthesized in our laboratory, strong [M - H]+ ion peaks were often observed for the molecules with tertiary amino groups. In this work, the MALDI TOF MS behavior of two groups of compounds that incorporate tertiary amino moieties was investigated. One group is bisurea dimethylanilines (BUDMAs) prepared for the study of molecular recognition in thermoplastic elastomers, and the other group is the poly(propylene imine) diaminobutane dendrimers. The results clearly demonstrate the appearance of the [M - H]+ ions. In order to understand the possible mechanisms for the generation of these ions, a series of model compounds, ranging from primary to tertiary amines, were investigated. Unlike the tertiary amines, no [M - H]+ ion peaks were recorded for the primary amines, and only barely detectable ones, if any, for some secondary amines. It appears that the tertiary amino groups play an important role in the formation of these ions. In addition to MALDI TOF MS analysis, these samples were also applied to electrospray ionization (ESI) MS where no [M - H]+ ions were observed. The results indicate that the generation of [M - H]+ ion is due to the unique MALDI conditions and is likely to be formed via dehydrogenation of a protonated tertiary amine resulting in an N=C double bond. The absence of [M - H]+ ion peaks for the primary and secondary amines is probably because upon their formation these ions could easily transfer one proton to the corresponding amines in the MALDI gas-phase plume, yielding neutral imines that cannot be detected by MS.     

Detection of benzopyrene-deoxyguanosine adducts by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for the detection of BPDE-d guanosine adducts using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described and illustrated. The results indicate that MALDI is capable of detecting two other DNA benzopyrene adducts, which are trace products formed during the synthesis of BPDE-d guanosine. This MALDI-TOFMS method offers the potential for the detection of DNA adducts in human tissue using very limited sample purification and preparation.     

Ionization mechanism of oligonucleotides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The ionization of nucleosides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was systematically investigated using adenine (A), thymine (T), guanine (G) and cytosine (C) with several common matrices. Experimental results of the protonation and deprotonation of the bases of A, T, G and C in the matrices 2,5-dihydroxybenzoic acid (2,5-DHB), alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA) and 3-hydroxypicolinic acid (3-HPA) provide an insight into the ionization mechanism of oligonucleotides in MALDI. It was found that the low ion signal from DNA in poly-G in MALDI as reported in earlier work could be attributed to the fact that the base of G is difficult to ionize. Our results suggest that the ionization of DNA in MALDI is dominated by the protonation and deprotonation of bases and it is basically independent of the backbone of DNA. Both the protonation and deprotonation are strongly structure dependent. The protonation is dominated by pre-protonation before laser ablation, while the deprotonation is controlled by the thermal reaction.     

Characterization of some synthetic Ru and Ir complexes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied to the analysis of Ru(OCOCF(3))(2)(CO)(PPh(3))(2), Ru(OCOC(3)F(7))(2)(CO)(PPh(3))(2), Ir(tBuppy)(3) and Ir(ppy)(2)(acac) complexes. A troublesome problem in the MALDI-TOFMS characterization of these metal complexes is the possible replacement of complex ligands by matrix. In this contribution, 10 matrices, ranging from acidic to basic, were investigated: alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), dithranol, 2,4,6-trihydroxyactophenone (THAP), 6-azo-2-thiothymine (ATT), norharman, 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB), 4-nitroaniline (NA) and 2-amino-5-nitrophyridine (ANP). With most of the matrices, including the neutral and basic ones, matrix substitution of ligand could clearly be detected. Based on the experimental results, possible mechanisms of matrix substitution were discussed. It was demonstrated that the ligand exchange process might also occur through the gas-phase reactions initiated by laser shots. Among the matrices tested, DCTB was found to be the best one for the complexes that are prone to ligand exchange by matrix.     

Quantitation of synthetic polymers using an internal standard by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

MALDI-TOF MS is utilized to perform quantitative analysis on synthetic polymers. Despite the inherent limitations of MALDI, good quantitative results have been obtained in the three sets of experiments described here. An internal standard with similar molecular properties as the analytes is introduced. Plots of relative integrated intensity ratios as a function of theoretical ratios of stoichiometry are drawn based on the results. The satisfactory slopes and correlation coefficients illustrated the practicality of quantitative measurement by MALDI-TOF MS.     

Towards a reliable molecular mass determination of intact glycoproteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Different matrices and sample-matrix preparation procedures have been tested in order to study their influence on the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of intact glycoproteins, which present different degrees of glycosylation (human transferrin; bovine fetuin; bovine alpha(1)-acid-glycoprotein; recombinant human erythropoietin; and the novel erythropoiesis stimulating protein). Using sinapinic acid (SA) and the fast evaporation method, the studied glycoproteins became susceptible to fragmentation at any laser intensity, suggesting that this 'hot' matrix is unsuitable for a reliable molecular mass determination of glycosylated compounds. In contrast, 2,5-dihydroxybenzoic acid (DHB) and 6-aza-2-thiothymine (ATT), with an adequate sample-matrix preparation, provided improved results. Samples containing DHB after crystallization by vacuum drying demonstrated the best performance because the labile functional groups from the glycoforms were apparently fragmented to a lower extent. The average molecular masses obtained using this methodology were in all cases a better estimation than those values reported in the literature. The results were reproducible, and sensitivity was similar to that obtained with SA and the fast evaporation method. These excellent results suggest that this MALDI-TOF-MS methodology could be useful for an improved determination of the average molecular mass values of microheterogeneous compounds such as glycoproteins, glycosylated compounds or, in general, molecular mass values of molecules with similar labile functional groups.