Matrix-assisted laser desorption/ionization tandem reflectron time-of-flight mass spectrometry of fullerenes

Atandem reflectron time-of-flight mass spectrometer developed in our laboratory provides a unique opportunity to investigate the collision-induced dissociation of fullerene ions formed by matrix-assisted laser desorption/ionization (MALDI). Specifically, this opportunity arises from the ability to utilize high energy collisional activation (normally available only on tandem sector instruments by using continuous ionization techniques) for ions formed by pulsed laser desorption, whereas most MALDI time-of-flight instruments record product ion mass spectra of ions formed by metastable or postsource decay. In this study we investigate the products of mass-selected and collisionally activated C ₆₀ ⁺ and C ₇₀ ⁺ ions by using different target gases over a range of target gas pressures. In general, heavier target gases produce more extensive fragmentation and improve the mass resolution of lower mass ionic products because a greater portion of these ions are formed by single collisions. Additionally, the tandem time-of-flight instrument utilizes a nonlinear (curved-field) reflectron in the second mass analyzer that enables high energy collision-induced dissociation spectra to be recorded without scanning or stepping the reflectron voltage.     

Characterization of poly(butylene terephthalate) by size-exclusion chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Poly(butylene terephthalate) (PBT) samples have been analyzed with size-exclusion chromatography (SEC) using a mixed solvent of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and chloroform as the mobile phase. Several matrices and different sample deposition methods have been investigated to analyze PBT with matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Optimum results have been acquired by depositing PBT on top of a 2,4,6-trihydroxyacetophenone matrix. The found MALDI-TOF-MS method can be used to analyze the end group functionalities of PBT, as demonstrated with the samples at hand. By combining SEC (off-line) with MALDI-TOF-MS, absolute molecular masses of PBT can be measured, and these have been found to be considerably lower than those determined with SEC using polystyrene standards.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of some conducting polymers

The application of laser desorption/ionization and matrix-assisted laser desorption/ionization mass spectrometry to the study of the structure of some conducting polymers was investigated. A methodology was developed and the experimental conditions for the characterization of these polymers were established. The parameters chosen to be changed for the method optimization are the matrix material (seven different matrices were tested depending on the polymer studied) and the solvent used in the preparation of the samples. The method seems to be suitable for the study of the structure of two oxidation states of polyaniline: emeraldine and pernigraniline. The analysis of the polymers derived from m-aminophenylbenzothiazole, benzothiazole and tetrathiopentalenes was also performed.} Copyright 2000 John Wiley & Sons, Ltd.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of dextran and dextrin derivatives

Application of matrix-assisted laser desorption/ionization (MALDI) to the analysis of dextran and dextrin derivatives, specifically glucose saccharides, by time-of-flight (TOF) mass spectrometry has been reported. MALDI-TOF analysis was carried out on alpha-, beta- and gamma-cyclodextrin, two O-methylated-beta-cyclodextrins of differing degrees of substitution (DS) and dextrans (a linear glucose saccharide), as pure and doped solutions and as mixtures of two or more of these. Doping was carried out with trace amounts of inorganic salts. The purpose of the analysis of the cyclodextrins was to determine whether they would form inclusion complexes with the various cations added, or whether less specific cation addition/exchange was occurring either prior to desorption or in the gas phase.     

Sequence determination in aliphatic poly(ester amide)s by matrix-assisted laser desorption/ionization time-of-flight and time-of-flight/time-of-flight tandem mass spectrometry

Poly(ester amide)s from dimethyl sebacate or sebacic acid and 2-aminoethanol or 4-amino-1-butanol were characterized by post-source decay matrix-assisted laser desorption/ionization time-of-flight (PSD-MALDI-TOF) and time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS). Sodiated oligomers were selected as precursor ions for dissociation studies. PSD analysis was performed on dimethyl sebacate, dicarboxylic, carboxylic and amino alcohol, and diamino alcohol terminated oligomers. PSD-MALDI-TOF mass spectra yielded information on the fragmentation mechanisms of the poly(ester amide) chains, showing that the main cleavages proceed through a beta-hydrogen transfer rearrangement. MALDI-TOF/TOF-MS/MS provided structural information concerning ester/amide sequences in the polymer chains. As expected, together with the ions appearing in the PSD-MALDI mass spectrum, several new abundant fragment ions in the low-mass range are present in MALDI-TOF/TOF-MS/MS spectra. These new product ions proved to be diagnostic and made it possible to establish the presence of random sequences of ester and amide bonds in the poly(ester amide)s samples.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipids: ionization and prompt fragmentation patterns

Ionization and prompt fragmentation patterns of triacylglycerols, phospholipids (PLs) and galactolipids were investigated using matrix-assisted laser desorption/ionization (MALDI). Positive ions of non-nitrogen-containing lipids appeared only in the sodiated form, while nitrogen-containing lipids were detected as both sodiated and protonated adducts. Lipids containing acidic hydroxyls were detected as multiple sodium adducts or deprotonated ions in the positive and negative modes, respectively, with the exception of phosphatidylcholines. The positive MALDI spectra of triacylglycerols contained prompt fragments equivalent to the loss of RCOO(-) from the neutral molecules. Prompt fragment ions [PL-polar head](+) were observed in the positive MALDI spectra of all phospholipids except phosphatidylcholines. The phosphatidylcholines produced only a minor positive fragment corresponding to the head group itself (m/z 184). Galactolipids did not undergo prompt fragmentation. Post-source decay (PSD) was used to examine the source of prompt fragments. PSD fragment patterns indicated that the lipid prompt fragment ions did not originate from the observed molecular ions (sodiated or protonated), and suggested that the prompt fragmentation followed the formation of highly unstable, probably protonated, precursor ions. Pathways leading to the formation of prompt fragment ions are proposed.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry studies of low molecular weight dendrimers

We report the application of matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry, with delayed extraction in the reflectron mode, for the characterization of low molecular weight dendrimers. 20 dendrimer samples were measured and 4 typical dendrimers, as examples, are discussed in detail. Several factors that affect the analysis including the matrices used, the concentrations of sample, the solvents and cationization reagent used, were investigated in detail. Experimental results indicate that the type of solvent can greatly influence exact mass measurement. However, sample preparation is generally not very critical for dendrimer analysis using MALDI-ToF since many kinds of matrices and a wide range of sample concentrations can be used efficiently. In addition, the Cs(+) ion can be used to enhance the efficiency of cationization. Some reasons for this behavior are discussed on the basis of results of calculations using Gaussian94 software (a connected system of programs for performing a variety of semi-empirical and ab initio molecular orbital (MO) calculations).     

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of underivatized and permethylated gangliosides

Underivatized and permethylated gangliosides have been studied by the matrix-assisted laser desorption (MALD) ionization technique. The samples investigated included commercially available and highly purified gangliosides from the human brain containing up to five sialic acid residues. Several permethylated gangliosides have also been studied, and MALD has proven successful in analyzing multicomponent mixtures of glycolipids with different fatty acyl residues. During the studies a variety of matrix and wavelength combinations have been tested in both the positive and negative ion modes. The best results have been obtained with the matrices 2,5-dihydroxybenzoic acid, 4-hydrazinobenzoic acid, 1,5-diaminonaphthalene, and 6-aza-2-thiothymine. Negative ion mass spectra of the underivatized gangliosides have always been of better quality than the positive ion mass spectra, exhibiting better signal-to-noise ratio, better resolution, less fragmentation, and less adduct formation with Na⁺ and K⁺. With increasing number of sialic acid substituents the molecular ion region became less and less resolvable, leading to broadened peaks even in the negative ion mode. Fragmentation could frequently be observed in the negative ion mode, and it was pronounced in the positive ion mode. The major fragmentation pathways corresponded to loss of sialyl group(s) and to decarboxylation of one of the sialyl residues. For underivatized gangliosides the typical sample amount used was 10–20 pmol. Permethylation led to a significant improvement in sensitivity (two orders of magnitude); the detection limit of permethylated gangliosides was about 10 fmol. The higher stability of the permethylated compounds was indicated by the fact that positive ion mass spectra exhibited only a marginal extent of fragmentation.     

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the analysis of polydienes

An analytical method based on matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry has been developed to provide information on oligomer structure, average molecular weight, and molecular weight distributions of polydienes (e.g., polybutadiene and polyisoprene), an important class of industrial polymers. This MALDI method involves the use of all-trans-retinoic acid as the matrix, copper (II) nitrate as the cationization reagent, and tetrahydrofuran as the solvent. The incorporation of this copper salt generates Cu⁺ adducts with the polymer chains. It also improves the signal strength and extends the upper mass range when used with all-trans-retinoic acid, as compared to silver nitrate. With this formulation, it is demonstrated that polybutadienes of narrow polydispersity with masses up to 300,000 u and polyisoprenes of narrow polydispersity with masses up to 150,000 u can be analyzed. The upper molecular weight limit is set by the requirement of using higher matrix-to-polymer ratios with increasing polymer molecular weight, to the point where the instrument can no longer detect the small quantity of polymer present in the matrix host. It is also shown that this sample preparation generates previously unreported adduction behavior. The practical implications of this adduction behavior on polymer structural analysis, accuracy of molecular weight determination, and the upper molecular weight limit of oligomer resolution are discussed. It is illustrated that, in a linear time-lag focusing MALDI instrument, oligomer resolution can be obtained for polydienes with molecular weights up to 24,000, providing structural confirmation of the end-groups and the repeat unit. The average molecular weights of a number of polydienes of narrow polydispersity determined by MALDI are compared to those obtained by gel permeation chromatography, and discrepancies are noted.     

Matrix-assisted laser desorption/ionization directed nano-electrospray ionization tandem mass spectrometric analysis for protein identification

In those cases where the information obtained by peptide mass fingerprinting or matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is not sufficient for unambiguous protein identification, nano-electrospray ionization (nano-ESI) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis must be performed. The sensitivity of nano-ESI/MS, however, is lower than that of MALDI-MS, especially at very low analyte concentrations and/or in the presence of contaminants, such as salt and detergents. Moreover, to perform ESI-MS/MS, the peptide masses of the precursor ions must be known. The approach described in this paper, MALDI-directed nano-ESI-MS/MS, makes use of information obtained from the more sensitive MALDI-MS experiments in order to direct subsequent nano-ESI-MS/MS experiments. Peptide molecular ions found in the MALDI-MS analysis are then selected, as their (+2) precursor ions, for nano-ESI-MS/MS sequencing, even though these ions cannot be detected in the ESI-MS spectra. This method, originally proposed by Tempst et al. (Anal. Chem. 2000, 72: 777-790), has been extended to provide better sensitivity and shorter analysis times; also, a comparison with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been performed. These experiments, performed using quadrupole time-of-flight instruments equipped with commercially available nano-ESI sources, have allowed the unambiguous identification of in-gel digested proteins at levels below their ESI-MS detection limits, even in the presence of salts and detergents.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry investigations of polystyrene and poly(methyl methacrylate) produced by monoacylphosphine oxide photoinitiation

The chain-end-group composition was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of low-molecular-weight polystyrene (PS) and poly(methyl methacrylate) (PMMA) produced by free-radical polymerization with a monoacylphosphine oxide, (2,4,6-trimethylbenzoyl) diphenylphosphine oxide (TPO), as a photoinitiator. Gel permeation chromatography (GPC) fractionation of the PS and PMMA samples with initial polydispersities of 1.81 and 2.81, respectively, yielded improved MALDI-TOF MS spectra. Spectral analyses of the PS fractions showed distributions attributable to PS having two diphenylphosphinyl ends and PS having one diphenylphosphinyl end and/or one 2,4,6-trimethylbenzoyl end, indicating that a combination of PS radicals with the highly reactive diphenylphosphine oxide group at one end of the chains was the predominant mode of termination. MALDI-TOF MS results for PMMA fractions provided evidence for termination primarily by disproportionation, but structure determination was confounded by the presence of isobaric peaks. Discernible peaks were obtained by MALDI-TOF MS analyses of GPC fractions of TPO-initiated poly(methyl-d₃ methacrylate-d₅), in which the major product was PMMA with a diphenylphosphinyl end group and an abstracted deuterium end group, whereas the minor combination product had two diphenylphosphinyl chain ends. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 2161–2171, 2007     

Matrix-assisted laser desorption/ionization mass spectrometry of taxanes

Taxanes are biologically active compounds that have been extensively used in pharmacology for their powerful anticancer properties. High specificity and low level sensitivity for analysis of these compounds have been obtained with reversed-phase high-pressure liquid chromatography/mass spectrometry (RP-HPLC/MS), but the number of applications of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for low molecular weight analytes is rapidly growing. A new MALDI-MS approach for the rapid screening of a variety of taxanes and a tandem mass spectrometric (MS/MS) analysis of the most important and diagnostic taxane fragmentation pathways are proposed. A solid-phase extraction method followed by preliminary quantification is also reported.     

Matrix-assisted laser desorption/ionization mass spectra reflect solution-phase zinc finger peptide complexation

The complexation between an 18-residue zinc finger peptide of CCHC type (CCHC=Cys-X₂-Cys-X₄-His-X₄-Cys, X=variable amino acid) from the gag protein p55 of human immunodeficiency virus type 1 (HIV-1) and various transition metal ions was studied by means of circular dichroism spectroscopy and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A correlation between the complexation behavior in solution and in MALDI-MS could be established. It was shown that MALDI-MS is a fast method suitable for studying metal binding properties of zinc finger complexes.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for metabolic flux analyses using isotope-labeled ethanol

We describe a novel method for the determination of the concentration and labeling degree of ethanol originating from 1-13C-labeling experiments. This method is suitable for high-throughput metabolic flux analysis because of the possible parallel sample preparation and fast final analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In a closed vial containing culture supernatant, ethanol is enzymatically oxidized to acetaldehyde. The acetaldehyde formed evaporates and is readily trapped in a second enclosed but open vial containing acidified 2,4-dinitrophenylhydrazine (DNPH). The 2,4-acetaldehyde dinitrophenylhydrazone (Ac-DNPH) that is formed is insoluble under these conditions. This leads to a constant conversion rate of the acetaldehyde produced from ethanol after 14 h minimum incubation time. MALDI-TOFMS was used to quantify the formed Ac-DNPH with [13C2]-ethanol as internal standard. The relative signal intensities of the unlabeled ethanol derivative as well as of [1-13C]-ethanol were linearly related to the ethanol concentration within a range of 1 to 50 mM with a limit of detection of 0.6 mM, a range which is sufficient for flux analysis in microtiter plate fermentation experiments. The method allows the estimation of the [1-13C]-ethanol originating from 1-13C-labeling experiments of Saccharomyces cerevisiae strains. In experiments where the expected flux range was exceeded, unlabeled ethanol was determined with a linear range from 30 to 500 mM. Ethanol quantification using this method was compared with enzymatic analysis and exhibited differences of less than 3.3% on average. Comparison of flux partitioning ratios between glycolysis and the pentose-phosphate pathway (PPP) based on MALDI-TOFMS and gas chromatography (GC)/MS methods showed good agreement, with differences for ethanol and alanine labeling of only 4.3%.     

Intact protein analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Direct tandem mass spectrometric (MS/MS) analysis of small, singly charged protein ions by tandem time-of-flight mass spectrometry (TOFMS) is demonstrated for proteins up to a molecular mass of 12 kDa. The MALDI-generated singly charged precursor ions predominantly yield product ions resulting from metastable fragmentation at aspartyl and prolyl residues. Additional series of C-terminal sequence ions provide in some cases sufficient information for protein identification. The amount of sample required to obtain good quality spectra is in the high femtomolar to low picomolar range. Within this range, MALDI-MS/MS using TOF/TOF trade mark ion optics now provides the opportunity for direct protein identification and partial characterization without prior enzymatic hydrolysis.     

New fragmentation mechanisms in matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry of carbohydrates

The spectra recorded by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) of complex carbohydrates from human milk are presented. Besides ions originating from glycosidic cleavages and from sugar ring fragmentations, these spectra show intense peaks that may be assigned to ions produced by three new fragmentation pathways involving a six-atom rearrangement. These ions, together with the A fragments from sugar ring fragmentations, open the possibility of obtaining a complete mapping of the linkage positions present in the carbohydrates investigated by MALDI-TOF/TOF.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of atom transfer radical polymerization macroinitiators derived from poly(3-ethyl-3-hydroxymethyloxetane)

The matrix-assisted laser desorption/ionization time-of-flight analysis of macroinitiators for atom transfer radical polymerization prepared by the functionalization of a multihydroxyl, highly branched polyether, poly(3-ethyl-3-hydroxymethyloxetane), allows the exact determination of the macroinitiator functionality, providing information on the number of bromine atoms in individual macromolecules, in contrast to NMR analysis, which gives average values. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 608–614, 2004     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of self-assembled monolayers of ruthenium complexes on gold

Self-assembled monolayers (SAMs) of three ruthenium complexes, [Ru(L)(2)](PF(6))(2), [Ru(L)(tpyPO(3))](PF(6))(2), and [Ru(L18)(tpyPO(3))](PF(6))(2), were prepared on evaporated gold films on glass or stainless steel plates; where L = 2, 6-bis(benzimidazoyl)pyridine, tpyPO(3) = 2,6-bis(2,2':6', 2"-terpyridyl)pyridine phosphanate, and L18 = 2, 6-bis(N-octadecylbenzimidazoyl)pyridine. Structures of these SAM complexes were studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The SAMs were either prepared by direct binding of Ru-complexes to Au films by alkanethiol or by the multilayer method. In the multilayer method 1,4-thiobutylphosphate was used to form a base layer on an Au film, and the base layer was then chemically bridged to the Ru-complexes by zirconium phosphate. MALDI-TOFMS of SAM1, that had been prepared by direct binding of [Ru(L)(2)](PF(6))(2) to the Au film by an octanethiol group, showed cleavage at the S-Au linkages and elimination of the counter anion to yield a molecular ion and its dimeric ion. On the other hand, SAM2 and SAM3, which had been prepared by bridging Ru-complexes [Ru(L)(tpyPO(3))](PF(6))(2) or [Ru(L18)(tpyPO(3))](PF(6))(2) to the base layers with zirconium phosphate, showed dissociation from the base layers and elimination of the counter anion to give ions of the Ru complex molecules and their fragmentation ions. No molecular ion containing the base layer resulting from the S-Au bond cleavage was observed. Copyright 2000 John Wiley & Sons, Ltd.     

Matrix-assisted laser desorption/ionization mass spectrometry using a visible laser

Visible matrix-assisted laser desorption/ionization (VIS-MALDI) was performed using 2-amino-3-nitrophenol as matrix. The matrix is of near-neutral pH, and has an optical absorption band in the near-UV and visible region. A frequency-doubled Nd:YAG laser operated at 532 nm wavelength was used for matrix excitation and comparisons were made with a frequency-tripled Nd:YAG laser (355 nm). Visible and ultraviolet (UV)-MALDI produce similar mass spectra for peptides, polymers, and small proteins with comparable sensitivities. Due to the smaller optical absorption coefficient of the matrix at 532 nm wavelength, the optical penetration depth is larger, and the sample consumption per laser shot in VIS-MALDI is higher than that of UV-MALDI. Nevertheless, VIS-MALDI using 2-amino-3-nitrophenol as matrix may offer a complementary technique to the conventional UV-MALDI method in applications where deeper laser penetration is required.     

Differentiating structural isomers of sialylated glycans by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry

Using model acidic glycans, we demonstrate the benefits of permethylation for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI/TOF-TOF) tandem mass spectrometry. With both the linear and branched structures, extensive cross-ring fragmentation product ions were generated, yielding valuable information on sugar linkages. Elimination of the negative charges commonly associated with sialylated structures through permethylation allowed their structural analysis in the positive ion mode. Extensive A- and X-type ions were observed for the linear structures, and slightly weaker signals for the branched sialylated structures. The diagnostic cross-ring fragments, permitting a distinction between alpha2-3 and alpha2-6 linkages of the sialic acid residues, were seen in abundance. Importantly, the cross-ring fragmentation with the branched structures provides adequate information to assign sialic acid residues, with a specific linkage, to a particular antenna.