Matrix-assisted Laser Desorption/ionization Conditions Research Articles

New suitable deprotonating matrices for the analysis of carboxylic acids and some acidic compounds by matrix-assisted laser desorption/ionization mass spectrometry in negative ion mode.

Direct non-derivatization analysis of organic acids and acidic compounds by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) in positive ion mode is not always possible due to the low ionization efficiency of analytes. Some new efficient deprotonating matrices were suggested that allowed the production of negative ions from acidic compounds during MALDI-MS experiments. Various tested carboxyl-containing compounds as well as compounds with acidic properties were mixed with the suggested deprotonating matrices [4-dimethylaminobenzaldehyde (DMABA), N,N-dimethylamino-p-phenylenediamine or 3-aminoquinoline] and applied on a standard MALDI target followed by recording MALDI mass spectra in negative ion mode. All the tested acidic compounds mixed with the suggested deprotonating matrices produced abundant [M - H]- ions under MALDI conditions. DMABA produced the strongest signals reflecting greater sensitivity of analysis. The suggested deprotonating matrices are commercially available compounds and are good alternatives to well-known matrices of this kind and, in particular, the often used 9-aminoacridine. DMABA is the best of the tested potential matrices and is suitable for the detection of low molecular weight carboxyl-containing compounds, substituted phenols, and mixtures of naphthenic acids by (-)MALDI-MS.

Features of Tryptic Peptides Providing Their Detection and Identification by MALDI Mass Spectrometry

Characteristics of tryptic peptides that provide the detection and identification of these compounds and corresponding proteins by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with α-cyano-4-hydroxycinnamic acid as a matrix are determined and discussed. Twenty-five proteins are identified; the features of reliably identified peptides are compared to those of the compounds that might be produced by trypsinolysis but were not detected. Two key factors enhancing a possibility of the detection of tryptic peptides are determined. One refers to the basicity of peptides in the gas phase, which is the highest for peptides with the C-terminal arginine residue; this reason for the emergence of significant analytical signals is well known. Another factor contributing to the ionization of peptides in study under MALDI conditions is their hydrophilicity. This result did not correlate with the most conclusions of earlier relevant studies discussed in the article.

Combining Salt Doping and Matrix Sublimation for High Spatial Resolution MALDI Imaging Mass Spectrometry of Neutral Lipids.

The combination of sodium salt doping of a tissue section along with the sublimation of the matrix 2,5-dihydrobenzoic acid (DHB) was found to be an effective coating for the simultaneous detection of neutral lipids and phospholipids using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry in positive ionization mode. Lithium, sodium, and potassium acetate were initially screened for their ability to cationize difficult to analyze neutral lipids such as cholesterol esters, cerebrosides, and triglycerides directly from a tissue section. The combination of sodium salt and DHB sublimation was found to be an effective cation/matrix combination for detection of neutral lipids. Further experimental optimizations revealed that sodium carbonate or sodium phosphate followed by DHB sublimation increases the signal intensity of the neutral lipids studied depending on the specific lipid family and tissue type by 10-fold to 140-fold compared with that of previously published methods. Application of sodium carbonate tissue doping and DHB sublimation resulted in crystal sizes ≤2 μm. We were thus able to image a mouse brain cerebellum at a high spatial resolution and detected 37 cerebrosides in a single run using a MALDI-TOF instrument. The combination of sodium doping and DHB sublimation offer a targeted and sensitive approach for the detection of neutral lipids that do not typically ionize well under normal MALDI conditions.

Toward understanding the ionization mechanism of matrix-assisted ionization using mass spectrometry experiment and theory.

Matrix-assisted ionization (MAI) mass spectrometry does not require voltages, a laser beam, or added heat to initiate ionization, but it is strongly dependent on the choice of matrix and the vacuum conditions. High charge state distributions of nonvolatile analyte ions produced by MAI suggest that the ionization mechanism may be similar to that of electrospray ionization (ESI), but different from matrix-assisted laser desorption/ionization (MALDI). While significant information is available for MAI using mass spectrometers operating at atmospheric and intermediate pressure, little is known about the mechanism at high vacuum. Eleven MAI matrices were studied on a high-vacuum time-of-flight (TOF) mass spectrometer using a 266 nm pulsed laser beam under otherwise typical MALDI conditions. Detailed comparisons with the commonly used MALDI matrices and theoretical prediction were made for 3-nitrobenzonitrile (3-NBN), which is the only MAI matrix that works well in high vacuum when irradiated with a laser. Screening of MAI matrices with good absorption at 266 nm but with various degrees of volatility and laser energies suggests that volatility and absorption at the laser wavelength may be necessary, but not sufficient, criteria to explain the formation of multiply charged analyte ions. 3-NBN produces intact, highly charged ions of nonvolatile analytes in high-vacuum TOF with the use of a laser, demonstrating that ESI-like ions can be produced in high vacuum. Theoretical calculations and mass spectra suggest that thermally induced proton transfer, which is the major ionization mechanism in MALDI, is not important with the 3-NBN matrix at 266 nm laser wavelength. 3-NBN:analyte crystal morphology is, however, important in ion generation in high vacuum. The 3-NBN MAI matrix produces intact, highly charged ions of nonvolatile compounds in high-vacuum TOF mass spectrometers with the aid of ablation and/or heating by laser irradiation, and shows a different ionization mechanism from that of typical MALDI matrices.

Novel Reactive Matrices for the Analysis of Alcohols by Matrix-Assisted Laser Desorption/Ioization Mass Spectrometry

A possibility of using a number of aromatic and heteroaromatic carboxylic acids and their halogen anhydrides as reactive matrix compounds for the analysis of alcohols of different structures by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been studied. It is shown that the acylation of alcohols with nicotinic and quinoline-6-carboxylic acid chlorides gives derivatives with high desorption/ionization efficiency under MALDI conditions, and that the free acids formed as a result of the hydrolysis of anhydrides act as matrix compounds. The proposed approach is tested on a number of aliphatic, alicyclic, and aromatic alcohols.

Letter: α,ω,N,N-Dimethylaminoalkylamines as possible derivatization agents for the analysis of small carbonyl compounds by low energy mass spectrometry.

Reaction with α,ω-N,N-dimethylaminoalkylamines (2-dimethylaminoethylamine, 3- dimethylaminopropylamine, 4-dimethylaminobutylamine) to form Schiff bases followed by quaternization of the N,N-dimethylamino group by alkyl (deuteroalkyl) halides to generate fixed-charge fragments is suggested for the characterization of carbonyl compounds by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. As model objects, some aliphatic aldehydes and alicyclic and steroid ketones were involved in the modification. Using gas chromatography mass spectrometry, the first modification stage proved to be quantitative. Not only the MALDI conditions but also the nanostructurized target provided spectra that revealed peaks for the cationic parts of derivatives. It was shown that the use of deuterated alkyl halides allows one to prepare deuterium-labeled standards for possible quantitative analysis.

Reactive matrices for matrix-assisted laser desorption/ionization mass spectrometry of primary amines.

Some aromatic carbonyl compounds [2,4-dihydroxybenzaldehyde (2,4-DHBA), 2,5- dihydroxyacetophenone (DHAP), 2,3,5-trihydroxybenzaldehyde and 2,4-dinitrobenzaldehyde] were examined as potential reactive matrices for the analysis of mono-, di-, and polyamines by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Although all the above compounds readily and quantitatively react with primary amines to form Schiff bases (the completeness of the reactions was proved by gas chromatography MS of derivatized aliphatic amines), only DHBA and DHAP provide efficient desorption/ionization under MALDI conditions. This means that two these aromatic carbonyl compounds can simultaneously exhibit properties of both derivatization agents and efficient matrices for MALDI-MS analysis. The use of such reactive matrices eliminates the need to perform additional derivatization experiments. With the aid of DHBA and DHAP, it was confirmed that each individual oligomer of polypropylene glycols and co- polyethylene/propylene glycols contained two terminal amino groups. To calculate the number of NH2 groups, mass increments of the introduced groups and a difference in the masses of cations attached to the initial and derivatized amines were used. The suggested approach, involving the application of the same reactive matrices, was applied to the determination of a number of NH(2) groups in individual oligomers of branched polyethyleneimines (PEIs) by MALDI-MS. In this case, DHAP appeared to be the best reactive matrix. The dependence of the number of NH(2) groups on the quantity of monomeric units and branching of each individual PEI oligomer is shown.

Matrix-assisted laser desorption/ionization, nanostructure-assisted laser desorption/ionization and carbon nanohorns in the detection of antineoplastic drugs. 1. The cases of irinotecan, sunitinib and 6-alpha-hydroxy paclitaxel.

The development of surface-assisted laser desorption/ionization (SALDI) methodologies in mass spectrometry allows, in principle, the development of new analytical approaches to qualitative and quantitative measurements on small molecules. Some of these methods have been applied to characterize two antineoplastic drugs: irinotecan (1) and sunitinib (2), and also 6-α-hydroxy-paclitaxel (3), the main metabolite of paclitaxel. Three different SALDI approaches have been tested employing nanostructure- assisted laser desorption/ionization (NALDI), carbon nanohorns (NHs) and carbon nanohorns covered by liquid additives. The results so obtained have been compared to those observed under matrix-assisted laser desorption/ionization (MALDI) conditions. Compounds 1 and 2 show the easy formation of protonated molecular species under all the experimental conditions, but the highest absolute intensity was achieved by NALDI. On the contrary, ionic species of low intensity are present for 3, among which are those that exhibit the highest intensity caused by [M+K](+) ions. After a critical evaluation of the obtained data, the linear response of the [M+H](+) ion intensity of 1 versus different deposited sample amounts was investigated, and the best results (R(2) = 0.9889) were obtained under MALDI conditions. The analysis of plasma samples spiked with 1 showed, again, that the MALDI approach was the best one (R(2) = 0.9766). The failure of NALDI measurements could be rationalized by the presence of ion suppression effects.

Simple approach to derivatization of alcohols and phenols for the analysis by matrix(surface)-assisted laser desorption/ionization time-of-flight mass spectrometry.

Direct analysis of hydroxyl-containing compounds by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) methods is not always possible due to the neutral character of analytes. The suggested fixed-charge derivatization may increase the ionization efficiency for various alcohols and phenols in specific matrix- and surface-activated LDI conditions. Aliphatic and steroid alcohols, as well as chlorophenols, were converted into various ammonioacetyl derivatives, containing a covalently bonded charged group, by reaction with bromoacetyl chloride and amine-type compounds such as triethylamine, pyridine or quinoline. The derivatives are suitable for MALDI-time-of-flight (TOF)MS analysis. Triethylammoniumacetyl, pyridyliumacetyl and quinoliniumacetyl derivatives were prepared from aliphatic alcohols, some sterols and chlorinated phenols in one stage with quantitative yields. The derivatives produced characteristic MALDI and SALDI mass spectra. The suggested derivatization approach for the modification of alcohols is simple and does not require any expensive reagents. The derivatives include a fixed charge and produce intense signals in MALDI (preferentially non-acidic matrices) and matrix-free SALDI (nanostructured target) conditions. Corresponding mass spectra are suitable for the determination of molecular mass and profiling of alcohols.

Contribution of thermal energy to initial ion production in matrix-assisted laser desorption/ionization observed with 2,4,6-trihydroxyacetophenone.

Although several reaction models have been proposed in the literature to explain matrix-assisted laser desorption/ionization (MALDI), further study is still necessary to explore the important ionization pathways that occur under the high-temperature environment of MALDI. 2,4,6-Trihydroxyacetophenone (THAP) is an ideal compound for evaluating the contribution of thermal energy to an initial reaction with minimum side reactions. Desorbed neutral THAP and ions were measured using a crossed-molecular beam machine and commercial MALDI-TOF instrument, respectively. A quantitative model incorporating an Arrhenius-type desorption rate derived from transition state theory was proposed. Reaction enthalpy was calculated using GAUSSIAN 03 software with dielectric effect. Additional evidence of thermal-induced proton disproportionation was given by the indirect ionization of THAP embedded in excess fullerene molecules excited by a 450 nm laser. The quantitative model predicted that proton disproportionation of THAP would be achieved by thermal energy converted from a commonly used single UV laser photon. The dielectric effect reduced the reaction Gibbs free energy considerably even when the dielectric constant was reduced under high-temperature MALDI conditions. With minimum fitting parameters, observations of pure THAP and THAP mixed with fullerene both agreed with predictions. Proton disproportionation of solid THAP was energetically favorable with a single UV laser photon. The quantitative model revealed an important initial ionization pathway induced by the abrupt heating of matrix crystals. In the matrix crystals, the dielectric effect reduced reaction Gibbs free energy under typical MALDI conditions. The result suggested that thermal energy plays an important role in the initial ionization reaction of THAP.

Screening Dyrk1A inhibitors by MALDI-QqQ mass spectrometry: systematic comparison to established radiometric, luminescence, and LC–UV–MS assays

Enzyme-catalyzed reactions play key roles in disease pathology, thus making them relevant subjects of therapeutic inhibitor screening experiments. Matrix-assisted laser desorption/ionization (MALDI) assays have been demonstrated to be able to replace established screening approaches. They offer increased sample throughput, but care must be taken to avoid instrumental bias from differences in ionization efficiencies. We compared a MALDI-triple-quadrupole (QqQ) method for the Dyrk1A peptide substrate woodtide to LC-MS, liquid chromatography with ultraviolet detection (LC-UV), luminescence, and radiometric assays. MALDI measurements were performed on a MALDI-QqQ instrument in the multiple-reaction monitoring mode. Different MALDI conditions were investigated to address whether matrix type, sample support, and MRM- or SIM-based detection conditions can be used to accommodate the molar responses of substrate peptide and its phosphorylated form. UV detection served as a reference method. The impact of MALDI matrix on IC50 values was small, even considering that matrix preparations were used that are known to alleviate response differences. IC50 values determined by MALDI were ca. 2-fold lower than those determined by LC-UV. Although MALDI generated lower ion yields for the phosphorylated peptide than for the peptide substrate, we found that a correction of compound potencies was readily possible using correction factors based on unbiased LC-UV results. A thorough method development delivered a robust assay with excellent performance (Z' > 0.91) that was close to that seen for LC-UV.

Ion‐to‐neutral ratio of 2,5‐dihydroxybenzoic acid in matrix‐assisted laser desorption/ionization

In most previous studies, the ratios of desorbed ions and neutrals from matrix-assisted laser desorption/ionization (MALDI) were measured outside the common MALDI conditions. In this work, we measured the ratios under common MALDI conditions. Ions were detected using a time-of-flight mass spectrometer in combination with a time-gated ion imaging detector. Mass-resolved desorbed neutral molecules at different angles and velocities were measured using a modified crossed molecular beam apparatus. The upper limit of the ion-to-neutral ratio from pure 2,5-dihydroxybenzoic acid (25DHB) is 4 × 10(-9) at laser fluence 40 J/m(2), it increases to 3 × 10(-7) at laser fluence 250 J/m(2). The ratios of matrix from the mixture of 25DHB and analyte remain in the same order of magnitude as pure 25DHB. However, the ratio of analyte depends strongly on the analyte. Values as large as 10(-3)-10(-4) for bradykinin and as small as <10(-8) for glycine were observed at laser fluence ~100 J/m(2). The ion-to-neutral ratios of 25DHB matrix measured in this work are much smaller than some of the values reported in previous work using different methods and/or under different MALDI conditions.

Characteristic features of mass-spectra of some nitroxide radicals

Some compounds containing mono- and polynitroxide radicals have been investigated and characterized by electron ionization (EI), chemical ionization (CI), and matrix-assisted laser desorption-ionization (MALDI) mass spectrometry. Some characteristics of the mass spectra of these radicals are demonstrated. It was found that, under CI and MALDI conditions, ions [M+nH]+ are formed along with monoprotonated molecules [M+H]+, depending on the type of the radical n = 2–5.

Application and Evaluation of Solvent-Free Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for the Analysis of Derivatized Fullerenes

A variety of derivatized fullerenes have been studied by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Of particular emphasis has been the evaluation of a recently introduced solvent-free sample/target preparation method. Solvent-free MALDI is particularly valuable in overcoming adverse solvent-related effects, such as insolubility and/or degradation of the sample. The method was applied to fullerene derivatives susceptible to decomposition under insufficiently "soft" MALDI conditions. Analytes included the hydrofullerene: C(60)H(36), fluorofullerenes: C(60)F(x) where x = 18, 36, 46, 48 and C(70)F(x) where x = 54, 56, methano-bridged amphiphilic ligand adducts to C(60) and the [4 + 2] cycloadduct of tetracene to C(60). The new solvent-free sample preparation is established as an exceedingly valuable addition to the repertoire of preparation protocols within MALDI. The MALDI mass spectra were of very high quality throughout, providing a testimony that "soft" MALDI conditions could be achieved. Using the [4 + 2] cycloadduct of tetracene to C(60) as the model analyte for direct comparison with solvent-based MALDI, the solvent-free approach led to less fragmentation and more abundant analyte ions. Applying solvent-free sample preparation, different matrix compounds have been examined for use in the MALDI of derivatized fullerenes, including sulfur, tetracyanoquinodimethane (TCNQ), 9-nitroanthracene (9-NA) and trans-2-[3-(4-tert-butylphenyl)-2-methyl-2- propenylidene]malononitrile (DCTB). DCTB was confirmed as the best performing matrix, reducing unwanted decomposition and suppression effects.

Combining Fluorescence Detection and Mass Spectrometric Analysis for Comprehensive and Quantitative Analysis of Redox-Sensitive Cysteines in Native Membrane Proteins

Monobromobimane (MBB) is a lipophilic reagent that selectively modifies free cysteine residues in proteins. Because of its lipophilic character, MBB is capable of labeling cysteine residues in membrane proteins under native conditions. Reaction of MBB with the sulfhydryl groups of free cysteines leads to formation of highly fluorescent derivatives. Here we describe a procedure for the detection and relative quantitation of MBB-labeled cysteines using fluorescence and mass spectrometric analyses, which allow determination of free cysteine content and unambiguous identification of MBB-modified cysteine residues. We have applied this approach to the analysis of the free and redox-sensitive cysteine residues of a large membrane protein, the sarcoplasmic reticulum Ca2+ release channel with a molecular mass of 2.2 million Da. Labeling was performed under physiologic conditions where the channel complex is in its native environment and is functionally active. The purified MBB-labeled channel complex was enzymatically digested, and the resulting peptides were separated by reversed-phase high-performance chromatography. MBB-labeled peptides were detected by fluorescence and identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Under MALDI conditions, partial photolytic fragmentation of the MBB-peptide bound occurred, thus allowing convenient screening for the MBB-modified peptides in the MS spectrum by detection of the specific mass increment of 190.07 Da for MBB-modified cysteine residues. Modification of the peptides was further confirmed by tandem mass spectrometric analysis, utilizing sequencing information and the presence of the specific immonium ion for the MBB-modified cysteine residues at m/z 266.6. Quantitative information was obtained by comparison of both fluorescence and MS signal intensities of MBB-modified peptides. Combination of fluorescence with MS detection and analysis of MBB-labeled peptides supported by a customized software program provides a convenient method for identifying and quantifying redox-sensitive cysteines in membrane proteins of native biological systems. Identification of one redox-sensitive cysteine (2327) in the native membrane-bound sarcoplasmic reticulum Ca2+ release channel is described.

Solvent-Free Mass Spectrometry for Hydrophobic Peptide Sequence Analysis and Protein Conformation Studies

Solvent-Free Mass Spectrometry for Hydrophobic Peptide Sequence Analysis and Protein Conformation Studies

Bimetallic silver-gold clusters by matrix-assisted laser desorption/ionization

Pure gold clusters (Aun+) were produced up to the cluster size of n = 100 by matrix-assisted laser desorption/ionization (MALDI). The mass spectrum of the resulting clusters showed alteration in the ion intensity at odd-even clusters size. On the other hand, intensity drops at cluster size predicted by the jellium model theory was also observed. Positively and negatively charged bimetallic silver-gold clusters were produced under MALDI conditions from the mixture of HAuCl4/silver trifluoroacetate and the 2-(4-hydroxyphenylazo)benzoic acid (HABA) matrix. A linear correlation was found between the intensity ratio of AunAgm+ to Au(n+1)Ag(m-1)+ cluster ions and the molar ratio of the gold to silver salt. It was observed that the composition and the distribution of the clusters can be varied with the molar ratio of the silver and gold salts. It was also found that the resulting cluster sizes obey the lognormal distribution.

Unprecedented Matrix-Induced Reduction of Flavins Observed Under FAB and MALDI Conditions

Flavin-derived compounds such as riboflavin, riboflavin 5-phosphate (flavin mononucleotide, FMN) and flavin-adenine dinucleotide (FAD), under matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB) conditions produce unprecedented reduced species that can be attributed to [M+2H]+. and [M+3H]+ in addition to the anticipated [M+H]+ ions. On the other hand, electrospray ionization (ESI) conditions generate only the expected [M+H]+ as the sole molecular-related ion. A protonation from the matrix accompanied by a concomitant electron transfer, rather than the transfer of hydrogen atom(s), [H. of CH groups], is proposed to explain the observed reduction process. The reduction site expands to N(1)=C(10a)-C(4a)=N(5), a 1,4-diaza-1,3-butadiene system, in the isoalloxazine ring, resembling the behavior of prosthetic groups of aerobic dehydrogenases. Interestingly, CID-MS / MS of most abundant molecular-related ions generated by the FAB and ESI modes, namely [M+2H]+. for FAB and [M+H]+ for ESI, produce the identical even-electron fragment ion of isoalloxazine moiety, indicating a radical loss vs. the evenelectron molecule loss from the individual precursor ions. The observed phenomena are compared with those previously reported for FABMS, thermospray mass spectrometry (TSPMS) and ESIMS. Keywords: Matrix-Induced Reduction, Flavins, thermospray, prosthetic groups, aerobic dehydrogenases

Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix

We describe two approaches employing electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and matrix assisted laser desorption/ionization (MALDI) post-source decay (PSD) for determining the location of an abasic site in modified oligodeoxynucleotides (ODNs). With MS/MS, we found both complementary fragment ions (a n′ and w n′) produced at the abasic site were predominant in the mass spectra and allowed the location to be determined. Under MALDI conditions, most ODNs carrying an abasic site are singly charged, and PSD gives predominately w n′ ions at the abasic sites, revealing their location. We also describe another approach for identifying and locating abasic sites in model ODNs; namely, an “in situ” derivatization coupled with MALDI mass spectrometry (MS). In general, an ODN n-mer containing an abasic site at the m-th position from the 5′-terminus can react with the matrix component, anthranilic acid, to form a Schiff base. The adduct upon MALDI breaks into 3′ and 5′ fragments (w n−m, b m, a m, d m−1) at the abasic site, revealing its location. ESI MS methods are also applicable for detecting the hydrazone derivatives of abasic sites, and the fragmentation of hydrazones shows the location of the abasic site.

MALDI-TOF mass spectrometry methods for evaluation of in vitro aminoacyl tRNA production

Unnatural amino acid mutagenesis requires the in vitro production of aminoacyl tRNAs. Bacteriophage T4 RNA ligase is used to ligate a-amino-protected dCA amino acids to 74mer tRNA. Previously, there has been no facile method for evaluating the efficiency of this reaction prior to using the tRNA in translation. We report a novel use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in monitoring the formation of aminoacyl 76mer tRNA. This method is more efficient and precise than the traditional technique of gel electrophoresis. These MALDI conditions should also prove useful for analyzing aminoacyl tRNAs produced through aminoacyl tRNA synthetases and other methods.