Solid state nuclear magnetic resonance as a tool to explore solvent-free MALDI samples

Solid-state Nuclear magnetic resonance (NMR) was used here to explore structural characteristics of samples to be subjected to matrix-assisted laser desorption/ionization (MALDI) and prepared without the use of any solvent. The analytical systems scrutinized in NMR were mixtures of a 2,5-dihydroxybenzoic acid (2,5-DHB) matrix and caesium fluoride (CsF), used as the cationization agent in synthetic polymer MALDI mass analysis, at different molar ratios (1:1, 5:1, and 10:1). Complementary information could be obtained from ¹³C, ¹³³Cs, and ¹⁹F NMR spectra. Grinding the matrix together with the salt in the solid state was shown to induce a strong modification in the molecular organization within the MALDI sample. The evidenced mechano-induced reactions allow strong interactions between the matrix and the cation, up to the formation of a salt, and only occur in the presence of some water molecules. Addition of a poly(ethylene oxide) polymer as the analyte did not further modify the observed molecular organizations. Although relative matrix and salt concentrations in the scrutinized samples were unusual for MALDI analysis, mass spectra of good quality could be obtained and revealed that cation attachment on polymers during the MALDI process is not a matrix-independent event since a lower ionization efficiency was obtained from highly organized solid samples, mostly consisting of 2,5-DHB caesium salt species.     

Use of meso- tetrakis(pentafluorophenyl)porphyrin as a matrix for low molecular weight alkylphenol ethoxylates in laser desorption/ ionization time-of-flight mass spectrometry

The use of UV-absorbing molecules as matrices in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is well documented. The matrices that are currently used have low molecular weights (<300 Da) and thus, for a typical MALDI-TOF spectrum, the low-mass range (m/z 100-500) is dominated by matrix ions. Consequently, the applications of MALDI-TOFMS have been restricted mostly to the analysis of high molecular weight analytes. This report demonstrates the use of meso-tetrakis(pentafluorophenyl)porphyrin (F20TPP, MW 974.57) as a matrix in the MALDI-TOF mass spectrometric analysis of some commercial nonylphenol ethoxylates (4-(C(9)H(19))-C(6)H(4)-(OCH(2)CH(2))(n)-OH), in which the ethoxymer ion distribution ranges from 331-771 Da. When F20TPP was used without a sodium ion dopant, there were no MALDI signals for the ethoxylates. However, addition of sodium acetate to the sample produced MALDI spectra in which the ethoxymer molecules were sodiated to form [M + Na](+) ions. A comparison of the mass spectrometric data with those obtained when alpha-cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix indicated that the F20TPP-induced spectra provided comparable data, with the advantage of having less matrix interference in the low-mass range (m/z 100-500). Thus, the use of F20TPP and similar porphyrins may provide the means to apply MALDI-TOF to the analysis of low molecular weight molecules with minimum interference from matrix signals. Copyright 1999 John Wiley & Sons, Ltd.     

Deep‐ultraviolet laser ablation electrospray ionization mass spectrometry

A 193‐nm wavelength deep ultraviolet laser was used for ambient laser ablation electrospray ionization mass spectrometry of biological samples. A pulsed ArF excimer laser was used to ablate solid samples, and the resulting plume of the desorbed material merged with charged electrospray droplets to form ions that were detected with a quadrupole time‐of‐flight mass spectrometer. Solutions containing peptide and protein standards up to 66‐kDa molecular weight were deposited on a metal target, dried, and analyzed. No fragmentation was observed from peptides and proteins as well as from the more easily fragmented vitamin B₁₂ molecule. The mass spectra contained peaks from multiply charged ions that were identical to conventional electrospray. Deep UV laser ablation of tissue allowed detection of lipids from untreated tissue. The mechanism of ionization is postulated to involve absorption of laser energy by a fraction of the analyte molecules that act as a sacrificial matrix or by residual water in the sample.     

Thin-layer chromatography--postsource-decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of small drug molecules

The structural analysis of small drug molecules by directly coupling thin-layer chromatography (TLC) with postsource-decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is reported. The applicability of this technique is shown using two examples: the TLC-PSD MALDI analysis of two representatives of nonsteroidal antiinflammatory drugs (tenoxicam and piroxicam) and the analysis of the pharmaceutically active compound UK-137,457 and one of its related substances UK-124,912. The matrices alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA) and graphite are used to investigate the effect of the precursor ion selection on the TLC-PSD MALDI spectra of the drug molecules studied. Although alpha-CHCA enhances the [M+H]+ ion formation graphite produces in general only sodium adducts. Structural differentiation of tenoxicam and piroxicam is possible only by selecting the sodium adduct of both drug molecules as precursor ions. In the case of the TLC-PSD MALDI analysis of UK-137,457 and its related substance UK-124,912 at the 1% level, the PSD spectra obtained in alpha-CHCA by selecting the protonated adduct of the small molecules as precursor ions shows distinguishable dissociation patterns containing structurally significant information.     

Incubation with Cu(II) and Zn(II) salts enhances MALDI‐TOF mass spectra of amyloid‐beta and α‐synuclein toward in vivo analysis

Insoluble senile plaque aggregates are indicative of Alzheimer's disease pathology. A similar phenomenon occurs in Parkinson's disease with the build‐up of Lewy bodies. The analysis of senile plaques, and other brain samples, from Alzheimer's disease and Parkinson's disease patients by matrix‐assisted laser desorption/ionization mass spectrometry has advantages but also presents obstacles because of the nature of the processes utilized in isolation procedures and storage. Salts, buffers, and detergents necessary in the isolation of biological species may cause adducts and ion suppression that convolute the spectra obtained. We previously determined that amyloid‐beta from isolated senile plaque deposits fragment similarly to the synthetic 40 and 42 amino acid peptide when analyzed by matrix‐assisted laser desorption/ionization mass spectrometry. In addition, α‐synuclein also fragments predictably by in‐source decay. This provides information that may be applied to the identification and localization of amyloid‐beta and α‐synuclein in senile plaques and intact tissue sections. Ion suppression must still be accounted for when analyzing biological samples, which makes identifying fragments at lower abundance difficult. The addition of certain transition‐metal salts (Cu(II), Zn(II)) to the sample prior to analysis serves to “clean” the spectra and allow the peptide fragments produced to be observed with a much higher signal to noise and occasionally, improved resolution. We present a systematic study of incubation with different metal salts and their impact on the quality of the spectra, as well as the role of the binding of the metals to the model biological compounds, obtained for synthetic amyloid‐beta, synthetic α‐synuclein, and isolated senile plaques. The optimized sample preparation methods presented will provide for simpler and more thorough identification of these biologically relevant species in human‐derived samples.     

Copper(I) chloride: a simple salt for enhancement of polystyrene cationization in matrix-assisted laser desorption/ionization mass spectrometry

The possibility of using copper(I) chloride as a doping salt to enhance the cationization of polystyrene in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was investigated. It was shown that copper(I) chloride possesses sufficient solubility in tetrahydrofuran. The parameters of the MALDI mass spectra of different polystyrene samples, such as the number-average (M(n)) and mass-average (M(w)) molecular mass values, obtained by copper(I) cationization were compared with those obtained by means of silver(I) cationization, and good agreement was found. It was also shown that application of copper(I) chloride as a doping salt, and dithranol as a matrix, ensured good MALDI mass spectra of the sample spots even after storage for 1 month.     

Matrix-assisted laser desorption ionization mass spectrometry with 2-(4-hydroxyphenylazo)benzoic acid matrix

A novel matrix substance, 2-(4-hydroxyphenylazo) benzoic acid, or HABA, has been found to be very advantageous for matrix-assisted ultraviolet laser desorption ionization mass spectrometry. This compound has been successfully used for the desorption of peptides, proteins, and glycoproteins up to approximately 250 kDa. For these materials, the most abundant analyte-related peaks correspond to [M + H]⁺ ions and multiply protonated molecules. Comparisons with sinapic acid, 2,5-dihydroxybenzoic acid, and α-cyano-4-hydroxycinnamic acid indicate that the new matrix provides comparable sensitivity for peptides and smaller proteins but results in better sensitivity for larger proteins and glycoproteins in protein mixtures. Other matrices discriminate against the higher mass components in these cases. Somewhat reduced mass resolution has been found for smaller proteins, but for larger proteins and glycoproteins the best mass resolution can often be obtained with the new matrix. For other classes of compounds that form ions predominantly via cation attachment, at least as good sensitivity and even better resolution have been obtained. Derivatized glycolipids and synthetic polymers have been studied in detail. For the analysis of many synthetic polymers, the best performance in terms of sensitivity and mass resolution has been observed with HABA matrix. Mass resolution was higher for cation adducts than for the protonated peptide molecules in the same mass range. The new matrix exhibits greatly extended (in time) analyte ion production and reproducibility. Owing to the uniform sample surface with this matrix, barely any spatial variation of the ion signal could be observed. In addition, many hundreds of single-shot mass spectra could be accumulated from the same spot, even for larger proteins.     

Artifact-free matrix-assisted laser desorption ionization time-of-flight mass spectra of tert.-butyldimethylsilyl ether derivatives of cyclodextrins used for the synthesis of single-isomer, chiral resolving agents for capillary electrophoresis

Artifact-free, high-resolution matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectra have been obtained for the labile, single-isomer, tert.-butyldimethylsilyl ether derivatives of alpha-, beta- and gamma-cyclodextrins by optimizing the MALDI sample preparation method. 2,5-Dihydroxybenzoic acid, a 3:1 mixture of 2,5-dihydroxybenzoic acid and 1-hydroxyisoquinoline, and 2,4,6-trihydroxyacetophenone were investigated as MALDI matrices with methanol and acetonitrile as matrix solvents. Partial-to-complete loss of the tert.-butyldimethylsilyl groups was observed when the commonly used 2,5-dihydroxybenzoic acid was the MALDI matrix and/or methanol was the solvent, both with and without trifluoroacetic acid as additive. Loss of the labile tert.-butyldimethylsilyl groups was avoided with 2,4,6-trihydroxyacetophenone as MALDI matrix and acetonitrile as matrix solvent. Good ion intensities were achieved for the (M+Na)+ and (M+K)+ quasimolecular ions in the positive-ion mode. Minor byproducts were observed in some of the samples and the information was used to aid the optimization of the synthetic work.     

Ion-pair assisted recovery of matrix-assisted laser desorption/ionization mass spectral signals from SDS-containing peptide-protein mixtures

We have developed a simple and effective means of using alkylammonium ion-pairing agents, such as cetyltetramethylammonium bromide, to recover matrix assisted laser desorption/ionization mass spectrometric (MALDI-MS) signals from sodium dodecyl sulfate (SDS)-containing protein and peptide samples. A two-layer method of matrix preparation, with a bottom matrix layer of ion-pairing agents and a top matrix layer of SDS protein samples, is essential for reproducible MALDI mass spectra with good recovery. Both buffer ions and ion-pairing agents have profound effects on signal recovery and can be rapidly and systematically optimized. This practical technique, termed ion-pair assisted recovery (IPAR), is compatible with major SDS-based biotechniques and can be easily incorporated into high-throughput proteomic analysis.     

Mass spectrometric investigation of the side-arm lariat effect of ortho- and para-methoxyphenoxymethyl-15-crown-5 in the gas phase

Mixtures of unsubstituted 15-crown-5 and its analogues containing ortho- and para-methoxyphenoxymethyl substituents with sodium salts were investigated by matrix assisted laser desorption/ionization (MALDI) mass spectrometry. Peaks of cationized molecules [M+Na]⁺ and cluster ions [2M+2Na+An]⁺, where M is the crown ether molecule and An is monobasic acid anion, were observed in the mass spectra. It was shown that an increase of the shielding degree of the sodium cation in complexes with crown ethers, i.e., the lariat effect, led to a significant decrease in the intensity of peaks of the cluster ions.     

High-sensitivity negative-ion laser spray for liquid chromatography/mass spectrometry.

A negative-ion mode laser spray interface for use in liquid chromatography/mass spectrometry (LC/MS) has been developed and investigated. The laser spray gave sensitivities that were orders of magnitude better than the negative-ion electrospray for all samples investigated with the exception of sugars. Fragment ions, HSO(-4)and SO(-3), were formed from the laser-sprayed aqueous solution of cholesterol 3-sulfate sodium salt. This suggests that structural information may be obtained directly from the laser-spray mass spectra for thermally labile compounds.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an inorganic particle matrix for small molecule analysis

Fine metal or metal oxide powder as an alternative to conventional organic matrices in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been utilized successfully for lower molecular mass analytes, poly(ethylene glycol) 200 (PEG 200) and methyl stearate. Eleven kinds of particle, Al, Mn, Mo, Si, Sn, SnO2, TiO2, W, WO3, Zn and ZnO, were evaluated. The analyte was mixed with a metal or metal oxide powder (inorganic matrix) with particle diameter of tens of micrometers and liquid dispersant, followed by application to the sample target. Using a commercial MALDI-TOFMS instrument equipped with an internal 337 nm pulsed nitrogen laser, the analytes, PEG 200 and methyl stearate, were ionized as the alkali metal ion adducted molecules [M+Na]+ or [M+K]+ when the inorganic matrices Mn, Mo, Si, Sn, TiO2, W, WO3, Zn or ZnO were used. In the case of an Al matrix, PEG 200 was ionized as [M+K]+, whereas methyl stearate was ionized as [M+H]+ and [M+Al]+. These particles have potential as the matrix for MALDI. During our examination, however, only SnO2 particles did not ionize either PEG 200 or methyl stearate. Based on our protocol, when TiO2 powder was suspended with liquid paraffin, PEG 200 and methyl stearate gave their MALDI-TOF mass spectra with the lowest background noise and highest intensity. TiO2 powder seemed to be a broad potential matrix for low molecular mass polar or non-polar analytes. The results suggested that bulk particles caused rapid heating/vaporization processes and ionized analyte molecules under irradiation with a pulsed UV laser. The present method can be readily applied to obtain the low background noise MALDI-TOF mass spectra of small-sized compounds.     

3-Mercaptopropionic acid modified ZnSe quantum dots as the matrix for direct surface-assisted laser desorption/ionization mass spectrometric analysis of peptides/proteins from sodium salt solution

This study describes a strategy of using zinc selenium quantum dots (ZnSe QDs) modified with 3-mercaptopropionic acid (3-MPA) as the matrix for direct analysis of peptides and proteins from sodium salt solution in surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). The enhancement of detection sensitivity for these biomolecules was due to the adsorption of positively charged peptides or proteins onto the surfaces of negatively charged ZnSe-3MPA QDs via electrostatic interactions resulting in an increase in ionization efficiency for sodium adduct ions ([M+Na](+)). The applicability of the current approach was demonstrated for a variety of peptides, including leucine-enkephalin, methione-enkephalin, HW6, substance P and angiotensin II, and proteins (cytochrome c, myoglobin and lysozyme). Signal intensities of these peptides or proteins can be enhanced by 25-95 times compared with those obtained by LDI-MS in the absence of ZnSe-3MPA QDs. Applying ZnSe-3MPA QDs to serve as the matrix in SALDI-MS is a simple and effective approach for direct analysis of peptide and protein molecules from sodium salt solution without any pretreatment as the peptides and proteins can be successfully detected as sodium adduct ions ([M+Na](+)).     

Size exclusion chromatography/mass spectrometry applied to the analysis of polysaccharides.

A novel method for analysing polysaccharide materials is described which employs size-exclusion chromatography (SEC) followed by detection by on-line electrospray ionisation mass spectrometry (ESI-MS) and off-line matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). It is demonstrated through SEC/ESI ion trap mass spectrometry that the formation of multiply charged oligomer ions, which bind up to five sodium cations, allows the rapid analysis of polysaccharide ions with molecular weights in excess of 9 kDa. MALDI spectra generated from fractionation of the effluent collected from the same SEC separation are shown to be in good agreement with the ESI spectra with respect to molecular weight distributions and types of ions generated. ESI and MALDI mass spectra of samples obtained from sequential graded ethanol precipitation and SEC fractionation of acid and enzymatically digested arabinoxylan polysaccharides show important structural differences between polysaccharide fragments. In addition, a comparison is made between the mass spectra of native and permethylated SEC-separated fragments of acid and enzymatically treated arabinogalactan. Linkage information of the permethylated arabinogalactan oligomers can be rapidly established through the use of on-line SEC/ESI-MS( n) experiments.     

Characterization of inorganic coordination complexes by matrix-assisted laser desorption/ionization mass spectrometry

We report the direct laser desorption/ionization (LDI) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis of four inorganic coordination complexes: monometallic [Ir(dpp)(2)Cl(2)](PF(6)), homonuclear trimetallic ([(bpy)(2)Ru(dpp)](2)RuCl(2))- (PF(6))(4), and heteronuclear [(tpy)Ru(tpp)Ru(tpp)RhCl(3)](PF(6))(4) and ([(bpy)(2)Ru(dpp)](2)IrCl(2))(PF(6))(5) (dpp = 2,3-bis-(2'-pyridyl)pyrazine, bpy = 2,2'-bipyridine, tpy = 2,2',6',2"-terpyradine, tpp = 2,3,5,6,-tetrakis-(2'-pyridyl)pyrazine). Spectral intensities and fragmentation patterns are compared and evaluated for instrument parameters, matrix selection, and matrix-to-analyte ratio. Direct LDI and MALDI mass spectra of the monometallic complex showed the same ion peaks and differed only in the relative peak intensities. Direct LDI of the trimetallic complexes produced only low-mass fragments containing one metal at most. MALDI spectra of the trimetallic complexes exhibited little fragmentation in the high-mass region (>1500 Da) and less fragmentation in the low-mass region compared to direct LDI. Significant fragments of the molecules were detected and identified, including ligand fragments, intermediate-mass fragments such as [Ru(tpy)](+), and molecular ions with varying degrees of PF(6)(-) loss ([M - n(PF(6))](+), where n = 1-3). A correlation exists between the solution-phase electrochemistry and the observed [M - n(PF(6))](+) series of peaks for the trimetallic complexes. Proper matrix selection for MALDI analysis was vital, as was an appropriate matrix-to-analyte ratio. The results demonstrate the applicability of MALDI-TOFMS for the structural characterization of labile inorganic coordination complexes.     

Structural Characterization of Normal and Modified Oligonucleotides by Matrix-assisted Laser Desorption Fourier Transform Mass Spectrometry

Matrix-assisted UV laser desorption Fourier transform mass spectrometry (266 nm, nicotinic acid matrix) can be used for the detailed structural characterization of normal and modified oligonucleotides. The negative ion spectra for these compounds revealed abundant (M ? H) ^- ions as well as fragment ions that provided the information necessary to determine oligomer sequence and to differentiate isomers. The nicotinic acid matrix was required for the production of (M ? H) ^- ions for the oligonucleotide dimers, trimers, tetramers, and hexamers examined in this study. Elimination of the nicotinic acid matrix resulted in complete loss of the (M ?H) ^- ions as well as most of the larger fragment ions for the oligomers. The primary fragmentation pathway was observed to be phosphate ester bond cleavage with the resulting charge retained on the 3’ end of the oligomer and enabled isomeric differentiation of compounds such as d(S’-CGCG-3’) and d(S’-CCGG-3’). Collisioninduced dissociation experiments of the (M ? H) ^- ions for these compounds confirmed the preferential loss of nucleotides from the 5’ end of the oligomers. The presence and location of modifications such as methyl and ethyl alkyl groups to the oligonucleotides could also be identified.     

Electrospray tandem mass spectrometry for structural characterization of aporphine- benzylisoquinoline alkaloids

Electrospray mass spectrometry and tandem mass spectrometry techniques were utilized to elucidate the structures of ten aporphine-benzylisoquinoline alkaloids, consisting of monoether link between aporphine and benzyltetrahydroisoquinoline units, which were isolated and identified previously from a variety of Thalictrum sp. (Ranunculaceae family) based mainly on the UV, IR, CD, NMR, EI-MS, CI-MS, derivatization, and chemical degradation techniques. In this investigation, protonated molecules, [M+H]+ ions, for nine tertiary alkaloids, a molecular ion, [M+'] ion, for a quaternary alkaloid, and very intense doubly- protonated molecules, [M+2H]2+ ions (100% of relative abundance) in Q1 Scan MS spectra, and prominent as well as diagnostic product ions for structural information in the tandem MS/MS spectra were observed for all investigated alkaloids each in nanogram quantities. More than 10 microg quantities of each investigated alkaloid or other isoquinoline and aporphine analogs needed for the CI-MS, EI-MS and FAB-MS analysis from the previous studies.     

Measuring salty samples without adducts with MALDI MS

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been used for the discovery of hundreds of novel cell to cell signaling peptides. Beyond its advantages of sensitivity and minimal sample preparation requirements, MALDI MS is attractive for biological analyses as high quality mass spectra may be obtained directly from specific locations within prepared tissue sections. However, due to the large quantity of salts present in physiological tissues, these mass spectra often contain many adducts of cationic salts such as sodium and potassium, in addition to the molecular ion [M + H]⁺. To reduce the presence of cation adducts in MALDI mass spectra obtained directly from tissues, we present a methodology that uses a slow condensation procedure to enable the formation of distinct regions of matrix/analyte crystals and cation (salt) crystals. Secondary ion mass spectrometric imaging suggests that the salts and MALDI matrix undergo a mutually exclusive crystallization process that results in the separation of the salts and matrix in the sample.     

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.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of phospholipase A2 and fibrinolytic enzyme, two enzymes obtained from Chinese Agkistrodon blomhoffii Ussurensis venom

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to analyze two enzymes, phospholipase A2 and fibrinolytic enzyme isolated from Chinese Agkistrodon blomhoffii Ussurensis venom. Using sinapinic acid as the matrix, positive ion mass spectra of the enzymes were obtained. In addition to the dominant protein [M + H]+ ions, multimeric and multiply charged ions were also observed in the mass spectra. The higher the concentration of the enzymes, the more multiply charged polymer and multimeric ions were detected. Our results indicate that MALDI-TOFMS can provide a rapid and accurate method for molecular weight determination of snake venom enzymes. Mass accuracies of 0.1 and 0.3% were achieved by analysis of highly dialyzed phospholipase A2 and fibrinolytic enzyme, and these results are much better than those obtained using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MALDI-TOFMS thus provides a reliable method to determine the purity and molecular weight of these enzymes, which are of potential use as therapeutants.