Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.     

Characterization of plant oligosaccharides by matrix-assisted laser desorption/ionization and electrospray mass spectrometry

Structural characterization of arabinoxylans from wheat by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry using a Q-TOF mass analyser (ESI-Q-TOF) or an ion trap (IT) mass analyser is presented. An arabinoxylan sample digested with endoxylanase A was analysed using MALDI-TOF mass spectrometry (MS), resulting in the identification of molecular ions for structures with up to 22 monosaccharide residues. As the two-component monosaccharides xylose and arabinose are isobaric, structures differing in the number of arabinose branching residues were indistinguishable based on molecular mass and also fragmentation pattern upon collision-induced dissociation (CID). Permethylation followed by ESI-CID analyses using ITMS was performed to obtain structural information regarding the number of arabinose branching residues and their spatial arrangement along the xylose backbone. Analysis of the signal corresponding to an oligomer with six monosaccharide residues showed the presence of at least four isomeric structures differing in degree of branching and position of the branched residue relative to the cleavage site of the enzyme. This is the first demonstration of the use of ESI-ITMS for the structural characterization of arabinoxylan mixtures.     

Characterization of mouse switch variant antibodies by matrix-assisted laser desorption ionization mass spectrometry and electrospray ionization mass spectrometry

The amino acid sequences of mouse monoclonal antibodies have been characterized completely by mass spectrometry. Antibodies used in the present study were derived from mouse switch variant cell lines that produce four kinds of immunoglobulin Gs (IgGs). The amino acid sequences of these antibodies had not been estimated from the corresponding DNA sequence, so the sequences of IgGs derived from other strains were used as references in this study. Intra- and interchain disulfide bonds of the IgGs were reduced and carboxymethylated and the products were subjected to proteolytic digestion. The existence of N-linked oligosaccharides also was taken into account. The capabilities and limitations of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and capillary liquid chromatography-electrospray ionization mass spectrometry are discussed in the structural characterization of the antibodies. Based on our results, allotypes of the antibodies examined are discussed. This study shows that amino acid sequences of proteins, such as IgG, can be investigated without information about the corresponding DNA sequence if appropriate reference sequences derived from other strains can be used.     

Structural features of polyacylated anthocyanins using matrix-assisted laser desorption/ionization and electrospray ionization time-of-flight mass spectrometry

In our continuing studies to isolate water-soluble vacuolar pigments, we expect to elucidate more structural details using mass spectrometry (MS). Because of its sensitivity, only a small amount of pigment extracted from natural plants is required for MS measurement. Nuclear magnetic resonance is also a useful spectroscopic method for structural determination. In this study, two soft ionization techniques, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), on time-of-flight (TOF) mass spectrometers, were used to analyze five polyacylated anthocyanins with more than two aromatic acid molecules in the side chains. ESI is advantageous for the detection of individual molecular ions, while MALDI is essential for the detection of characteristic fragment ions originating from the anthocyanidin. Although 2,5-dihydroxybenzoic acid (DHBA) is an effective matrix in MALDI-TOFMS to obtain informative fragment ions of polyacylated anthocyanins, α-cyano-4-hydroxycinnamic acid (CHCA) is the preferred matrix for the identification of aglycones. In particular, in measurements of polyacylated anthocyanins with two acylated glycoside chains, fragment ions originating from anthocyanidin can only be observed in MALDI-TOFMS using CHCA as the matrix.     

Normalization techniques for high-throughput screening by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry

Mass spectrometry (MS) is an effective analytical tool for high-throughput screening (HTS) in the drug discovery field. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MS is a high-throughput platform that has achieved analysis times of sub-seconds-per-sample. Due to the high-throughput analysis speed, methods are needed to increase the analyte signal while decreasing the variability in IR-MALDESI-MS analyses to improve data quality and reduce false-positive hits. The Z-factor is used as a statistic of assay quality that can be improved by reducing the variation of target ion abundances or increasing signal. Herein we report optimal solvent compositions for increasing measured analyte abundances with direct analysis by IR-MALDESI-MS. We also evaluate normalization strategies, such as adding a normalization standard that is similar or dissimilar in structure to the model target drug, to reduce the variability of measured analyte abundances with direct analyses by IR-MALDESI-MS in both positive and negative ionization modes.     

Solid-phase derivatization of tryptic peptides for rapid protein identification by matrix-assisted laser desorption/ionization mass spectrometry

Solid-phase sulfonation of tryptic peptides adsorbed to C18 muZipTips has been carried out to facilitate de novo sequencing with mass spectrometry. Peptides are reacted with the sulfonation reagent while they are still adsorbed to the solid phase. Excess reagent passes through the ZipTip to waste. Washing the products before subsequent elution from the mini-column also affords sample cleanup prior to analysis. Near quantitative N-terminal sulfonation can be achieved reliably at room temperature in only a few seconds. The method has been applied successfully to model peptides and to solution or in-gel digests of proteins. Current sequencing limits are about 100 fmol of protein. Multiplexed sample sulfonation reactions have been carried out with a manual 8-position micropipettor or using centrifugal force to reliably pass reagents and wash solutions over sample-loaded ZipTips. With multiplexing, overall preparation times have been reduced to about 1 min per sample. The solid-phase format facilitates efficient use of precious digest samples by enabling them to be recovered from the matrix-assisted laser desorption/ionization (MALDI) sample stage after mass fingerprinting, derivatized and re-analyzed by MALDI postsource decay mass spectrometry.     

Development of a dual-spray electrospray deposition system for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A new method of matrix-assisted laser desorption/ionization (MALDI) sample preparation using a dual-spray electrospray deposition system is demonstrated and employed for the investigation of gas-phase cationization reactions in the MALDI plume. The dual-spray electrospray system is found to increase the homogeneity of the sample similarly to that of a conventional single-spray electrospray system. The dual-spray electrospray system allows for intimate mixing of separately prepared sample components and results in improved quantitative results. The development of this device also leads to the possibility of mixing sample components prepared in different solvents without the need to be concerned with solvent miscibility.     

Miniaturizing sample spots for matrix-assisted laser desorption/ionization mass spectrometry

The trend of miniaturization in bioanalytical chemistry is shifting from technical development to practical application. In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), progress in miniaturizing sample spots has been driven by the needs to increase sensitivity and speed, to interface with other analytical microtechnologies, and to develop miniaturized instrumentation.We review recent developments in miniaturizing sample spots for MALDI-MS. We cover both target modification and microdispensing technologies, and we emphasize the benefits with respect to sensitivity, throughput and automation.We hope that this review will encourage further method development and application of miniaturized sample spots for MALDI-MS, so as to expand applications in analytical chemistry, protein science and molecular biology.     

Simplifying sample handling for protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

An ultrasonic bath, an ultrasonic probe and a sonoreactor were used to speed up the kinetics of the reactions involved in each step of the sample handling for in-gel protein identification by peptide mass fingerprint, PMF, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The following steps were successfully accelerated using ultrasonic energy: gel washing, protein reduction, and protein alkylation. As a result, a reduction comprising 80% to 90% of the total time involved in the classic approach was achieved. In addition the sample handling was also drastically simplified. The number of peptides identified and the protein sequence coverage obtained for the new procedure were comparable to those obtained with the traditional sample treatment for the following protein standards: glycogen phosphorylase b, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and alpha-lactalbumin. Finally, as a proof of the procedure, specific proteins were identified from complex protein mixtures obtained from three different sulphate-reducing bacteria: Desulfovibrio desulfuricans G20, Desulfuvibrio gigas NCIB 9332, and Desulfuvibrio desulfuricans ATCC 27774.     

On-line atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

A new technique is presented for the coupling of atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) mass spectrometry with liquid delivery systems. Mass measurements of polymers and peptides are demonstrated using a co-dissolved matrix, e.g. alpha-cyano-4-hydroxycinnamic acid (HCCA). Improvements in terms of sensitivity are achieved by optimizing the shape und control of the exit capillary and by using a laser (355 nm) at a 1 kHz repetition rate. Two calibration experiments promise a good applicability of the presented coupling method for quantitative measurements. The limit of detection achieved so far is 500 nM for peptides in methanol solution containing 25 mM HCCA.     

Quantitative aspects in electrospray ionization ion trap and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of malto-oligosaccharides

Mass spectrometry is widely applied in carbohydrate analysis, but still quantitative evaluation of data is critical due to different ionization efficiencies of the constituents in a mixture. Different size and chemical structure of the analytes cause their uneven distribution in droplets (electrospray ionization, ESI) or matrix spots (matrix-assisted laser desorption/ionization, MALDI). In addition, instrumental parameters affect final ion yields. In order to study and optimize the latter, an equimolar mixture of malto-oligosaccharides (DP1-6) was analyzed using varying target masses for ESI as well as different matrices and laser power for MALDI. The sodium adducts and derivatives for positive ion mode (hydrazones with Girard's T Reagent, GT) and negative ion mode (reductively aminated with o-aminobenzoic acid, oABA) were studied. Negatively charged oABA-labeled malto-oligosaccharides turned out to be unsuitable for quantification of the malto-oligomeric composition. Best agreement was achieved when applying target masses in the range of the highest homolog in the mixture in electrospray ionization ion trap (ESI-IT) (1-2% deviation with GT label or as Na(+) adducts). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) gave best results when the laser power was adjusted significantly over the desorption/ionization threshold (1% deviation with GT label). Both parameters show significant influence on the determined oligomeric composition. Consequently, estimation and even quantitative determination of amounts of oligosaccharides in a mixture can be achieved when the analytes are labeled and the proper instrumental parameters are used.     

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.     

Whole-cell matrix-assisted laser desorption/ionization mass spectrometry for rapid identification of bacteriocin/lantibiotic-producing bacteria

The potential of whole-cell matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) for the discovery of microorganisms that produce lantibiotics and/or bacteriocins in the mass range 3-5 kDa is demonstrated. Cells of the antibiotic-producing model strain Bacillus subtilis ATCC 6633 were grown on Landy agar plates, and prepared with a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid (DHBs) as matrix using the bottom-layer method. Time-course analyses showed that cells grown for 1-2 days on agar plates gave the best results in terms of intensity of the bacteriocin/lantibiotic signals. Even after storage of matrix-crystal-embedded cells for several days high-resolution spectra (>10,000) could be obtained. To verify the applicability of this technique, different Bacillus wild-type and gene deletion mutant cells were analyzed. In most of the cases the known antibiotic phenotype of the producer could be corroborated by whole-cell MALDI-TOFMS, particularly for the lantibiotics subtilin and ericin. The preparation recipe for whole-cell analysis by MALDI-TOF was also highly sensitive for hydrophobic lipopeptide antibiotics like surfactin, mycosubtilin, fengycin, and bacillomycin in the lower mass range (1-1.5 kDa).     

Fast protein sequence determination with matrix-assisted laser desorption and ionization mass spectrometry

Measurement and identification of digested peptides by matrix-assisted laser desorption and ionization mass spectrometry (LDI-MS) is demonstrated. Synthetic human parathyroid hormone, pTH (1-34), with a molecular mass of 4117.8 Da was digested with carboxypeptidases Y and B and the sequence of 14 amino acids from the C-terminus of the peptide was determined by analyzing the molecular mass of the truncated peptides. Furthermore, a tryptic digestion of pTH (1-34) was carried out and a molecular mass map of pTH (1-34) was obtained. With the results of the proteolytic digestion a rapid confirmation of the amino-acid sequence of the protein was possible. It is shown that the results of the tryptic digestion can be used for the unambiguous identification of the amino acid residues Lys and Arg, which cannot be distinguished with a mass spectrometer because of their equal nominal masses. Several advantages of amino acid sequence determination by the combination of digestion and LDI-MS are obvious: high sensitivity in the low pmol range, fast digestion time due to high enzyme/substrate ratios, quantification is unnecessary because the amino acids are identified by their molecular mass differences, the low chemical expenditure for the digestions and the accuracy of the sequence determination. Measurements with LDI-MS are fast: sample preparation and the measurement take only a few min. The mass determination and amino acid sequence is completely unimpaired by amino acid contaminations or impurities in the sample. The sensitivity of the method is in the low pmol to fmol range and thus comparable to other analytical methods.     

Differentiation of enantiomers using matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has successfully been used to differentiate pseudo-enantiomeric (isotopically labelled) amino acids by using cyclodextrin as complexing host. By using different pseudo-enantiomeric mixtures (i.e. R(Dn) + S; and R + S(Dn)), it has been demonstrated that the preference of cyclodextrin for S-enantiomers is not due to the size differences caused by the hydrogen/deuterium substitution. It is postulated that this method can be extended to differentiate enantiomers (and determine enantiomeric excess) by using a pair of enantiomeric hosts, as demonstrated previously using other ionization techniques, but with much higher sensitivity.     

Automated protein identification using atmospheric-pressure matrix-assisted laser desorption/ionization

Atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) ion trap mass spectrometry (ITMS) has been evaluated for automated protein identification. By using signal averaging and long ion-injection times, protein identification limits in the 50-fmol range are achieved for standard protein digests. Data acquisition requires 7.5 min or less per sample and the MS/MS spectra files are automatically processed using the SEQUEST database searching algorithm. AP-MALDI-ITMS was compared with the widely used methods of microLC/MS/MS (ion trap) and automated MALDI-TOF peptide mass mapping. Sample throughput is 10-fold greater using AP-MALDI compared with microcapillary liquid chromatography/tandem mass spectrometry (microLC/MS/MS). The protein sequence coverage obtained from AP-MALDI-MS/MS spectra matched by SEQUEST is lower compared with microLC/MS/MS and MALDI-TOF mass mapping. However, by using the AP-MALDI full-scan peptide mass fingerprint spectrum, sequence coverage is increased. AP-MALDI-ITMS was applied for the analysis of Coomassie blue stained gels and was found to be a useful platform for rapid protein identification.     

Hydrogen exchange studies on Alzheimer's amyloid-beta peptides by mass spectrometry using matrix-assisted laser desorption/ionization and electrospray ionization

The conformation and aggregation behavior of synthetic Alzheimer's amyloid peptides (Abeta) has been investigated using hydrogen-deuterium exchange measured by electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. Mass spectrometric fragmentation of deuterated Abeta peptides was carried out by collision-induced dissociation, inlet fragmentation, and post-source decay. In contrast to the C-terminally truncated peptides Abeta(1-40) and Abeta(1-36) showing full hydrogen-deuterium exchange, Abeta(1-42) and the pyroglutamyl peptide Pyr(3)-Abeta(3-42) produced more complex signal patterns resulting from the formation of beta-sheet-structured oligomers having 18-20 strongly protected protons. Using mass spectrometric fragmentation the results show that the reduced isotope exchange of Abeta(1-42) can be attributed to the central part of the chain comprising residues 8-23. This confirms involvement of the hydrophobic binding domain LVFFA in the course of Abeta aggregation and demonstrates that hydrogen-deuterium exchange in combination with mass spectrometry is well suited for structural analysis of monomeric and reversibly associated amyloid peptides using picomole quantities of material.     

Analysis of carrageenans by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. I. kappa-Carrageenans.

Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectra of small kappa-carrageenans are reported and discussed. MALDI spectra can be obtained in both positive and negative ion mode. In the absence of extraneous metal ions, positive ions are formed by the attachment of one Na(+) ion to the carrageenan, whereas for negative ions one Na(+) ion is detached from the sulfate group. Multiply charged species are not observed in MALDI. Intense ESI spectra can be obtained in negative ion mode and now multiply charged species are seen. Alkali exchange experiments show that in these small carrageenan anions one, but only one, alkali metal ion is bound in a bidentate coordination with two ionic sulfate groups. G2-type ab initio calculations on model ions HO(-) [M(+)] (-)OH (M = Li, Na, K, Cs), as well as arguments based on a simple Coulombic interaction model, show that the bidentate stabilization energy drops rapidly as the size of the alkali cation increases. Exchange of Na(+) with Li(+) leads to expulsion of the Na(+) ion generating, in ESI, intense multiply charged anions. An attempt is made to rationalize this behavior in terms of hydration effects.     

In-situ monitoring of protein labeling reactions by matrix-assisted laser desorption/ionization mass spectrometry

Taking the labeling reaction of horse heart cytochrome c or ubiquitin with biotinamidocaproate N-hydroxysucchinimide ester (biotin-NHS) as test cases, this report demonstrates the usefulness of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for in-situ monitoring of the labeling process and for determining the composition of the labeled products without the need for prior separation. The effects of pH and starting materials concentration on the labeling process were investigated in detail. Our MALDI MS results show that: (1) labeled products are always mixtures of different conjugates, which may explain peak broadening found in chromatographic studies of labeling reactions; (2) the higher conjugate fractions become more prominent as the labeling reaction proceeds, with a concomitant exponential decline of the lower conjugate fractions; (3) biotin-NHS can be incorporated into peptides and protein in a stepwise and controlled manner simply by adjusting the molar ratio of the starting materials.