Characterization of polyethylenimine by electrospray ionization and matrix-assisted laser desorption/ionization

Branched polyethylenimines (PEIs) with lower average molecular weights (600, 1200 and 1800 Da) have been studied by Electrospray Ionization (ESI) and Matrix‐Assisted Laser Desorption/Ionization (MALDI) mass spectrometry. In both, ESI and MALDI mass spectra, the main distribution arises from protonated PEI oligomers with NH₂ end groups, [PEI + H]⁺, which are observed at m/z 43n + 18. A trace of sodium contamination in the PEI samples results in the presence of a series that appears at m/z 43n + 40 [PEI + Na]⁺. However, only the MALDI mass spectra show a [PEI + K]⁺ series at m/z 43n + 56, because of matrix contamination with potassium, and a series generated by condensation of the matrix with PEI at m/z 43n + 30. Collisionally activated dissociation tandem mass spectrometry (CAD (MS/MS)) of protonated PEI oligomers is shown to yield three fragment ion series b_n, and K_n. The experiments have demonstrated the capabilities of these mass spectrometry techniques, along with CAD MS/MS to detect and characterize such polar synthetic polymers. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

Estimation of the proton affinity values of fifteen matrix-assisted laser desorption/ionization matrices under electrospray ionization conditions using the kinetic method

The kinetic method, which is known to be simple, fast and precise, is used for the measurement of proton affinity values of fifteen selected matrix-assisted laser desorption/ionization matrices under electrospray ionization conditions. The stabilization of [M - H](-)/[M + Matrix - H](-) ions for beta-cyclodextrin in negative ion mode has been rationalized based on the proton affinity values of the matrices.     

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.     

Infrared matrix-assisted laser desorption and ionization by using a tunable mid-infrared free-electron laser

Initial results of infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry of proteins by using the Vanderbilt free-electron laser as the source of selective vibrational excitation are reported. The ability of this laser to initiate desorption and ionization by excitation of specific vibrational modes is demonstrated. For the first time it is shown that IR-MALDI mass spectrometry at wavelengths other than those available from conventional fixed-frequency IR lasers, that is, 2.79 (Er:YSGG), 2.94 (Er:YAG), and 9.3-10.6 μm (CO₂), is feasible and exhibits similar performance. IR-MALDI mass spectra were taken in the wavelength ranges 2.8-4 and 5.5-6.5 μm, covering the absorption bands of the O-H and C=O stretch vibrations typical of many organic compounds such as succinic acid, fumaric acid, or nicotinic acid, which were used as matrices in these studies. A comparison between these results and Er:YAG/YSGG MALDI data are given. The potential of IR-MALDI at wavelengths near the C=O stretch vibration and the possibilities for studies of the IR-MALDI mechanisms by using this kind of tunable source are discussed.     

Quantitative analysis of small pharmaceutical drugs using a high repetition rate laser matrix-assisted laser/desorption ionization source

In this work, a high repetition rate laser matrix-assisted laser desorption/ionization (MALDI) source is studied on a quadrupole-time-of-flight (QqTOF) and a triple quadrupole (QqQ) mass spectrometer for rapid quantification of small pharmaceutical drugs. The high repetition rate laser allows an up to 100-fold higher pulse frequency as compared with regular MALDI lasers, resulting in much larger sample throughput and number of accumulated spectra. This increases the reproducibility of signal intensities considerably, with average values being around 5% relative standard deviation after taking into account the area ratio of the analyte to an internal standard. Experiments were conducted in MS/MS mode to circumvent the large chemical background due to MALDI matrix ions in the low mass range. The dynamic range of calibration curves on the QqTOF mass spectrometer extended over at least two orders of magnitude, whereas on the QqQ it extended over at least three orders of magnitude. Detection limits ranged from 60-400 pg/microL on the QqTOF and from 6-70 pg/microL on the QqQ for a series of benzodiazepines. The benzodiazepine content of commercial pill formulations was quantified, and less than 5% error was obtained between the present method and the manufacturer's certified values. Furthermore, a high sample throughput was achieved with this method, so that a single MALDI spot could be quantitatively scanned in as little as 15 s, and an entire 96-well MALDI plate in 24 min.     

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.     

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.     

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.     

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.     

Differentiation of isomeric photomodified oligodeoxynucleotides by fragmentation of ions produced by matrix-assisted laser desorption ionization and electrospray ionization

UV irradiation of oligodeoxynucleotides at 254 nm generates several different types of DNA photoproducts, including cis-syn cyclobutane pyrimidine dimers, pyrimidine[6-4] pyrimidone photoproducts and their Dewar valence isomers, and thymine-adenine photoproducts (TA*). Studies of photoproducts in oligodeoxynucleotides require the development of suitable structure determination methods such as mass spectrometry. In an earlier study (Vollmer et al. Int. J. Mass Spectrom. Ion Processes 1997, 165/166, 487-496), we showed that fast atom bombardment and tandem sector mass spectrometry can be used to locate the site of photomodification and identify most of the photoproducts of d(TATTAT). One goal of the present research was to expand the method to the more sensitive electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) methods. A second goal was to test the generality of the methods by investigating not only the photoproducts of d(TATTAT) but also those of three other oligodeoxynucleotides, d(GTATTAT), d(GGCTATAA), and d(AATTAA). The photoproducts of these sequences were separated by HPLC and gave characteristic fragment ions in postsource decompositions of MALDI-produced ions and collisionally activated decompositions of ESI-produced ions.     

Wavelength dependence of matrix-assisted laser desorption and ionization using a tunable mid-infrared laser

Matrix-assisted laser desorption and ionization by infrared laser (IR-MALDI) is expected to be an effective methods for soft-ionization of high-molecular weight proteins and intracellular proteins. IR-MALDI is not widely used because its low sensitivity, complexity, high cost, and as it does not work well on commercial MALDI time-of-flight mass spectrometers (TOFMSs). We employed a tunable mid-infrared (MIR) laser as a light source for MALDI to investigate the IR-MALDI. The laser wavelength can be tuned within a range from 5.5 to 10.0 μm, and included several biomaterial group vibration modes. We evaluated the wavelength dependence of ionization in IR-MALDI for four matrices: succinic acid, urea, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid) and 2,5-dihydroxybenzoic acid (DHB). These matrices contained various groups of vibration modes, and absorbed an infrared (IR) energy at a specific wavelength. The mass spectra of angiotensin II was obtained at a specific wavelength corresponding to the CO stretching and benzene ring vibration mode. In IR-MALDI, we considered the strong molecular bond attracting an electron from a neighboring hydrogen atom, possibly protonating the hydrogen atom.     

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.     

Protein analysis by desorption electrospray ionization mass spectrometry and related methods

Desorption electrospray ionization mass spectrometry (DESI‐MS) requires little to no sample preparation and has been successfully applied to the study of biologically significant macromolecules such as proteins. However, DESI‐MS and other ambient methods that use spray desorption to process samples during ionization appear limited to smaller proteins with molecular masses of 25 kDa or less, and a decreasing instrumental response with increasing protein size has often been reported. It has been proposed that this limit results from the inability of some proteins to easily desorb from the surface during DESI sampling. The present study investigates the apparent mass dependence of the instrumental response observed during the DESI‐MS analysis of proteins using spray desorption collection and reflective electrospray ionization. Proteins, as large as 66 kDa, are shown to be quantitatively removed from surfaces by using spray desorption collection. However, incomplete dissolution and the formation of protein–protein and protein–contaminant clusters appear to be responsible for the mass‐dependent loss in sensitivity for protein analysis. Alternative ambient mass spectrometry approaches that address some of the problems encountered by spray desorption techniques for protein analysis are also discussed. Copyright © 2013 John Wiley & Sons, Ltd.     

基质辅助激光解吸电离质谱和电喷雾电离质谱在辣根过氧化物酶糖肽结构分析中的应用

糖链结构的质谱解析是今后糖蛋白分析中的重要研究内容,其中完整糖肽的分析,由于可以同时获得糖基化位点和对应糖链的结构信息,更具有重要意义和研究前景。本工作对质谱软电离技术在完整糖肽分析中的应用进行了研究,其中包括了基质辅助激光解吸电离(matrix-assisted laser desorption ionization, MALDI)和电喷雾电离(electrospray ionization, ESI)技术。通过平行使用两种串联质谱(tandem mass spectrometry, MS/MS)分析策略: MALDI-MS/MS和ESI-MS/MS对目标糖蛋白——辣根过氧化物酶进行分析,并讨论了其互补性。结果表明,MALDI和ESI技术各有优劣,结合串联质谱分析,可获得糖肽的糖链结构信息;两条路线互补使用,在揭示蛋白质糖基化修饰(位点和结构)的研究中十分必要。     

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.