Fragmentation of vitamin B12 in aerosol matrix-assisted laser desorption ionization

Aerosol matrix-assisted laser desorption ionization (MALDI) with a reflection time-of-flight mass spectrometer was used to study fragmentation of vitamin B₁₂. Six MALDI matrices were used: 2,5-di-hydroxy benzoic acid (gentisic acid), 4-nitroaniline, 3,5-dimethoxy-4-hydroxy cinnamic acid (sinapic acid), 3,4-di-hydroxy cinnamic acid (caffeic acid), trans-4-hydroxy-3-methoxy cinnamic acid (ferulic acid), and α-cyano-4-hydroxy cinnamic acid (4-HCCA). Mass spectra were obtained with a 355-nm pulsed Nd:YAG laser at irradiances between 0. 1 and 5 GW/cm² (between 3- and 150-mJ pulse energy). Loss of CN was a major product of prompt ion source fragmentation and the ratio of fragmented to intact analyte was found to be strongly dependent on matrix and weakly dependent on laser irradiance. Additionally, free cobalt ions and cobalt ions bound to small methanol clusters were observed in the mass spectra. The cobalt removal from the corrin ring of vitamin B₁₂ results from direct photon absorption by vitamin B₁₂, but is enhanced by the presence of matrix.     

Matrix-free infrared soft laser desorption/ionization

Infrared soft laser desorption/ionization was performed using a 2.94 µm Er : YAG laser and a commercial reflectron time-of-flight mass spectrometer. The instrument was modified so that a 337 nm nitrogen laser could be used concurrently with the IR laser to interrogate samples. Matrix-assisted laser desorption/ionization (MALDI), laser desorption/ionization and desorption/ionization on silicon with UV and IR lasers were compared. Various target materials were tested for IR soft desorption ionization, including stainless steel, aluminum, copper, silicon, porous silicon and polyethylene. Silicon surfaces gave the best performance in terms of signal level and low-mass interference. The internal energy resultant of the desorption/ionization was assessed using the easily fragmented vitamin B₁₂ molecule. IR ionization produced more analyte fragmentation than UV-MALDI analysis. Fragmentation from matrix-free IR desorption from silicon was comparable to that from IR-MALDI. The results are interpreted as soft laser desorption and ionization resulting from the absorption of the IR laser energy by the analyte and associated solvent molecules. Copyright © 2004 John Wiley & Sons, Ltd.     

Oxidation of ferrocene derivatives in desorption/lonization on porous silicon.

In matrix-assisted laser desorption/ionization (MALDI), the true molecular structures of some analytes are not represented by the observed ions due to a redox reaction. In earlier reports, electron transfer from analyte to chemical matrix has been proposed for the oxidation of ferrocene derivatives in MALDI. To address such a redox phenomenon in laser desorption/ionization processes, two ferrocene derivatives, FcCH2CH2Fc and FcCH2NMe2 [Fc:(CsHs)Fe(CsH4)], were analyzed by a matrix-free method, desorption/ionization on porous silicon (DIOS). The oxidized species, Fc+CH2NMe2 and FcCH2CH2Fc+, were detected in the DIOS mass spectra. The results suggested that electron transfer from the analytes to the sample target occurs during the ionization process.     

Surface modification and laser pulse length effects on internal energy transfer in DIOS

Benzyl-substituted benzylpyridinium (BP) chloride salts were used as a source of thermometer ions to probe the internal energy (IE) transfer in desorption/ionization on porous silicon (DIOS). To modify their wetting properties and the interaction energies with the thermometer ions, the DIOS surfaces were silylated to produce trimethylsilyl- (TMS), amine- (NH2), perfluoroalkyl- (PFA), and perfluorophenyl-derivatized (PFP) surfaces. Two laser sources--a nitrogen laser with pulse length of 4 ns and a mode locked 3 x omega Nd:YAG laser with a pulse length of 22 ps--were utilized to induce desorption/ionization and fragmentation at various laser fluence levels. The corresponding survival yields were determined as indicators of the IE transfer and the IE distributions were extracted. In most cases, with increasing the laser fluence in a broad range (approximately 20 mJ/cm2), no change in IE transfer was observed. For ns excitation, this was in remarkable contrast with MALDI, where increasing the laser fluence resulted in sharply (within approximately 5 mJ/cm2) declining survival yields. Derivatization of the porous silicon surface did not affect the survival yields significantly but had a discernible effect on the threshold fluence for ion production. The IE distributions determined for DIOS and MALDI from alpha-cyano-4-hydroxycinnamic acid reveal that the mean IE value is always lower for the latter. Using the ps laser, the IE distribution is always narrower for DIOS, whereas for ns laser excitation the width depends on surface modification. Most of the differences between MALDI and DIOS described here are compatible with the different dimensionality of the plume expansion and the differences in the activation energy of desorption due to surface modifications.     

Internal energy transfer in laser desorption/ionization from silicon nanowires

Laser-induced desorption/ionization from silicon nanowires (SiNW) is an emerging method for mass spectrometry of small to medium-size molecules. In this new technique, we examined the internal energy transfer to seven benzylpyridinium thermometer ions and extracted the corresponding internal energy distributions. To explore the effect of the energy-deposition rate on the internal energy transfer, two lasers with significantly different pulse lengths (4 ns vs 22 ps) were utilized as excitation sources. A comparison of ion yields indicated that the SiNW substrates required 5-8 times less laser fluence for ion production than either matrix-assisted laser desorption/ionization (MALDI) or desorption/ionization on silicon (DIOS). In contrast however, the survival yield (SY) values showed that the internal energy transferred to the thermometer ions was more than (ps laser) or comparable to (ns laser) MALDI but it was significantly less than in DIOS. The internal energy transfer was only slightly dependent on laser fluence and on wire density. These effects were rationalized in terms of the confinement of thermal energy in the nanowires and of unimpeded three-dimensional plume expansion. Unlike in MALDI from CHCA and in perfluorophenyl-derivatized DIOS, for desorption from SiNWs the effect of laser pulse length on the internal energy transfer was found to be negligible.     

Determination of vitamin B<Subscript>12</Subscript> using a chemiluminescence flow system

A method to determine vitamin B₁₂ by measuring the chemiluminescence (CL) intensities using a flow injection (FI) system has been proposed. It is based on the catalytic effect of cobalt(II) in vitamin B₁₂ on the CL reaction between luminol and hydrogen peroxide in a basic medium. The increment of the CL intensity is proportional to the concentration of vitamin B₁₂ in the range 8.68–86.9 ng/mL (r ² = 0.9984) with a detection limit (3σ) of 0.89 ng/mL. The CL response is obtained in 10 s at a flow rate of 3.0 mL/min with a relative standard deviation (RSD) of less than 2.5% (n = 6). The method has been successfully applied to the determination of vitamin B₁₂ in pharmaceutical injections. The text was submitted by the authors in English.     

Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ionization and laser desorption/ionization on porous silicon mass spectrometry

Cysteine sulfonic acid-containing peptides, being typical acidic peptides, exhibit low response in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, matrix conditions and the effect of diammonium hydrogencitrate (DAHC) as additive were investigated for ionization of cysteine sulfonic acid-containing peptides in MALDI. A matrix-free ionization method, desorption/ionization on porous silicon (DIOS), was also utilized to evaluate the effect of DAHC. When equimolar three-component mixtures of peptides carrying free cysteine, cysteine sulfonic acid, and carbamidomethyl cysteine were measured by MALDI using a common matrix, alpha-cyano-4-hydroxycinnamic acid (CHCA), no signal corresponding to cysteine sulfonic acid-containing peptide could be observed in the mass spectrum. However, by addition of DAHC to CHCA, the peaks of cysteine sulfonic acid-containing peptides were successfully observed, as well as when using 2,4,6-trihydroxyacetophenone (THAP) and 2,6-dihydroxyacetophenone with DAHC. In the DIOS mass spectra of these analytes, the use of DAHC also enhanced the peak intensity of the cysteine sulfonic acid-containing peptides. On the basis of studies with these model peptides, tryptic digests of oxidized peroxiredoxin 6 were examined as a complex peptide mixture by MALDI and DIOS. In MALDI, the peaks of cysteine sulfonic acid-containing peptides were observed when using THAP/DAHC as the matrix, but this was not so with CHCA. In DIOS, the signal from cysteine sulfonic acid-containing peptides was suppressed; however, the use of DAHC significantly enhanced the signal intensity with an increase in the number of observed peptides and increased signal-to-noise ratio in the DIOS spectra. The results show that DAHC in the matrix or on the DIOS chip decreases discrimination and suppression effects in addition to suppressing alkali-adduct ions, which leads to a beneficial effect on protonation of peptides containing cysteine sulfonic acid.     

Reduction of organic dyes in matrix-assisted laser desorption/ionization and desorption/ionization on porous silicon

Reduction of analytes in matrix-assisted laser desorption/ionization (MALDI) often obscures the actual determination of molecular structure. To address the redox reactions in laser desorption/ionization processes, the organic dyes Methylene Blue, Janus Green B, Crystal Violet and Rhodamine B were analyzed by MALDI or by desorption/ionization on porous silicon (DIOS). Susceptibility to reduction in MALDI was dependent on both the reduction potentials of analytes and the molar ratio of analyte to matrix molecules. Addition of Cu(II) ions as an electron scavenger suppressed the reduction of Methylene Blue in MALDI. The results suggested that electron transfer to analytes from the sample target and/or from the matrix contributed to the reduction. In DIOS, the reductions of organic dyes were more prominent than in MALDI, and were not prevented by Cu(II) ion doping, probably due to direct contact of the analytes with silicon which had little electric resistance.     

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

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

Matrix-assisted ionization vacuum for protein detection,fragmentation and PTM analysis on a high resolution linear ion trap-orbitrap platform

Matrix-assisted ionization vacuum (MAIV) is a novel ionization technique that generates multiply charged ions in vacuum without the use of laser ablation or high voltage. MAIV can be achieved in intermediate-vacuum and high-vacuum matrix-assisted laser desorption/ionization (MALDI) sources and electrospray ionization (ESI) sources without instrument modification. Herein, we adapt MAIV onto the MALDI-LTQ-Orbitrap XL platform for biomolecule analysis. As an attractive alternative to MALDI for in solution and in situ analysis of biomolecules, MAIV coupling to high resolution and accurate mass (HRAM) MS instrument has successfully expanded the mass detection range and improved the fragmentation efficiency due to the generation of multiply charged ions. Additionally, the softness of MAIV enables potential application in labile post-translational modification (PTM) analysis. In this study, proteins as large as 18.7 kDa were detected with up to 18 charges; intact peptides with labile PTM were well preserved during the ionization process and characterized MS/MS; peptides and proteins in complex tissue samples were detected and identified both in liquid extracts and in situ. Moreover, we demonstrated that this method facilitates MS/MS analysis with improved fragmentation efficiency compared to MALDI-MS/MS.     

MALDI-TOF MS stability study of model poly(p-phenylene terephthalamide)s

In the present study, we address the possibility of matrix-assisted laser desorption/ionization (MALDI)–time-of-flight MS analysis-induced chain fragmentation in poly(p-phenylene terephthalamide) (PPD-T) by considering two possible sources: (1) grinding-induced fragmentation resulting from the evaporation–grinding MALDI sample preparation method (E-G method) and (2) in-source/metastable fragmentation induced by the MALDI laser. An analysis of variance (ANOVA) statistical study found, with a high probability, that obtaining MALDI spectra with the effective laser area as large as possible (the “fanned-out” setting) did not cause any chain fragmentation due to the E-G MALDI sample preparation method, even when three additional grinding steps were used. However, the effect of laser fluence was less clear. A significant effect of laser fluence was observed for lower mass oligomers (<1,400 Da), but there was essentially no effect for higher mass species up to our limit of ANOVA measurement (∼2,300 Da). Plausible explanations are presented to explain these observations. The most likely scenario is that “unexpected” end-group modifications occur during PPD-T synthesis, producing small quantities of low mass species, which are amplified by the MALDI-EG extraction procedure.     

Determination of vitamin B<Subscript>1</Subscript> in seawater and microalgal fermentation media by high-performance liquid chromatography with fluorescence detection

A highly sensitive high-performance liquid chromatographic method with fluorescence detection has been developed for determination of vitamin B₁. Vitamin B₁ was converted into a fluorescent compound by treatment with hydrogen peroxide–horseradish peroxidase and the derivative was subsequently analyzed by HPLC on a Waters Spherisorb ODS2 column (250 mm×4.6 mm ID, 5 μm) with 40:60 methanol–pH 8.5 acetate buffer solution as mobile phase and fluorescence detection at 440 nm (with excitation at 375 nm). The calibration graph was linear from 5.00×10⁻¹⁰ mol L⁻¹ to 5.00×10⁻⁷ mol L⁻¹ for vitamin B₁ with a correlation coefficient of 0.9991 (n=9). The detection limit was 1.0×10⁻¹⁰ mol L⁻¹. The method was successfully used for determination of vitamin B₁ at pg mL⁻¹ levels in microalgal fermentation media and seawater after solid-phase extraction. Recovery was from 89 to 110% and the relative standard deviation was in the range 1.1 to 4.3%.     

Study on the redox reactions for organic dyes and S‐nitrosylated peptide in electrospray droplet impact

Reduction of analytes in ionization processes often obscures the determination of molecular structure. The reduction of analytes is found to take place in various desorption/ionization methods such as fast atom bombardment (FAB), secondary ion mass spectrometry (SIMS), matrix‐assisted laser desorption/ionization (MALDI) and desorption ionization on porous silicon (DIOS). To examine the extent of the reduction reactions taking place in electrospray droplet impact (EDI) processes, reduction‐sensitive dyes and S‐nitrosylated peptide were analyzed by EDI. No reduction was observed for methylene blue. While methyl red has a lower reduction potential than methylene blue, the reduction product ions were detected. For S‐nitrosylated peptide, protonated molecule ion [M + H]⁺ and NO‐eliminated molecular ion [M − NO + H]^+• were observed but reduction reactions are largely suppressed in EDI compared with that in MALDI. As such, the analytes examined suffer from little reduction reactions in EDI. Copyright © 2008 John Wiley & Sons, Ltd.     

Matrix Effects on the Fragmentation of Vitamin B12 in Plasma Desorption Mass Spectrometry

A continuing effort in mass spectrometry is to optimize desorption/ionization processes in order to enhance analyte ion yields and reduce fragmentation. The effect of small carbohydrate and amino acid matrices on the yield of secondary ions from vitamin B₁₂ (cyanocobalamin) was examined using plasma desorption mass spectrometry. The extent of the corrin decomposition is dependent upon the matrix-to-analyte ratio. The enhanced yields of the high-mass fragment ion [M–CN + H]⁺ and the ions corresponding to protonated molecules, however, are dependent upon both the matrix-to-analyte ratio and the nature of the matrix. © 1997 John Wiley & Sons, Ltd.     

Local structure and photoluminescent characteristics of Eu<Superscript>3+</Superscript> in ZnO–SiO<Subscript>2</Subscript> glasses

The photoluminescence properties of xZnO–(100−x)SiO₂ (x = 0, 5, 10, 20) containing 1% Eu₂O₃ prepared by a sol–gel method were systematically investigated. The results indicated that the relative proportion of f–f transitions to charge transfer (CT) absorption decreased with the increase of ZnO concentration. The intensity of ⁵D₀–⁷F_J transitions of Eu³⁺ ions was enhanced with the increase of ZnO content due to local structure changes and decreased quantities of Eu³⁺ ions clusters. The results of fluorescence line narrow (FLN) spectra indicated that Eu³⁺ ions occupied one site in SiO₂ glass and two sites in ZnO–SiO₂ glasses. The second-order crystal field parameters were calculated. B₂₀ and B₂₂ for site 1 increased with excitation energy, while ones hardly changed for site 2.     

Effect of metal substitution for cobalt on the oxygen adsorption properties of YBaCo<Subscript>4</Subscript>O<Subscript>7</Subscript>

YBaCo₄O₇ compound is capable to intake and release a large amount of oxygen in the temperature range of 200–400°C. In the present study, the effect of Zn, Ga and Fe substitution for Co on the oxygen adsorption/desorption properties of YBaCo₄O₇ were investigated by thermogravimetry (TG) method. Due to fixed oxidation state of Zn2+ ions, the substitution of Zn²⁺ for Co²⁺ suppresses the oxygen adsorption of YBaCo_4−xZn_xO₇. The substitution of Ga³⁺ for Co³⁺ also decreases the oxygen absorption capacity of YBaCo_4−xGa_xO₇. This can be explained by the strong affinity of Ga³⁺ ions towards the GaO₄ tetrahedron. Compared with Zn- and Ga-substituted samples, the drop of oxygen adsorption capacity is smallest for Fe-substituted samples because of the similar changeability of oxidation states of Co and Fe ions.     

Laser Desorption Ionization Versus Electrospray Ionization Mass Spectrometry: Applications in the Analysis of Cluster Anions

Mass spectra of transition metal carbonyl cluster anions were recorded using laser desorption ionization time-of-flight (LDI-TOF) and electrospray ionization (ESI) techniques. The LDI spectra generally contain peaks corresponding the intact cluster together with extensive CO loss fragments ions whereas the ESI spectra exhibit peaks corresponding the intact cluster together with few (if any) CO loss fragment ions. The parameters of both techniques can be modified to vary the extent of fragmentation. In all cases no fragmentation of the metal core is observed. Overall, ESI is a more informative method for the analysis of these types of cluster anions.     

Quantitative MALDI-MS<Superscript><Emphasis Type="Italic">n</Emphasis></Superscript> analysis of cocaine in the autopsied brain of a human cocaine user employing a wide isolation window and internal standards

Detection of drugs in tissue typically requires extensive sample preparation in which the tissue is first homogenized, followed by drug extraction, before the extracts are finally analyzed by LC/MS. Directly analyzing drugs in intact tissue would eliminate any complications introduced by sample pretreatment. A matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS ⁿ ) method as been developed for the quantification of cocaine present in postmortem brain tissue of a chronic human cocaine user. It is shown that tandem mass spectrometry (MS² and MS³) increase selectivity, which is critical for differentiating analyte ions from background ions such as matrix clusters and endogenous compounds found in brain tissue. It is also shown that the use of internal standards corrects for signal variability during quantitative MALDI, which can be caused by inhomogeneous crystal formation, inconsistent sample preparation, and laser shot-to-shot variability. The MALDI-MS ⁿ method developed allows for a single MS³ experiment that uses a wide isolation window to isolate both analyte and internal standard target ions. This method is shown to provide improved precision [∼10–20 times reduction in percent relative standard deviation (%RSD)] for quantitative analysis compared to using two alternating MS³ experiments that separately isolate the target analyte and internal standard ions.     

Effective methods for the measurement of CsI cluster ions using MALDI‐MS with suitable solvent combinations and additives

A method to measure CsI cluster ions ((CsI)_nCs⁺, (CsI)_nI^?) from CsI samples in matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) was developed with a 2‐[(2E)‐3‐(4‐tert‐butylphenyl)‐2‐methylprop‐2‐enylidene] malononitrile (DCTB) matrix and additives. Solvent combinations in which the CsI and DCTB solutions were miscible were effective in detecting CsI cluster ions at a mass range of over m/z 2000 and are associated with a characteristic spread of DCTB within the CsI/DCTB mixture. The addition of saccharides or sugar alcohols to the CsI/DCTB mixture improved the DCTB distribution and widened the mass distribution of CsI cluster ions up to m/z 10 000 in the linear mode. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterizing Enzyme Cooperativity with Imaging SAMDI-MS

This paper describes a method that combines a microfluidic device and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI) mass spectrometry to calculate the cooperativity in binding of calcium ions to peptidylarginine deiminase type 2 (PAD2). This example uses only 120 μL of enzyme solution and three fluidic inputs. This microfluidic device incorporates a self-assembled monolayer that is functionalized with a peptide substrate for PAD2. The enzyme and different concentrations of calcium ions are flowed through each of eight channels, where the position along the channel corresponds to reaction time and position across the channel corresponds to the concentration of Ca²⁺. Imaging SAMDI (iSAMDI) is then used to determine the yield for the enzyme reaction at each 200 μm pixel on the monolayer, providing a time course for the reactions. Analysis of the peptide conversion as a function of position and time gives the degree of cooperativity (n) and the concentration of ligand required for half maximal activity (K_0.5) for the Ca²⁺ – dependent activation of PAD2. This work establishes a high-throughput and label-free method for studying enzyme-ligand binding interactions and widens the applicability of microfluidics and matrix-assisted laser desorption/ionization mass spectrometry (MALDI) imaging mass spectrometry.