Matrix-assisted Laser Desorption/ionization Plume Research Articles

Matrix-dependent a/x pair and overdegraded w/y/z ions generated by radical-directed dissociation of peptide radical cations [M]+ in matrix-assisted laser desorption/ionization in-source decay.

Fragmentation of peptide radical cations [M]. + has been examined using matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) with hydrogen-abstracting nitro-substituted matrices. The ISD spectra of peptides containing an arginine (Arg) residue at carboxyl (C)-termini showed preferential [w]+ ions when 4-nitro-1-naphthol (4,1-NNL) matrix was used, whereas the use of 3,5-dinitrosalicylic acid (3,5-DNSA) resulted in preferential [x]+ ions. Minor or some [d]+ , [x]+ , [y]+ , and [z]+ ions were also observed. For peptides containing Arg residue at amino (N)-termini, the ISD spectra showed preferential [a]+ ions independent of matrix used. The observed [a]+ , [w]+ , [x]+ , [y]+ , and [z]+ ions can be rationally explained by radical-directed dissociation (RDD) of the peptide radical cations [M]. + , although [d]+ ions may be formed via Norrish Type I cleavage and/or by RDD of [M]. + ions. The formation of overdegraded [d]+ , [w]+ , [y]+ , and [z]+ ions is discussed from the standpoint of the internal energy of radical cations [M]. + and radical fragment ions [a + H]. + and [x + H]. + deposited via collisional interactions with excited matrix molecules in the MALDI plume. The radical site of the peptide cations [M]. + was presumed to be backbone amide nitrogen, from MALDI-ISD data with three different deuterated amino acids.

Combining MALDI-2 and transmission geometry laser optics to achieve high sensitivity for ultra-high spatial resolution surface analysis.

A transmission geometry optical configuration allows for smaller laser spot size to facilitate high-resolution matrix-assisted laser/desorption ionization (MALDI) mass spectrometry. This increase in spatial resolution (ie, smaller laser spot size) is often associated with a decrease in analyte signal. MALDI-2 is a post-ionization technique, which irradiates ions and neutrals generated in the initial MALDI plume with a second orthogonal laser pulse, and has been shown to improve sensitivity. Herein, we have modified a commercial Orbitrap mass spectrometer to incorporate a transmission geometry MALDI source with MALDI-2 capabilities to improve sensitivity at higher spatial resolutions.

Laser induced post-desolvation of MALDI clusters

Despite being controversial for many years, it is now widely accepted that the Matrix Assisted Laser Desorption/Ionization (MALDI) process leads to the ejection of aggregates or clusters in addition to isolated particles and ions. Clusters composed of analyte surrounded by matrix molecules exhibit a distribution in size and are relatively stable. Several studies have demonstrated that these clusters do not necessarily fully desolvate before ion extraction, but rather progressively along the ion path. This leads to a decrease in analytical performances since these non-desolvated clusters do not contribute to the ion signal but also generate an important chemical background noise. Therefore, proper cluster desolvation before ion extraction in order to release naked analyte ions in the gas phase holds great promise of improved mass spectra sensitivity. This work presents a set-up allowing cluster desolvation using nanosecond pulses of a near-infrared laser that intercepts the expanding MALDI plume before ion extraction.

Investigation and optimization of parameters affecting the multiply charged ion yield in AP-MALDI MS

Liquid matrix-assisted laser desorption/ionization (MALDI) allows the generation of predominantly multiply charged ions in atmospheric pressure (AP) MALDI ion sources for mass spectrometry (MS) analysis. The charge state distribution of the generated ions and the efficiency of the ion source in generating such ions crucially depend on the desolvation regime of the MALDI plume after desorption in the AP-to-vacuum inlet. Both high temperature and a flow regime with increased residence time of the desorbed plume in the desolvation region promote the generation of multiply charged ions. Without such measures the application of an electric ion extraction field significantly increases the ion signal intensity of singly charged species while the detection of multiply charged species is less dependent on the extraction field. In general, optimization of high temperature application facilitates the predominant formation and detection of multiply charged compared to singly charged ion species. In this study an experimental set-up and optimization strategy is described for liquid AP-MALDI MS which improves the ionization efficiency of selected ion species up to 14 times. In combination with ion mobility separation, the method allows the detection of multiply charged peptide and protein ions for analyte solution concentrations as low as 2fmol/μL (0.5μL, i.e. 1fmol, deposited on the target) with very low sample consumption in the low nL-range.

Influence of initial velocity of analytes on in-source decay products in MALDI mass spectrometry using salicylic acid derivative matrices

The fragmentation and recombination process of peptide radicals during matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) with salicylic acid derivative matrices has been described. MALDI-ISD is initiated by hydrogen transfer between peptide and matrix. The use of reducing and oxidizing matrix leads to the formation of c′/z and a/x fragment pairs, respectively. The fragmentation of analyte radicals occurs via a uni-molecular dissociation processes and independently of the collision in MALDI plume. In contrast, hydrogen transfer between analyte radicals and matrix molecules occurs by collision in MALDI plume as a bi-molecular process. The rate of collision in MALDI plume could be estimated by initial velocity of analyte ions and it affect on the yields of ISD fragments. Considering the initial velocity of analyte ions allows better understanding the MALDI-ISD process.

Direct access to aptamer–protein complexes via MALDI-MS

We report on the direct detection of protein–aptamer complexes by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). By using optimized conditions, we were able to observe the complexes of thrombin and two different thrombin binding aptamers (TBAs) directly. We also detected the complex of PDGF-AB/BB with the specific PDGF binding aptamer (Apt-35) in a 1 : 2 stoichiometry. Detection of the complex between lysozyme and its corresponding aptamer further confirmed the capability of MALDI-MS for studying such systems. All these analyses could be performed with very low sample concentrations (1 pmol) and volumes (1–10 μL). Well-designed control experiments confirmed that the complex observation is due to specific non-covalent interactions, rather than non-specific clusters formed in the MALDI plume. The stronger thrombin–TBA29 complex showed a larger signal at the m/z of the intact complex than the weaker thrombin–TBA15 complex; the complex signal of Apt-35 and PDGF-BB was stronger in MALDI compared with that of PDGF-AB and PDGF-AA. These observations indicate that the noncovalent interaction strength in solution is reflected in the MALDI mass spectra.

Role of three-body recombination for charge reduction in MALDI process

Ions in Matrix-Assisted Laser Desorption/Ionization (MALDI) are predominantly singly charged for small analyte molecules. With the estimated high number density and low temperature of electrons, the three-body recombination mechanism is attractive and should be considered as an important cause for the charge reduction in the plume. Theoretical calculations indicate that the rate coefficient of the three-body recombination is about 50 times higher than that of the two-body recombination if the analyte molecule has insufficient degrees of freedom. Experimental results show that, for small analyte molecules, the ratio of AH2(2+)/AH(+) is close to the theoretical 5% value from the three-body recombination modeling and this ratio declines with the increasing electron and matrix molecule number density caused by greater laser irradiance. The ratio of [A + 2](+)/[A + 1](+) is higher than the theoretical isotopic value, and the excess [A + 2](+) could exclusively result from the three-body recombination collisions. Further evidence demonstrates that [A + 2](+)/[A + 1](+) increases with electron number density, which is in correspondence with the model. All of these theoretical and experimental results indicate that three-body recombination is an essential charge reduction mechanism for small molecules in the MALDI plume.

9,10‐Diphenylanthracene as a matrix for MALDI‐MS electron transfer secondary reactions

The most common secondary-ionization mechanism in positive ion matrix-assisted laser desorption/ionization (MALDI) involves a proton transfer reaction to ionize the analyte. Peptides and proteins are molecules that have basic (and acidic) sites that make them susceptible to proton transfer. However, non-polar, aprotic compounds that lack basic sites are more difficult to protonate, and creating charged forms of this type of analyte can pose a problem when conventional MALDI matrices are employed. In this case, forming a radical molecular ion through electron transfer is a viable alternative, and certain matrices may facilitate the process. In this work, we investigate the performance of a newly developed electron-transfer secondary reaction matrix: 9,10-diphenylanthracene (9,10-DPA). The use of 9,10-DPA as matrix for MALDI analysis has been tested using several model compounds. It appears to promote ionization through electron transfer in a highly efficient manner as compared to other potential matrices. Thermodynamic aspects of the observed electron transfers in secondary-ionization reactions were also considered, as was the possibility for kinetically controlled/endothermic, electron-transfer reactions in the MALDI plume.

MALDI-MS detection of noncovalent interactions of single stranded DNA with Escherichia coli single-stranded DNA-binding protein

The Escherichia coli single-stranded DNA binding protein (SSB) selectively binds single-stranded (ss) DNA and participates in the process of DNA replication, recombination and repair. Different binding modes have previously been observed in SSB•ssDNA complexes, due to the four potential binding sites of SSB. Here, chemical cross-linking, combined with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), is used to determine the stoichiometry of the SSB•ssDNA complex. SSB forms a stable homotetramer in solution, but only the monomeric species (m/z 19,100) can be detected with standard MALDI-MS. With chemical cross-linking, the quaternary structure of SSB is conserved, and the tetramer (m/z 79,500) was observed. We found that ssDNA also functions as a stabilizer to conserve the quaternary structure of SSB, as evidenced by the detection of a SSB•ssDNA complex at m/z 94,200 even in the absence of chemical cross-linking. The stability of the SSB•ssDNA complex with MALDI strongly depends on the length and strand of oligonucleotides and the stoichiometry of the SSB•ssDNA complex, which could be attributed to electrostatic interactions that are enhanced in the gas phase. The key factor affecting the stoichiometry of the SSB•ssDNA complex is how ssDNA binds to SSB, rather than the protein-to-DNA ratio. This further suggests that detection of the complex by MALDI is a result of specific binding, and not due to non-specific aggregation in the MALDI plume.

Fragmentation Processes of Hydrogen-Deficient Peptide Radicals in Matrix-Assisted Laser Desorption/Ionization In-Source Decay Mass Spectrometry

The mechanism of in-source decay (ISD) in matrix-assisted laser desorption/ionization (MALDI) has been described. The MALDI-ISD with an oxidizing matrix is initiated by hydrogen abstraction from peptides to matrix molecules, leading to hydrogen-deficient peptide radicals. Subsequently, the C(α)-C and C(α)-H bonds are cleaved, forming the a•/x fragment pair and [M-2H], respectively. Those reactions competitively occur during MALDI-ISD processes. Our results suggest that the C(α)-H bond cleavage to form [M-2H] was induced by collisions between hydrogen-deficient peptide radicals and matrix molecules in the MALDI plume. In contrast, the C(α)-C bond cleavages occur via a unimolecular dissociation process and independently of the collision rate in the MALDI plume. The formation mechanism of the a-, b-, and d-series fragments are also described. We report 2,5-bis(2-hydroxyethoxy)-7,7,8,8-tetracyanoquinodimethane (bisHE-TCNQ), being known as an organic semiconductor and an electron acceptor, as a novel suitable matrix for the MALDI-ISD of peptides via hydrogen abstraction.

Changes in Dissociation Efficiency and Kinetics of Peptide Ions Induced by Basic Residues and Their Mechanistic Implication

With matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry, total abundance of product ions formed by dissociation inside (in-source decay, ISD) and outside (post-source decay, PSD) the source was measured for peptide ions [Y(5)X + H](+), [XY(5) + H](+), [Y 2)XY(3) + H](+), and [XY(4)X + H](+) (X = tyrosine (Y), histidine (H), lysine (K), and arginine (R) with H for the ionizing proton). α-Cyano-4-hydroxycinammic acid was used as matrix. Product abundance became smaller in the presence of basic residues (H, K, and R), in the order Y > H ≈ K > R. In particular, product abundances in ISD of peptide ions with R were smaller than those with H or K by an order of magnitude, which, in turn, were smaller than that for [Y(6) + H](+) by an order of magnitude. Product abundance was affected by the most basic residue when more than one basic residue was present. A kinetic explanation for the data was attempted under the assumption of quasi-thermal equilibrium for peptide ions in MALDI plume which undergoes expansion cooling. Dramatic disparity in product abundance was found to arise from small difference in critical energy and entropy. Results indicate similar transition structures regardless of basic residues present, where the ionizing proton keeps interacting with a basic site. Further implication of the results on the dissociation mechanism along b-y channels is discussed.

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.

Clustering of Pentacene and Functionalized Pentacene Ions in a Matrix-Assisted Laser Desorption/Ionization Orthogonal TOF Mass Spectrometer

A high-performance orthogonal time-of flight (TOF) mass spectrometer, in combination with the matrix assisted laser desorption/ionization (MALDI) source operating at elevated pressure ( approximately 1 torr in N(2)), was used to perform MALDI-TOF analyses of pentacene and some of its derivatives with and without an added matrix. These molecules are among the most interesting semiconductor materials for organic thin film transistor applications (OTFT). The observation of ion-molecule reactions between "cold" analyte ions and neutral analyte molecules in the gas phase has provided some insight into the mechanism of pentacene cluster formation and its functionalized derivatives. Furthermore, some of the matrices employed to assist the desorption/ionization process of these compounds were observed to influence the outcome via ion-molecule reactions of analyte ions and matrix molecules in the gas phase. The stability and reactivity of the compounds and their clusters in the MALDI plume during gas-phase expansion were evaluated; possible structures of the resulting clusters are discussed. The MALDI-TOF technique was also helpful in distinguishing between two isomeric forms of bis-[(triisopropylsilyl)-ethynyl]-pentacene.

Fragmentation of Peptides in MALDI In-Source Decay Mediated by Hydrogen Radicals

In-source decay (ISD) in matrix-assisted laser desorption/ionization (MALDI) shares some similarities with the novel fragmentation technique electron capture dissociation (ECD). In both reactions, the otherwise strong N-C(alpha) bond is cleaved, forming fragment ions of the c and z types, while labile posttranslational modifications are preserved. Therefore, it is tempting to assume that ISD and ECD have some mechanistic aspects in common. Because electrons are present in the MALDI plume, we investigated the previously suggested possibility that ISD is a variation of ECD. However, experiments with peptides with only one site for efficient protonation revealed that ISD is not caused by electron capture. Instead, ICD seems to be induced by hydrogen atoms generated by a photochemical reaction of the matrix. We provide evidence for this reaction by hydrogen/deuterium exchange experiments with peptides containing a minimal number of exchangeable hydrogen atoms. The hydrogen atom model in ECD is indirectly supported by the proposed fragmentation mechanism for ISD, because our data suggest that hydrogen radicals can induce fragmentation by cleavage of the N-C(alpha) bond, independent from their origin.

Laser pulse length dependence of internal energy transfer in UV-MALDI-MS

Recent internal energy (IE) measurements for various analytes in matrix-assisted laser desorption ionization (MALDI) have indicated that the amount of IE transferred to analytes not only depends on the matrix but also on the nature of the analyte. Common matrixes, such as α-cyano-4-hydroxycinnamic acid (CHCA), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA), and 2,5-dihydroxy-benzoic acid (DHB), had been characterized as “cold” or “hot” according to the IEs of analyte ions produced in the corresponding MALDI plume. In this contribution, we present evidence that IE transfer in MALDI depends on the matrix, analyte, as well as on the laser pulse properties. A substituted benzylpyridinium salt as a thermometer molecule (TM) was investigated in CHCA, SA, and DHB matrixes. A nitrogen laser (4 ns pulse length) and a mode locked frequency tripled Nd:YAG laser (22 ps pulse length) were used as excitation sources at various fluences. Survival yields (SYs) of the analyte molecular ions were extracted from the spectra and the corresponding IEs were obtained from Rice–Ramsperger–Kassel–Marcus (RRKM) theory. The SYs indicate that the IEs of analyte ions in MALDI are analyte, matrix, and laser source dependent. The ion generation threshold fluences follow the same order for both lasers: CHCA<SA<DHB, but for the analyte the mode locked 3×ω Nd:YAG laser source requires a higher threshold fluence than the nitrogen laser. Despite the higher fluence, the SYs are generally higher (the corresponding IEs are lower) for the 3×ω Nd:YAG laser than for the nitrogen laser. The SYs of the TM molecular ions decrease with an increase of fluence for both the ns laser and the ps laser.

Thermodynamic control of final ion distributions in MALDI: in-plume proton transfer reactions

Gas-phase thermochemical data on matrix-assisted laser desorption/ionization (MALDI) matrix species were used to calculate the energetics of possible proton transfer reactions. These were investigated for the MALDI matrices 2,4,6-trihydroxyacetophenone and sinapic acid, with the tripeptide glycyl–glycyl–histidine as analyte. MALDI proton transfer product ion distributions were found to be predicted by the energetics of possible secondary ion–molecule reactions, at all laser fluences sufficient to generate a dense plume. Near the ion generation fluence threshold, the mass spectra deviate from the thermodynamic predictions. This shows that the MALDI plume exhibits both thermodynamically and kinetically controlled regimes, depending on desorption conditions. Possible thermal ground state proton transfer primary ionization pathways were also considered, and found to be inconsistent with the data.

Two-photon ionization thresholds of matrix-assisted laser desorption/ionization matrix clusters.

Direct two-photon ionization of the matrix has been considered a likely primary ionization mechanism in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. This mechanism requires that the vertical ionization threshold of matrix materials be below twice the laser photon energy. Because dimers and larger aggregates may be numerous in the early stages of the MALDI plume expansion, their ionization thresholds are important as well. We have used two-color two-photon ionization to determine the ionization thresholds of jet cooled clusters of an important matrix, 2,5-dihydroxy benzoic acid (DHB), and mixed clusters with the thermal decomposition product of DHB, hydroquinone. The thresholds of the clusters were reduced by only a few tenths of an eV compared to the monomers, to an apparent limit of 7.82 eV for pure DHB clusters. None of the investigated clusters can be directly ionized by two nitrogen laser photons (7.36 eV), and the ionization efficiency at the thresholds is low.

Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry of luteinizing hormone releasing hormone–metal ion complexes

Complexes of luteinizing hormone releasing hormone (LHRH) with divalent metal ions (Ni, Zn, Cu) have been studied by matrix-assisted laser desorption ionization (MALDI) and Fourier transform mass spectrometry. LHRH–metal complexes were detected in high abundance for all three metals from synthesized samples, particularly in negative ion mode. The mixture of the apopeptide with the metal salts yielded in most cases a very minor signal of metal–complex ions. As opposed to Ni and Zn, copper complex ions were mostly observed as Cu(I) adducts. This can be partly attributed to plume reactions of Cu(I) with the apopeptide. the Cu(II) complexes appeared only for the synthetic complex. We show how to distinguish between the contribution to the overall signal from desorbed complexes and from Cu(I) complexes formed in the MALDI plume.