Ultraviolet Matrix-assisted Laser Desorption Research Articles

Initial velocities of positive and negative protein molecule-ions produced in matrix-assisted ultraviolet laser desorption using a liquid matrix

The initial axial velocities for the positive and negative cytochrome c molecule-ions desorbed from a liquid matrix (3-nitro-benzyl alcohol) using a 17 ns ultraviolet laser (266 nm) have been determined. The method employed was based upon measurement of ion flight times through a field-free path co-axial with the ion optical axis. The possible interferences, such as energy deficits, due to the presence of electric fields were avoided by using a grid-electrode placed at a very short distance from the sample stage. The potential of this electrode was maintained at the same potential as the sample stage, thereby creating a narrow field-free region within which ion generation took place. Systematic variations of the potential at a second grid-electrode caused a gradual shift of the ion flight times, which could be related to the initial ion velocities. The ion velocities were obtained by correlating the measured time-shifts with values from theoretical analysis. The positive and negative molecule-ions were found to have axial velocities of 840±70 and 750±40 ms −1, respectively.

Analysis of oligodeoxynucleotides by negative-ion matrix-assisted laser desorption mass spectrometry

Thirty compounds were tested in combination with ammonium acetate for the ability to desorb and ionize oligodeoxynucleotides by ultraviolet matrix-assisted laser desorption mass spectrometry. Negative ion yields using matrices such as 2,5-dihydroxybenzoic acid and 3-hydroxy-4-methoxybenzaldehyde are enhanced by the addition of ammonium salts at a molar ratio of 1:1, pH 7. 3-Hydroxy-4-methoxybenzaldehyde was tested with 12 different ammonium, alkylammonium, and pyridinium salts for the ability to cocrystallize with oligodeoxynucleotides and to improve desorption and ionization. Ions of oligodeoxynucleotides 9, 10, and 11 nucleotides in length were observed with a matrix of 3-hydroxy-4-methoxybenzaldehyde and ammonium acetate, pH 7, at a mass resolution of 100–150 (fwhm). A small oligodeoxynucleotide (11-mer) was observed at the femtomole level with a combination of 2,5-dihydroxybenzoic acid and ammonium acetate as the matrix. Ions from single stranded DNA (60 nucleotides in length) were also observed using this same matrix combination. The results of these studies have shown that both sensitivity and desorption conditions need to be further improved before complex mixtures of large pieces of DNA can be effectively analyzed.

Acyl carrier proteins from developing seeds of Cuphea lanceolata Ait

The structure of acyl carrier protein (ACP) may determine the fate of the acyl moieties linked to it in the course of de-novo fatty acid synthesis in higher plants. To investigate a possible correlation between the structure of ACP and the synthesis of medium-chain fatty acids, we isolated and characterized ACP from the seeds of Cuphea lanceolata Ait. (subgenus Eucuphea/Section Heterodon), an annual crop that contains up to 90% decanoic (capric) acid in seed triacylglycerols. After a cell-free extract prepared from developing seeds was treated to 65% saturation with ammonium sulfate, two ACP-isoforms (ACP 1 and ACP 2) were identified in the supernatant that could be purified to homogeneity by anion-exchange chromatography and subsequent reversed-phase high-performance liquid chromatography. The molecular mass determined by matrix-assisted ultraviolet-laser desorption ionization mass spectrometry of ACP 1 was 9315 Da, whereas further heterogeneity was observed for ACP 2 with molecular masses of 8598 and 8703 Da. Aminoterminal sequencing was performed showing a high homology in the primary structures of ACP 1 and ACP 2. Both isoforms were present in the embryo, whereas in the chloroplast-containing seed coat ACP 2 was found in minute amounts, if at all. The expression of ACP 2 correlated with the production of capric acid during the phase of storage-lipid accumulation. These data indicate that ACP 2 is part of the machinery for the synthesis of medium-chain fatty acids, whereas ACP 1 appears to be a constitutive protein.

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

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

Matrix‐assisted laser desorption of peptides and proteins on a quadrupole ion trap mass spectrometer

The use of ultraviolet matrix-assisted laser desorption (MALD) to ionize peptides and proteins for analysis in a quadrupole ion trap is described. An ion source was modified to accommodate a fiber optic to transmit laser radiation from a nitrogen laser (337 nm) to the tip of the sample probe containing peptide or protein samples in a matrix of 2,5-dihydroxybenzoic acid (DHB) or 3,4-dimethoxy-4-hydroxy-cinnamic acid. Detection limits are demonstrated with 10 fmol of sperm-whale myoglobin. The dimer of sperm-whale myoglobin was also observed at m/z 34,430. A comparison is made of the tandem mass spectrum of (MS/MS) of human angiotensin I desorbed by MALD to that of the peptide desorbed by liquid secondary-ion mass spectrometry. Both spectra were found to contain abundant structural information.

Peptide-metal ion interactions in solution: Detection by laser desorption time-of-flight mass spectrometry and electrospray ionization mass spectrometry

The specific interaction of Cu(II) ions with metal-binding peptides in solution has been investigated by two different methods of soft ionization mass spectrometry, namely matrix-assisted ultraviolet laser desorption time-of-flight mass spectrometry (LDTOF) and electrospray ionization mass spectrometry (ES). The metal-binding peptide selected for these investigations is a 26-residue sequence found on the surface of the human plasma metal-transport protein histidine-rich glycoprotein. The peptide, (GHHPH)5 G, was synthesized and evaluated by ES and LDTOF before and after the addition of Cu(II) or Mn(II) ions in solution. In the absence of added metal ions, the peptide was observed to have a mass equal to within 0.5 Da of its calculated mass (2903.0 Da) by both LDTOF and ES. In the presence of Cu(II), up to five additional peaks were observed at mass increments of approximately 63.9 Da (LDTOF) or 62.3 Da (ES); Mn was not bound to the peptide under identical experimental conditions. By both LDTOF and ES, the maximum Cu-binding capacity observed (i.e., 5 g-atoms mol−1) demonstrated that up to 1 Cu could be bound per (GHHPH) internal repeat unit. This peptide-metal ion interaction stoichiometry was verified by direct titration in solution and, with immobilized peptide, by quantitative metal ion affinity chromatography. Thus, the ability to detect stable peptide-metal complexes did not appear to be differentially affected by the two different volatilization/ionization methods needed to generate charged intact molecular ions. The quantity and stoichiometry of bound Cu atoms was affected, however, by experimental conditions such as LDTOF matrix and ES solution composition. These results demonstrate the ability to verify directly the solution-phase binding capacity of metal-binding peptides by LDTOF and by ES. We conclude from these studies that other metallo-organic interactions may also be amenable to investigation by these rapid and sensitive techniques.

Suppression of matrix ions in ultraviolet laser desorption: Scanning electron microscopy and raman spectroscopy of the solid samples

AbstractSuppression of low‐mass ion peaks in matrix‐assisted ultraviolet laser desorption has been found to occur at low matrix‐to‐analyte molar ratios when using nicotinic acid as matrix, independent of the angle of illumination. Microscopic Raman scattering spectroscopy has been employed to investigate the matrix–analyte solid‐state composition. The matrix‐to‐analyte molar ratios employed in preparing the solutions are reliable guides to the relative amounts of matrix and analyte molecules in the solid crystals, given the method of sample preparation employed involving drying under a stream of nitrogen. A qualitative model based on the proton supply‐and‐demand argument is tentatively proposed to explain the suppression phenomenon.

Velocity distributions of intact high mass polypeptide molecule ions produced by matrix assisted laser desorption

The velocity distributions of polypeptide molecule ions (molecular mass 1000–15600 u) produced by matrix assisted ultraviolet laser desorption were measured using a modified time-of-flight mass spectrometer. The data demonstrate that polypeptide molecule ions produced by matrix assisted laser desorption at the ion production threshold irradiance have similar velocity distributions, with an average velocity of approximately 750 m/s. This result has important implications for the design of mass spectrometers that use the effect to generate polypeptide molecule ions and for the theoretical treatment of the laser desorption process. A jet expansion model for the desorption of high mass polymers is proposed to explain the results.

Investigation of UV matrix-assisted laser desorption Fourier transform mass spectrometry for peptides

Ultraviolet matrix-assisted laser desorption can be used to enhance formation of [M + H] +, [M + Na] +, and [M + K] + ions from small peptides for Fourier transform mass spectrometry (FTMS). In accord with laser desorption (LD) time-of-flight experiments, matrices such as nicotinic acid and 2-pyrazinecarboxylic acid exhibit strong enhancement effects (i.e., formation of abundant protonated and cationized molecules for the analyte with virtually no fragment ions) for 266 nm LD/FTMS, whereas pyrazinedicarboxylic acid provides no matrix enhancement at this wavelength. Both sinapinic acid and coumarin-120 provide strong matrix enhancement effects for the 355-nm LD of peptides. For the small peptides examined in this study, no significant differences in the abundance of fragment ions were observed between the 266- and 355-nm wavelengths. Matrix-assisted LD/FTMS is useful for the generation and characterization of ions corresponding to protonated and cationized molecules from virtually all biological compounds with molecular weights up to 2000. The lack of observation of biological ions with m/ z > 2500 may be related to inefficient trapping of these laser-desorbed ions or instrumental detection limitations of FTMS and is under further investigation.

Factors affecting the ultraviolet laser desorption of proteins

The production of high-mass quasimolecular ions from proteins by matrix-assisted ultraviolet laser desorption is described. A simple time-of-flight system using a Q-switched frequency-quadrupled Nd-YAG laser to desorb protein molecules is shown to have a mass range of up to 116,000 u by the observation of intact, singly charged quasimolecular ions from 700 fmol of beta-galactosidase subunit (mol.wt = 116,336 Da). Both positive- and negative-ion spectra of proteins are shown. Four new matrix materials, with properties as good as or better than nicotinic acid, are described. A mass resolution of approximately 500 (full width at half maximum definition) is demonstrated for proteins with mol.wt less than 20,000 Da. Product species, formed by fast photochemical reactions in the matrix, are observed to form adduct ions with protein molecules. These adduct ions are a significant cause of the observed broadness of protein quasimolecular ion peaks. The practical physical considerations in detection of large-mass quasimolecular ions from laser desorption, such as detector overloading, are discussed.