Matrix-assisted Laser Desorption/ionization Technique Research Articles

Speciation study of an imidazolate-bridged copper(II)–zinc(II) complex in aqueous solution

The solution equilibrium and the binding mode of the species in the five-component system containing two metal ions (copper(II) and zinc(II)) and three ligands ( A = diethylenetriamine, B = imidazole, C = tris(2-aminoethyl)amine) were investigated by pH-potentiometric titration, UV–visible spectrophotometry and EPR (electron paramagnetic resonance) spectroscopic titration in aqueous solution in the 2–11 pH range. An imidazolate-bridged heterobinuclear complex ( ACuBH − 1 ZnC) was found to evolve above pH = 7 and was stable between pH 7 and 11. The existence of the ACuBH − 1 ZnC complex (by determination of its molecular weight) was proved by mass spectrometry (ESI-MS (electrospray ionization mass spectrometry) and MALDI (matrix-assisted laser desorption/ionization) techniques). The electrochemical behaviour and the superoxide dismutase activity of this complex were also tested by cyclic voltammetry and the Riboflavin/NBT (nitro blue tetrazolium) assay, respectively.

An improvement of matrix-assisted laser desorption/ionization mass spectrometry using an infrared tunable free electron laser

Matrix-assisted laser desorption/ionization (MALDI) combined with time-of-flight mass spectrometry (TOFMS) is a powerful yet robust tool for protein identification, due to its high sensitivity and theoretically unlimited detectable mass range. A large part of functional proteins, such as membrane proteins, are insoluble as native forms in a matrix solution without a strong denaturing condition, hence are not amenable to the conventional MALDI–TOFMS analysis. Aiming at overcoming this difficulty, we have developed a novel MALDI technique (UV/FEL-MALDI). An infrared free electron laser (IR-FEL) has a wide tunability in a mid-IR range and is quite attractive as a source of selective vibrational excitation. The FEL wavelength can be tuned to activate a denaturant, which impedes the conventional MALDI process, without an excess heating of analyte molecules. This scheme lets a dense denaturant to be used for the MALDI sample preparation of insoluble proteins. A simultaneous use of the FEL with a nitrogen pulse laser for MALDI achieves spatially and temporally defined desorption, which is essential to TOFMS detection, while specificity and selectivity owing to an FEL wavelength can be conserved. Some attractive features of the protein clustering have been found in the application of UV/FEL-MALDI to hair keratins, which was chosen as a model of insoluble proteins.

An improvement of matrix-assisted laser desorption/ionization mass spectrometry using an infrared tunable free electron laser

Matrix-assisted laser desorption/ionization (MALDI) combined with time-of-flight mass spectrometry (TOFMS) is a powerful yet robust tool for protein identification, due to its high sensitivity and theoretically unlimited detectable mass range. A large part of functional proteins, such as membrane proteins, are insoluble as native forms in a matrix solution without a strong denaturing condition, hence are not amenable to the conventional MALDI–TOFMS analysis. Aiming at overcoming this difficulty, we have developed a novel MALDI technique (UV/FEL-MALDI). An infrared free electron laser (IR-FEL) has a wide tunability in a mid-IR range and is quite attractive as a source of selective vibrational excitation. The FEL wavelength can be tuned to activate a denaturant, which impedes the conventional MALDI process, without an excess heating of analyte molecules. This scheme lets a dense denaturant to be used for the MALDI sample preparation of insoluble proteins. A simultaneous use of the FEL with a nitrogen pulse laser for MALDI achieves spatially and temporally defined desorption, which is essential to TOFMS detection, while specificity and selectivity owing to an FEL wavelength can be conserved. Some attractive features of the protein clustering have been found in the application of UV/FEL-MALDI to hair keratins, which was chosen as a model of insoluble proteins.

Optical detection and charge-state analysis of MALDI-generated particles with molecular masses larger than 5 MDa.

Charged polystyrene nanoparticles are generated by matrix-assisted laser desorption/ionization (MALDI) and detected by laser-induced fluorescence (LIF) in a quadrupole ion trap. Employing the LIF technique, observations of individual fluorescent nanospheres (27 nm in diameter and containing 180 fluorescein dye equivalents) have been achieved with an average signal-to-noise ratio of approximately 10. With the trap operating at a frequency around 5 kHz, charge state analysis of the particles reveals that the number of charges carried by the spheres is between 1 and 10. It suggests a mass-to-charge ratio (m/z) in the range of 10(5)-10(6) for the MALDI-generated particles. To effectively trap such large particles (m > 5 MDa), damping of the particles' motions by using approximately 50 mTorr He buffer gas is absolutely required. Similar findings are obtained for particles with a nominal size of 1 microm in diameter, demonstrating that production of charged particles with a molecular mass as high as 10(12) Da is possible using the MALDI technique.

Optimization of a hydrophobic solid-phase extraction interface for matrix-assisted laser desorption/ionization.

Matrix-assisted laser desorption/ionization (MALDI) probe surfaces derivatized with octadecanethiol (C18) can be used as hydrophobic solid-phase extraction devices to isolate and desalt biopolymers directly on the probe surface. Using quantitative MALDI, it was possible to determine the approximate amount of peptide that bound to C18 surfaces and thus to calculate a surface density. It was determined that the amount of peptide bound at the probe surface was independent of the analyte concentration in the immersion solution (from high- to sub-ng ml-1 concentrations), but rather was dependent on the immersion time of the surface as it was exposed to the analyte. The capacity of C18-derivatized probes to bind biopolymers in fixed amounts frees the analyst from the necessity for adjusting analyte concentration through multiple step procedures such as serial dilution or vacuum drying. This time savings result in an overall increase in the efficiency of the MALDI technique.

P-Nitroaniline/glycerol: a binary liquid matrix for MALDI analysis

Liquid matrices for matrix-assisted laser desorption/ionization (MALDI) analysis offer advantages over solid matrices including increased shot-to-shot reproducibility and the ability to provide conditions more suitable for the retention of non-covalent complexes. Unfortunately, most of the liquid matrices available do not absorb at UV wavelengths. A second component to increase chromophoric absorption and to permit control over the chemical properties of the matrix can aid in overcoming some of the limitations currently faced by the MALDI technique. In the work reported here, a binary matrix that combines the advantages of the fluidity and low vapor pressure of glycerol with the weak basicity and chromophoric properties of p-nitroaniline is evaluated. Both positive-ion and negative-ion spectra are obtained for proteins, carbohydrates and oligonucleotides. The potential for analysis of chelates and noncovalent interactions using this liquid binary matrix is also explored.

Mass Resolution Improvement by Incorporation of Pulsed Ion Extraction in a Matrix-Assisted Laser Desorption/Ionization Linear Time-of-Flight Mass Spectrometer

A linear time-of-flight mass spectrometer has been modified to incorporate pulsed ion extraction of matrix-assisted laser desorption/ionization (MALDI) generated ions. A unique aspect of the experiments presented is the combination of pulsed extraction with very high source potentials (up to 25 kV) which allows improved mass resolution while maintaining excellent sensitivity for the large m/z ions generated by the MALDI technique. Mass resolution in excess of 1000 (fwhm) is demonstrated for cytochrome c (12,361.1 Da) with the pulsed ion extraction linear time-of-flight mass spectrometer described. The influence on obtainable mass resolution of experimental variables such as delay time between laser ionization and ion extraction, amplitude of the pulsed voltage employed, and the source bias voltage are presented. It is shown that, for any given source potential, the optimum pulsed extraction voltage is a linear function of the mass of the analyte. This is consistent with the observation that the initial ion velocity distribution for MALDI-generated ions is independent of mass.

Matrix-assisted laser desorption mass spectrometry of rhodopsin and bacteriorhodopsin

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for the integral membrane proteins bacteriorhodopsin and bovine rhodopsin desorbed from solubilized membrane preparations. Mass differences in the molecular weights measured for bleached and unbleached bacteriorhodopsin and rhodopsin indicate the removal of the retinal chromophores upon bleaching. The MALDI technique was also successful for determination of the major cleavage products obtained upon treatment of membrane bound rhodopsin with endoproteinase Asp-N and thermolysin. Our results indicate that the MALDI method is a useful means of obtaining accurate molecular weight information on hydrophobic proteins isolated in their native membranes.