Infrared Matrix-assisted Laser Desorption/ionization Research Articles

IR-MALDI Mass Spectrometry Imaging with Plasma Post-Ionization of Nonpolar Metabolites.

Ambient mass spectrometry imaging (MSI) methods come with the advantage of visualizing biomolecules from tissues with no or minimal sample preparation and operation under atmospheric-pressure conditions. Similar to all other MSI methodologies, however, ambient MSI modalities suffer from a pronounced bias toward either polar or nonpolar analytes due to the underlying desorption and ionization mechanisms of the ion source. In this study, we present the design, construction, testing, and application of an in-capillary dielectric barrier discharge (DBD) module for post-ionization of neutrals desorbed by an ambient infrared matrix-assisted laser desorption/ionization (IR-MALDI) MSI source. We demonstrate that the DBD device enhances signal intensities of nonpolar compounds by up to 104 compared to IR-MALDI without affecting transmission of IR-MALDI ions. This allows performing MSI experiments of mouse tissue and Danaus plexippus caterpillar tissue sections, visualizing the distribution of sterols, fatty acids, monoglycerides, and diglycerides that are not detected in IR-MALDI MSI experiments. The pronounced signal enhancement due to IR-MALDI-DBD compared to IR-MALDI MSI enables mapping of nonpolar analytes with pixel resolutions down to 20 μm in mouse brain tissue and to discern the spatial distribution of sterol lipids characteristic for histological regions of D. plexippus.

Optimized C-Trap Timing of an Orbitrap 240 Mass Spectrometer for High-Throughput Screening and Native MS by IR-MALDESI.

Infrared matrix-assisted laser desorption ionization (IR-MALDESI) is a hybrid mass spectrometry ionization source that combines the benefits of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) making it a great analytical tool for high-throughput screening (HTS) analyses. IR-MALDESI is coupled to an Orbitrap Exploris 240 mass spectrometer that utilizes a bent quadrupole (C-trap) to inject accumulated ions into the high-field Orbitrap mass analyzer. Here, we present a study on the optimized C-trap timing for HTS analyses by IR-MALDESI mass spectrometry. The timing between initial ion generation and the C-trap opening time was optimized to reduce unnecessary ambient ion accumulation in the mass spectrometer. The time in which the C-trap was held open, the ion accumulation time, was further optimized to maximize the accumulation of analyte ions generated using IR-MALDESI. The resulting C-trap opening scheme benefits small-molecule HTS analyses by IR-MALDESI by maximizing target ion abundances, minimizing ambient ion abundances, and minimizing the total analysis time per sample. The proposed C-trap timing scheme for HTS does not translate to large molecules; a NIST monoclonal antibody standard reference material was analyzed to demonstrate that larger analytes require longer ion accumulation times and that IR-MALDESI can measure intact antibodies in their native state.

In situ monitoring of photocatalyzed isomerization reactions on a microchip flow reactor by IR-MALDI ion mobility spectrometry

The visible-light photocatalytic E/Z isomerization of olefins can be mediated by a wide spectrum of triplet sensitizers (photocatalysts). However, the search for the most efficient photocatalysts through screenings in photo batch reactors is material and time consuming. Capillary and microchip flow reactors can accelerate this screening process. Combined with a fast analytical technique for isomer differentiation, these reactors can enable high-throughput analyses. Ion mobility (IM) spectrometry is a cost-effective technique that allows simple isomer separation and detection on the millisecond timescale. This work introduces a hyphenation method consisting of a microchip reactor and an infrared matrix-assisted laser desorption ionization (IR-MALDI) ion mobility spectrometer that has the potential for high-throughput analysis. The photocatalyzed E/Z isomerization of ethyl-3-(pyridine-3-yl)but-2-enoate (E-1) as a model substrate was chosen to demonstrate the capability of this device. Classic organic triplet sensitizers as well as Ru-, Ir-, and Cu-based complexes were tested as catalysts. The ionization efficiency of the Z-isomer is much higher at atmospheric pressure which is due to a higher proton affinity. In order to suppress proton transfer reactions by limiting the number of collisions, an IM spectrometer working at reduced pressure (max. 100 mbar) was employed. This design reduced charge transfer reactions and allowed the quantitative determination of the reaction yield in real time. Among 14 catalysts tested, four catalysts could be determined as efficient sensitizers for the E/Z isomerization of ethyl cinnamate derivative E-1. Conversion rates of up to 80% were achieved in irradiation time sequences of 10 up to 180 s. With respect to current studies found in the literature, this reduces the acquisition times from several hours to only a few minutes per scan.

Sub-ambient pressure IR-MALDI ion mobility spectrometer for the determination of low and high field mobilities.

A new ion mobility (IM) spectrometer, enabling mobility measurements in the pressure range between 5 and 500mbar and in the reduced field strength range E/N of 5-90 Td, was developed and characterized. Reduced mobility (K0) values were studied under low E/N (constant value) as well as high E/N (deviation from low field K0) for a series of molecular ions in nitrogen. Infrared matrix-assisted laser desorption ionization (IR-MALDI) was used in two configurations: a source working at atmospheric pressure (AP) and, for the first time, an IR-MALDI source working with a liquid (aqueous) matrix at sub-ambient/reduced pressure (RP). The influence of RP on IR-MALDI was examined and new insights into the dispersion process were gained. This enabled the optimization of the IM spectrometer for best analytical performance. While ion desolvation is less efficient at RP, the transport of ions is more efficient, leading to intensity enhancement and an increased number of oligomer ions. When deciding between AP and RP IR-MALDI, a trade-off between intensity and resolving power has to be considered. Here, the low field mobility of peptide ions was first measured and compared with reference values from ESI-IM spectrometry (at AP) as well as collision cross sections obtained from molecular dynamics simulations. The second application was the determination of the reduced mobility of various substituted ammonium ions as a function of E/N in nitrogen. The mobility is constant up to a threshold at high E/N. Beyond this threshold, mobility increases were observed. This behavior can be explained by the loss of hydrated water molecules.

Determination of Optimal Electrospray Parameters for Lipidomics in Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging.

Infrared matrix-assisted laser desorption ionization (IR-MALDESI) is an ambient mass spectrometry imaging (MSI) technique that relies on electrospray ionization (ESI) for ion generation of desorbed neutrals. Although many mechanisms in IR-MALDESI have been studied in depth, there has not yet been a comprehensive study of how the ESI parameters change the profiles of tissue specific lipids. Acetonitrile (ACN)/water and methanol (MeOH)/water solvent systems and compositions were varied across a series of applied ESI voltages during IR-MALDESI analysis of rat liver tissue. Gradients of 12 min were run from 5 to 95% organic solvent in both positive and negative polarities across 11 voltages between 2.25 and 4.5 kV. These experiments informed longer gradients (25-30 min) across shorter solvent gradient ranges with fewer voltages. Optimal ESI parameters for lipidomics were determined by the number and abundance of detected lipids and the relative proportion of background ions. In positive polarity, the best solvent composition was 60-75% ACN/40-25% H2O with 0.2% formic acid at 3.2 kV applied voltage. The best parameters for negative polarity analysis are 45-55% ACN/55-45% H2O with 1 mM of acetic acid for voltages between 2.25 and 3.2 kV. Using these defined parameters, IR-MALDESI positive polarity lipidomics studies can increase lipid abundances 3-fold, with 15% greater coverage, while an abundance increase of 1.5-fold and 10% more coverage can be achieved relative to commonly used parameters in negative polarity.

Atmospheric pressure laser desorption/ionization using a 6-7 µm-band mid-infrared tunable laser and liquid water matrix.

Due to the characteristic absorption peaks in the IR region, various molecules can be used as a matrix for infrared matrix-assisted laser desorption/ionization (IR-MALDI). Especially in the 6-7 µm-band IR region, solvents used as the mobile phase for liquid chromatography have absorption peaks that correspond to their functional groups, such as O-H, C=O, and CH3. Additionally, atmospheric pressure (AP) IR-MALDI, which is applicable to liquid-state samples, is a promising technique to directly analyze untreated samples. Herein we perform AP-IR-MALDI mass spectrometry of a peptide, angiotensin II, using a mid-IR tunable laser with a tunable wavelength range of 5.50-10.00 µm and several different matrices. The wavelength dependences of the ion signal intensity of [M + H](+) of the peptide are measured using a conventional solid matrix, α-cyano-4-hydroxycinnamic acid (CHCA) and a liquid matrix composed of CHCA and 3-aminoquinoline. Other than the O-H stretching and bending vibration modes, the characteristic absorption peaks are useful for AP-IR-MALDI. Peptide ions are also observed from an aqueous solution of the peptide without an additional matrix, and the highest peak intensity of [M + H](+) is at 6.00 µm, which is somewhat shorter than the absorption peak wavelength of liquid water corresponding to the O-H bending vibration mode. Moreover, long-lasting and stable ion signals are obtained from the aqueous solution. AP-IR-MALDI using a 6-7 µm-band IR tunable laser and solvents as the matrix may provide a novel on-line interface between liquid chromatography and mass spectrometry.

Water ice is a soft matrix for the structural characterization of glycosaminoglycans by infrared matrix-assisted laser desorption/ionization.

Glycosaminoglycans (GAGs) are a class of heterogeneous, often highly sulfated glycans that form linear chains consisting of up to 100 monosaccharide building blocks and more. GAGs are ubiquitous constituents of connective tissue, cartilage, and the extracellular matrix, where they have key functions in many important biological processes. For their characterization by mass spectrometry (MS) and tandem MS, the high molecular weight polymers are usually enzymatically digested to oligomers with a low degree of polymerization (dp), typically disaccharides. However, owing to their lability elimination of sulfate groups upon desorption/ionization is often encountered leading to a loss of information on the analyte. Here, we demonstrate that, in particular, water ice constitutes an extremely mild matrix for the analysis of highly sulfated GAG disaccharides by infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry. Depending on the degree of sulfation, next to the singly charged ionic species doubly- and even triply charged ions are formed. An unambiguous assignment of the sulfation sites becomes possible by subjecting sodium adducts of the GAGs to low-energy collision-induced dissociation tandem MS. These ionic species exhibit a remarkable stability of the sulfate substituents, allowing the formation of fragment ions retaining their sulfation that arise from either cross-ring cleavages or rupture of the glycosidic bonds, thereby allowing an unambiguous assignment of the sulfation sites.

Analysis of Noncovalent Chitinase-Chito-Oligosaccharide Complexes by Infrared-Matrix Assisted Laser Desorption Ionization and Nanoelectrospray Ionization Mass Spectrometry

Transferring noncovalently bound complexes from the condensed phase into the gas phase represents a challenging task due to weak intermolecular bonds that have to be maintained during the phase transition. Currently, electrospray ionization (ESI) is the standard mass spectrometric (MS) technique to analyze noncovalent complexes. Although infrared matrix-assisted laser desorption ionization (IR-MALDI)-MS also provides particular soft desorption/ionization conditions, this method has so far hardly been applied for the analysis of noncovalent complexes. In this study, we employed IR-MALDI orthogonal time-of-flight (o-TOF)-MS in combination with the liquid matrix glycerol to characterize the specific complex formation of chito-oligosaccharide (CHOS) ligands with two variants of Chitinase A (ChiA) from Serratia marcescens, the inactive E315Q mutant and the active W167A mutant, respectively. The IR-MALDI-o-TOF-MS results were compared to those obtained using nano-ESI-quadrupole (q)-TOF-MS and ultraviolet (UV)-MALDI-o-TOF-MS. Using IR-MALDI-o-TOF-MS, specific noncovalent complexes between ChiA and CHOS were detected with distributions between enzymes with bound oligosaccharides vs free enzymes that were essentially identical to those obtained by nano-ESI-q-TOF-MS. Chitinase-CHOS complexes were not detected when UV-MALDI was employed for desorption/ionization. The results show that IR-MALDI-MS can be a valuable tool for fast and simple screening of noncovalent enzyme-ligand interactions.

Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.

Infrared matrix-assisted laser desorption/ionization mass spectrometry for quantification of glycosaminoglycans and gangliosides

Infrared matrix-assisted laser desorption/ionization (IR-MALDI) is a “softer” ionization technique than UV-MALDI. In the present study, IR-MALDI using a 6 μm laser produced clean negative ion mass spectra of sulfated disaccharides, from heparin and chondroitin sulfate, without significant loss of sulfate groups. The sulfur content (wt%) of chondroitin sulfate could be calculated from the m/ z and the signal intensity of di- and tetra-saccharides derived from chondroitinase digestion. Similarly, no significant loss of sialic acid occurred in poly-sialylated gangliosides including GD1b, GT1b and GQ1b. Interestingly, multiply deprotonated [M− nH] n− ions were observed in their mass spectra, and were useful for relative quantification of gangliosides in complex mixtures. These features of IR-MALDI were anticipated to be useful for robust discrimination and quantification of different gangliosides by a mass imaging technique. The multiply deprotonated ions observed in the 6 μm laser IR-MALDI mass spectrum suggested that the mechanism underlying deprotonated ion generation might differ between the 2.94 μm and 6 μm laser wavelengths.

Matrix-assisted laser desorption ionization of infrared laser ablated particles

An infrared (IR) laser was used to ablate particles that were subsequently ionized by matrix-assisted laser desorption ionization (MALDI). Infrared light from a pulsed optical parametric oscillator (OPO) laser system was directed at a solid sample under vacuum containing a 2,5-dihydroxybenzoic acid (DHB) matrix and peptide or protein analyte. A pulsed 351 nm ultraviolet (UV) excimer laser that was directed 1.4 mm above and parallel to the sample surface was used to irradiate the ablated material in the desorption plume. Ions created by post-ablation ionization were detected with a linear time-of-flight (TOF) mass spectrometer. Mass spectra of the peptide bradykinin and proteins bovine insulin and cytochrome c, were recorded. Under these conditions, two simultaneous mass spectra were generated: an IR–MALDI mass spectrum from the OPO and a UV post-ablation spectrum generated by irradiating material in the plume. Factors affecting the two-laser ion yield were studied, including the delay time between the laser pulses and the fluence of the IR and UV laser.

赤外レーザーマトリックス支援レーザー脱離イオン化法の波長依存性に関する研究と応用

Infrared matrix-assisted laser desorption/ionization (IR-MALDI) using an infrared laser is promising, because most biomolecules have a specific absorption in the infrared range. IR-MALDI is expected to provide the following advantages: (1) use of various matrices; (2) use of biomolecules such as water and lipids as the matrix; and (3) super-soft ionization, i.e., high-molecular-weight protein analysis without fragmentation. However, IR-MALDI is not widely used because of its low sensitivity, complexity, high cost, and low-compatibility with commercial MALDI time-of-flight mass spectrometers. We evaluated the wavelength dependence of ionization in IR-MALDI to clarify the ionization mechanism. The molecular mass of angiotensin II was obtained at a wavelength between 5.8 and 6.2 μm, which corresponds to the >C=O stretching vibration mode. In IR-MALDI, we believe that the strong molecular bond attracts an electron from a neighboring hydrogen atom, possibly protonating it.

Resolution of infrared matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry using glycerol; enhancement with a disperse laser beam

Some experimental factors affecting the resolution in glycerol infrared matrix-assisted laser desorption/ionization (IR-MALDI) time-of-flight (TOF) mass spectrometry were investigated. Loading the sample inside a cavity covered with a grid was found to improve the resolving power as reported previously, although not to the extent attainable in UV-MALDI using the same instrument. The resolving power improved as the laser spot area at the sample position got larger, becoming almost comparable with that in UV-MALDI when the spot area was a little larger than the cavity size. Reduced concentration of the ablated materials in the acceleration region with the use of the grid and large irradiation area may be responsible for the enhanced resolution. In addition, the threshold laser fluences measured in this work were lower than those reported in the literature and tended to decrease more rapidly as the irradiation area increased than predicted previously. The implication of similar threshold fluences for matrix and analyte ions is discussed in relation to the analyte ion formation mechanism.

Characterization of Infrared Matrix-Assisted Laser Desorption Ionization Samples by Fourier Transform Infrared Attenuated TotalReflection Spectroscopy

Fourier transform infrared attenuated total reflection (FT-IR ATR) spectroscopy was used to characterize thin films of succinic acid, a matrix compound commonly used with infrared matrix-assisted laser desorption ionization (IR-MALDI) mass spectrometry. IR spectra of succinic acid thin films deposited alone and in combination with the analyte biomolecules insulin and cytochrome c were obtained by FT-IR ATR spectroscopy. Spectra of analyte and matrix alone were similar to those obtained previously from KBr pellets, Nujol mull, or thin-film absorption, although the ATR spectra have significantly lower background interferences. Thin films deposited from mixtures of water and methanol have additional peaks compared to films deposited from a methanol solution. These additional peaks are attributed to carboxylate groups stabilized by residual water molecules. No evidence was found to suggest that residual water absorption contributes to absorption at wavelengths typically used for IR-MALDI. Absorption of energy by analyte vibrational modes with rapid energy transfer to the matrix is suggested as a contributor to desorption and ionization consistent with the FT-IR ATR results.

Photoacoustic Analysis of Matrix-Assisted Laser Desorption/Ionization Processes with Pulsed Infrared Lasers

A fast piezoelectric detection system was utilized to record time-resolved photoacoustic signals reflecting the thermal expansion and the rate of material ablation in infrared matrix-assisted laser desorption/ionization (IR-MALDI). Glycerol was employed as a liquid and 2,5-dihydroxybenzoic acid (2,5-DHB) as a crystalline matrix. An Er:YAG laser (λ = 2.94 μm; τL ∼ 100 ns) and a wavelength-tunable optical parametric oscillator (OPO) laser system (λ = 1.4−4.0 μm; τL = 6 ns) were used for desorption/ionization. Material ejection and MALDI ion signals were recorded as a function of laser fluence, pulse duration, and wavelength. For glycerol as matrix, material ejection was found to be temporally confined to about the laser pulse duration when the OPO was employed. For excitation with the Er:YAG laser, a prolonged material ejection with an approximately exponential decay was observed with a characteristic time constant of ∼1 μs. For both lasers, material ejection was observed already at fluences substantially below the MALDI ion threshold. For excitation with the OPO, a correlation between a change in the desorption/ablation mechanism and the onset of ion generation was found. Crystalline 2,5-DHB preparations were investigated with the OPO only. Material ejection from this matrix was found to exhibit a significantly different dependence of the overall ejected material on laser fluence. The article also provides an introduction to the underlying photoacoustic theory adapted to the MALDI process.

Time-of-Flight Mass Spectrometry Using a Mid-Infrared Free-Electron Laser

A time-of-flight mass spectrometric apparatus was built for infrared matrix-assisted laser desorption/ionization (IR-MALDI) experiments using a free-electron laser (FEL). Preliminary experiments were carried out with a CO2 laser at 9.5 μm and a FEL at 10.6 μm. These mid-IR wavelengths were shown to produce light atomic ions from solid samples. The detected ion peaks were broadened by the FEL macropulse with 15 μs width. Applying a delayed extraction pulse to FEL-generated ions brought about a small improvement on the mass resolution. By chopping a flux of ions at exit of the ion source region, the ion peak width became narrower by a factor of more than 10.

Infrared matrix-assisted laser desorption/ionization of polycyclic aromatic hydrocarbons with a sulfolane matrix.

Infrared matrix-assisted laser desorption/ionization (IR-MALDI) of the polyaromatic hydrocarbons (PAHs) anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene was performed using a 10.6-microm CO2 laser and a liquid matrix. Sulfolane (tetrahydrothiophene 1,1-dioxide) was found to be an effective matrix for PAH ionization: mass spectra obtained with a sulfolane matrix contain an intense molecular ion peak; interference from PAH fragment and matrix peaks is negligible in all cases. The main limitation of the sulfolane matrix is sample evaporation after 3 to 5 min in vacuum. This sample lifetime can be increased to between 15 and 30 min using a 2:1 (v/v) mixture of sulfolane and glycerol, but the resulting spectra have greater matrix interference and decreased shot-to-shot signal stability.

Influence of the laser fluence in infrared matrix-assisted laser desorption/ionization with a 2.94 microm Er : YAG laser and a flat-top beam profile.

The dependence of the signal intensity of analyte and matrix ions on laser fluence was investigated for infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry using a flat-top laser beam profile. The beam of an Er : YAG laser (wavelength, 2.94 microm; pulse width, 90 ns) was coupled into a sapphire fiber and the homogeneously illuminated end surface of the fiber imaged on to the sample by a telescope. Three different laser spot sizes of 175, 350 and 700 microm diameter were realized. Threshold fluences of common IR matrices were determined to range from about 1000 to a few thousand J m(-2), depending on the matrix and the size of the irradiated area. In the MALDI-typical fluence range, above the detection threshold ion signals increase strongly with fluence for all matrices, with a dependence similar to that for UV-MALDI. Despite the strongly different absorption coefficients of the tested matrices, varying by more than an order of magnitude at the excitation laser wavelength, threshold fluences for equal spot sizes were found to be comparable within a factor of two. With the additional dependence of fluence on spot size, the deposited energy per volume of matrix at threshold fluence ranged from about 1 kJ mol(-1) for succinic acid to about 100 kJ mol(-1) for glycerol.

Matrix-assisted laser desorption/ionization time-of-flight analysis of Bacillus spores using a 2.94 microm infrared laser.

The performance of infrared (2.94 microm) and ultraviolet (337 nm) lasers were compared for analysis of purified spores of B. subtilis, B. cereus and B. globigii on a four-inch end-cap reflectron time-of-flight instrument. Infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectra of these microorganisms displayed a larger number of biomarker peaks above m/z 4000, compared with UV-MALDI. Biomarker peaks were observed at higher m/z values with the IR laser.

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.