The role of the laser pulse duration in infrared matrix-assisted laser desorption/ionization mass spectrometry

The role of the laser pulse duration in matrix-assisted laser desorption/ionization mass spectrometry with infrared lasers (IR-MALDI-MS) emitting in the 3 microm wavelength range has been evaluated. Mass spectrometric performance and characteristics of the IR-MALDI process were examined by comparing a wavelength-tuneable mid-infrared optical parametric oscillator (OPO) laser of 6 ns pulse duration, tuned to wavelengths of 2.79 and 2.94 microm, with an Er:YAG laser (lambda = 2.94 microm) with two pulse durations of 100 and 185 ns, and an Er:YSGG laser (lambda = 2.79 microm) with a pulse duration of 75 ns. Threshold fluences for the desorption of cytochrome C ions were determined as a function of the laser pulse duration for various common IR-MALDI matrices. For the majority of these matrices a reduction in threshold fluence by a factor of 1.2-1.9 was found by going from the 75-100 ns long pulses of the Erbium lasers to the short 6 ns OPO pulse. Within the experimental accuracy threshold fluences were equal for the 100 and the 185 ns pulse duration of the Er:YAG laser. Some pronounced pulse duration effects related to the ion formation from a glycerol matrix were also observed. The effect of the laser pulse length on the duration of ion emission was furthermore investigated.     

Time-resolved imaging of the plume dynamics in infrared matrix-assisted laser desorption/ionization with a glycerol matrix

The dynamics of the expanding material plume after irradiation of a matrix sample with two different infrared (IR) lasers, an Er:YAG laser of ca. 100 ns and an optical parametric oscillator (OPO) laser system of 6 ns pulse duration, were investigated by imaging the plumes with nanosecond time resolution. Both lasers emitted at an identical wavelength of 2.94 microm. Laser exposure parameters were typical for infrared matrix-assisted laser desorption/ionization mass spectrometry (IR-MALDI-MS); glycerol was employed as a liquid matrix to provide a homogeneous sample and reproducible plume formation. A Nd:YAG laser (532 nm; 8 ns) was used as the illumination source and a CMOS camera with a ten-bit dynamic range served for recording of the images. Dark-field as well as scattered light illumination was employed to preferentially image the gaseous and particulate components of the plume, respectively. During the initial phase of its expansion (ca. 1 micros) the plume appears to consist of a continuous cloud of material of varying density. At later times after exposure, individual particles of several micrometers in size dominate the images. For both laser pulse durations material ejection was observed for times as long as 100 micros postexposure. Subtle but distinct differences in the plume dynamics are observed for the two different pulse durations. They are related to a transition between the regimes below and above acoustic confinement. The experimental findings are compared to results obtained in two previous studies by photoacoustic analysis of the desorption process and IR-laser postionization of the plume.     

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.     

Analysis of the dynamics of laser induced plume propagation from liquid matrix using fast photography

Glycerol, a liquid matrix material for matrix-assisted laser desorption ionization mass spectrometry, was irradiated by a tunable pulsed infrared laser at wavelengths of 2.80 μ.m, 2.94 μ.m, 3.10 μ.m and 3.50 μ.m, covering the OH and CH stretch vibrations. A fast photography system was introduced to analyze the dynamic process of plume propagation induced by laser ablation up to 1000 μ.s of the delay time. Propagation distance of the plume front was measured and the corresponding velocities were calculated; they varied with the wavelength and decreased with the delay time. At the tunable wavelength of the peak of the OH absorption (3.0 μ.m), theoretical calculations indicate that energy deposition from the pulsed laser is in the regime of stress confinement. The mode of energy deposition depends on the wavelength of the OH vibration and its distance from the absorption maxima. However, stages after a 10 μ.s delay at various wavelengths show a certain similarity in the distance of plume propagation, which can be well fitted by a drag model.     

Tryptamine: a Reactive Matrix for MALDI Mass Spectrometry

A possibility of using tryptamine as a reactive matrix for the analysis of non-polar carbonyl compounds by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been shown. Presence of a terminal primary amine group in the tryptamine molecule predetermines the formation of Schiff bases from aliphatic and alicyclic carbonyl compounds. No additional matrix compounds are necessary to register MALDI mass spectra, because the excess of the derivatization agent plays the role of a matrix. MALDI mass spectra demonstrate high efficiency of desorption/ionization of the derivatives. To discover reactive matrices, a set of aromatic primary amines (mainly substituted anilines) has been tested, but they have not demonstrated matrix properties.     

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.     

Irradiation effects in MALDI,ablation, ion production,and surface modifications. Part II. 2,5-dihydroxybenzoic acid monocrystals

Irradiation effects at low and high laser fluence on 2,5-dihydroxybenzoic acid large crystals were investigated. Contrary to what was observed for matrices as cinnamic acid derivatives, no chemical degradation of matrix is evidenced and continuous ablation as well as ion production resulted of extended irradiation in all the fluence range corresponding to classical matrix-assisted laser desorption /ionization. Ripples are formed on the base of the crater for a limited number of laser shots under moderate fluence. For extended irradiation, conical shape craters are formed with the axis of the crater oriented along the incident direction of the laser beam. A study of the craters showed that ablation through the ablated volume slowly varied with the laser fluence when a strong increase of ion production (matrix and analyte) was recorded. Ablation volume was found to vary non-linearly with the number of laser shots. On a same spot, the ablated volume and the ion production were measured as a function of the laser energy. With an increasing laser energy (or fluence), the ablated volume slowly increases when the ion production strongly increases. This gives evidence of a decoupling between ablation and ionization. Interaction of the plume with the incoming beam is thus probable.     

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.     

MALDI-MS imaging of features smaller than the size of the laser beam

The feasibility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging of features smaller than the laser beam size has been demonstrated. The method involves the complete ablation of the MALDI matrix coating the sample at each sample position and moving the sample target a distance less than the diameter of the laser beam before repeating the process. In the limit of complete sample ablation, acquiring signal from adjacent positions spaced by distances smaller than the sample probe enhances image resolution as the measured analyte signal only arises from the overlap of the laser beam size and the non-ablated sample surface. Image acquisition of features smaller than the laser beam size has been demonstrated with peptide standards deposited on electron microscopy calibration grids and with neuropeptides originating from single cells. The presented MS imaging technique enables approximately 25 microm imaging spatial resolution using commercial MALDI mass spectrometers having irregular laser beam sizes of several hundred micron diameters. With appropriate sampling, the size of the laser beam is not a strict barrier to the attainable MALDI-MS imaging resolution.     

Benefits of 2.94 micron infrared matrix-assisted laser desorption/ionization for analysis of labile molecules by Fourier transform mass spectrometry

A 2.94 microm Er:YAG laser was used together with a commercial Fourier transform mass spectrometer to study labile biomolecules. The combination has shown superior performance over conventional 337 nm ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) Fourier transform mass spectrometry (FTMS), especially for the analysis of peptides with post-translational modifications. With succinic acid as a matrix, the sensitivity of the single-shot analysis was increased by an order of magnitude to the low femtomole level, with significantly less fragmentation observed. Intact molecular ions of a range of O-glycosylated and sulfated peptides were detected. Urea was found to induce even less fragmentation, although at the expense of the total ion yield. Molecular ions of a noncovalent complex (vancomycin + diacetyl-L-Lys-D-Ala-D-Ala) have been observed for the first time in MALDI-FTMS. 2.94 microm infrared (IR) MALDI also produced abundant molecular ions of a range of nonbiological samples, including C60 and C70 fullerenes as well as dimetal coordination complexes.     

Continuous flow infrared matrix‐assisted laser desorption electrospray ionization mass spectrometry

Continuous flow infrared matrix‐assisted laser desorption electrospray ionization (CF IR MALDESI) mass spectrometry was demonstrated for the on‐line analysis of liquid samples. Samples in aqueous solution were flowed through a 50 µm i.d. fused‐silica capillary at a flow rate of 1–6 µL/min. As analyte aqueous solution flowed through the capillary, a liquid sample bead formed at the capillary tip. A pulsed infrared optical parametric oscillator (OPO) laser with wavelength of 2.94 µm and a 20 Hz repetition rate was focused onto the capillary tip for sample desorption and ablation. The plume of ejected sample was entrained in an electrospray to form ions by MALDESI. The resulting ions were sampled into an ion trap mass spectrometer for analysis. Using CF IR MALDESI, several chemical and biochemical reactions were monitored on‐line: the chelation of 1,10‐phenanthroline with iron(II), insulin denaturation with 1,4‐dithiothreitol, and tryptic digestion of cytochrome c. Copyright © 2010 John Wiley & Sons, Ltd.     

New type of matrix for matrix-assisted laser desorption mass spectrometry of polysaccharides and proteins

3-Aminoquinoline is introduced as matrix for matrix-assisted laser desorption mass spectrometry of polysaccharides and proteins. The first MALD mass spectrum of the polysaccharide inulin is reported. 3-Aminoquinoline seems to be more effective than the commonly used acidic matrix 2,5-dihydroxybenzoic acid, assisting in the desorption and ionisation of inulin. A higher sensitivity, sharper peaks and a lower background were observed. 3-Aminoquinoline is also quite effective when measuring the protein myoglobin.     

基质与分析物的摩尔比和激光能量对分析物离子信号的影响

A method of aerosol introduction for matrix-assisted laser desorption/ionization (MALDI) is described. The aerosol particles containing matrix and analyte enter directly into the aerosol time-of-flight mass spectrometer (ATOFMS) at atmospheric pressure. The scattered light signals from the aerosol particles are collected by a photomultiplier tube (PMT) and are passed on to an external electronic timing circuit, which determines particle size and is used to trigger a 266 nm pulsed Nd:YAG laser. The aerosol MALDI mass spectra and aerodynamic diameter of single particles can be obtained in real-time. Compared with other methods of liquid sample introduction, this method realizes detection of single particles and, more importantly, the sample consumption is lower. The effects of matrix-to-analyte ratio and laser pulse energy on analyte ion yield are examined. The optimal matrix-to-analyte ratio and laser energy are 50-110:1 and 200-400 μJ respectively.     

Matrix-assisted laser desorption/ionization mass spectrometry using a visible laser

Visible matrix-assisted laser desorption/ionization (VIS-MALDI) was performed using 2-amino-3-nitrophenol as matrix. The matrix is of near-neutral pH, and has an optical absorption band in the near-UV and visible region. A frequency-doubled Nd:YAG laser operated at 532 nm wavelength was used for matrix excitation and comparisons were made with a frequency-tripled Nd:YAG laser (355 nm). Visible and ultraviolet (UV)-MALDI produce similar mass spectra for peptides, polymers, and small proteins with comparable sensitivities. Due to the smaller optical absorption coefficient of the matrix at 532 nm wavelength, the optical penetration depth is larger, and the sample consumption per laser shot in VIS-MALDI is higher than that of UV-MALDI. Nevertheless, VIS-MALDI using 2-amino-3-nitrophenol as matrix may offer a complementary technique to the conventional UV-MALDI method in applications where deeper laser penetration is required.     

IR-MALDESI Mass Spectrometry Imaging of Biological Tissue Sections Using Ice as a Matrix

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging of biological tissue sections using a layer of deposited ice as an energy-absorbing matrix was investigated. Dynamics of plume ablation were first explored using a nanosecond exposure shadowgraphy system designed to simultaneously collect pictures of the plume with a camera and collect the Fourier transform ion cyclotron resonance FT-ICR mass spectrum corresponding to that same ablation event. Ablation of fresh tissue analyzed with and without using ice as a matrix were compared using this technique. Effect of spot-to-spot distance, number of laser shots per pixel, and tissue condition (matrix) on ion abundance were also investigated for 50 μm-thick tissue sections. Finally, the statistical method called design of experiments was used to compare source parameters and determine the optimal conditions for IR-MALDESI of tissue sections using deposited ice as a matrix. With a better understanding of the fundamentals of ablation dynamics and a systematic approach to explore the experimental space, it was possible to improve ion abundance by nearly one order of magnitude.

Analysis of hydrocarbon dendrimers by laser desorption time-of-flight and fourier transform mass spectrometry

The first mass spectrometric analysis of a new class of hydrocarbon dendrimers that result from a convergent synthetic approach is reported. Molecular weights of a series of phenylacetylene dendrimers (715 to 14776 u MW) are characterized by ultraviolet matrix-assisted laser desorption (MALDltime-of-flight (TOF) mass spectrometry, direct and silver chemical ionization infrared laser desorption Fourier transform mass spectrometry @I’MSl, and ultraviolet matrix-assisted laser desorption silver chemical ionization Fourier transform mass spectromeby. New matrices and techniques were developed to facilitate analysis of the dendrimers. Mass measurement accuracies between 10 and 25 ppm are obtained for molecular ion species of the five dendrimers analyzed. Laser desorption time-of-flight and FI’MS techniques are shown to be complementary, with FTMS providing high mass resolution (27,000–67,000 resolving power) and accuracy for lower mass dendrimers (10–14 ppm) and MALD TOF yielding the highest resolution (1100 resolving power) and accuracy (25 ppm) for the largest dendrimer. These results are consistent with proposed empirical formulas.     

Production and fragmentation of multiply charged ions in 'electron-free' matrix-assisted laser desorption/ionization

An unusually large fraction of multiply charged ions is observed in 'electron-free' matrix-assisted laser desorption/ionization (MALDI). Here we investigate how the yield of multiply charged ions depends on experimental parameters in MALDI. It is found to increase if measures are taken to limit the number of electrons in the plume, for example, by using non-metallic MALDI targets or low laser pulse energies. The ionization energy of the matrix is another important parameter that affects the yield of multiply charged ions: matrices with high ionization energies lead to greater intensities of multiply charged ions. It is furthermore proposed that some of the fragment ions observed in MALDI are due to reactions of analyte with electrons in the plume. The possibility of electron capture dissociation of multiply charged ions produced by MALDI is shown.     

Analysis of native milk oligosaccharides directly from thin-layer chromatography plates by matrix-assisted laser desorption/ionization orthogonal-time-of-flight mass spectrometry with a glycerol matrix

We have recently presented a new method for direct coupling of high-performance thin-layer chromatography (HPTLC) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), illustrated by the analysis of a complex ganglioside mixture. In the current communication, an adaptation of this procedure to mixtures of native oligosaccharides from human and from elephant milk is described. The key features in this method are (1) glycerol as a liquid matrix, to provide a homogeneous wetting of the silica gel and a simple and fast MALDI preparation protocol, (2) an infrared (IR) laser for volume material ablation and particular soft desorption/ionization conditions, and (3) an orthogonal time-of-flight mass spectrometer for a high mass accuracy, independent of any irregularity of the silica gel surface. Chromatographic "mobility profiles" were determined by scanning the laser beam across the analyte bands. The current limit of detection for the MS analysis was determined to approximately 10 pmol of individual oligosaccharides spotted for chromatography. A liquid composite matrix, containing glycerol and the ultraviolet (UV-)MALDI matrix alpha-cyano-4-hydroxycinnamic acid, allows a direct HPTLC-MALDI-MS analysis with a 337 nm-UV laser as well. Compared to the IR-MALDI mode, the analytical sensitivity in UV-MALDI was found to be lower by one order of magnitude, whereas unspecific analyte ion fragmentation as well as adduct formation was found to be more extensive.     

Laserspray ionization (LSI) ion mobility spectrometry (IMS) mass spectrometry

A simple device is described for desolvation of highly charged matrix/analyte clusters produced by laser ablation leading to multiply charged ions that are analyzed by ion mobility spectrometry-mass spectrometry. Thus, for example, highly charged ions of ubiquitin and lysozyme are cleanly separated in the gas phase according to size and mass (shape and molecular weight) as well as charge using Tri-Wave ion mobility technology coupled to mass spectrometry. This contribution confirms the mechanistic argument that desolvation is necessary to produce multiply charged matrix-assisted laser desorption/ionization (MALDI) ions and points to how these ions can be routinely formed on any atmospheric pressure mass spectrometer.     

Multiphoton ionization of molecules: A comparison between femtosecond and nanosecond laser pulse ionization efficiency

Multiphoton ionization mass spectra of nonvolatile molecules laser desorbed into a supersonic beam are recorded. It is shown by indirect measurements that the laser desorption of neutrals is not mass limited, but lead to the formation of neutrals with intesities large enough for intense signals. To investigate the efficiency of the multiphoton ionization process with varying laser pulse durations, simultaneous laser pulses of 500 fs and 5 ns or 100 fs and 5 ns have been applied to the neutral beam. The energies of both femtosecond and nanosecond laser pulses are held in a comparable magnitude, and thus produce, in the resulting ion intensity, very large differences up to 4 orders of magnitude. For larger evaporated molecules (> 500 u) the ionization efficiency from nanosecond laser pulses drops significantly in comparison to femtosecond laser pulse excitation. A variety of possible reasons for the different ionization and dissociation behavior in femtosecond and nanosecond laser pulse excitations are discussed in this paper. It is rationalized that even with very short laser pulses and large molecules the “ladder switching model” for ionization and fragmentation is valid.