Ion yields in UV-MALDI mass spectrometry as a function of excitation laser wavelength and optical and physico-chemical properties of classical and halogen-substituted MALDI matrixes.

The laser wavelength constitutes a key parameter in ultraviolet-matrix-assisted laser desorption ionization-mass spectrometry (UV-MALDI-MS). Optimal analytical results are only achieved at laser wavelengths that correspond to a high optical absorption of the matrix. In the presented work, the wavelength dependence and the contribution of matrix proton affinity to the MALDI process were investigated. A tunable dye laser was used to examine the wavelength range between 280 and 355 nm. The peptide and matrix ion signals recorded as a function of these irradiation parameters are displayed in the form of heat maps, a data representation that furnishes multidimensional data interpretation. Matrixes with a range of proton affinities from 809 to 866 kJ/mol were investigated. Among those selected are the standard matrixes 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA) as well as five halogen-substituted cinnamic acid derivatives, including the recently introduced 4-chloro-α-cyanocinnamic acid (ClCCA) and α-cyano-2,4-difluorocinnamic acid (DiFCCA) matrixes. With the exception of DHB, the highest analyte ion signals were obtained toward the red side of the peak optical absorption in the solid state. A stronger decline of the molecular analyte ion signals generated from the matrixes was consistently observed at the low wavelength side of the peak absorption. This effect is mainly the result of increased fragmentation of both analyte and matrix ions. Optimal use of multiply halogenated matrixes requires adjustment of the excitation wavelength to values below that of the standard MALDI lasers emitting at 355 (Nd:YAG) or 337 nm (N(2) laser). The combined data provide new insights into the UV-MALDI desorption/ionization processes and indicate ways to improve the analytical sensitivity.

Rapid metabolic profiling of Nicotiana tabacum defence responses against Phytophthora nicotianae using direct infrared laser desorption ionization mass spectrometry and principal component analysis.

BACKGROUND: Successful defence of tobacco plants against attack from the oomycete Phytophthora nicotianae includes a type of local programmed cell death called the hypersensitive response. Complex and not completely understood signaling processes are required to mediate the development of this defence in the infected tissue. Here, we demonstrate that different families of metabolites can be monitored in small pieces of infected, mechanically-stressed, and healthy tobacco leaves using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry. The defence response was monitored for 1 - 9 hours post infection. RESULTS: Infrared laser desorption ionization orthogonal time-of-flight mass spectrometry allows rapid and simultaneous detection in both negative and positive ion mode of a wide range of naturally occurring primary and secondary metabolites. An unsupervised principal component analysis was employed to identify correlations between changes in metabolite expression (obtained at different times and sample treatment conditions) and the overall defence response.A one-dimensional projection of the principal components 1 and 2 obtained from positive ion mode spectra was used to generate a Biological Response Index (BRI). The BRI obtained for each sample treatment was compared with the number of dead cells found in the respective tissue. The high correlation between these two values suggested that the BRI provides a rapid assessment of the plant response against the pathogen infection. Evaluation of the loading plots of the principal components (1 and 2) reveals a correlation among three metabolic cascades and the defence response generated in infected leaves. Analysis of selected phytohormones by liquid chromatography electrospray ionization mass spectrometry verified our findings. CONCLUSION: The described methodology allows for rapid assessment of infection-specific changes in the plant metabolism, in particular of phenolics, alkaloids, oxylipins, and carbohydrates. Moreover, potential novel biomarkers can be detected and used to predict the quality of plant infections.

Matching IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay and its clinical application for tracing tumor-associated glycosphingolipids in hepatocellular and pancreatic cancer.

Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including the development of cancer. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of modern mass spectrometry. Here we introduce direct coupling of IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay for the structural characterization of GSLs. We matched three complementary methods including (i) TLC separation of GSLs, (ii) their detection with oligosaccharide-specific proteins, and (iii) in situ MS analysis of protein-detected GSLs. The high specificity and sensitivity is demonstrated by use of antibodies, bacterial toxins, and a plant lectin. The procedure works on a nanogram scale, and detection limits of less than 1 ng at its best of immunostained GSLs were obtained. Furthermore, only crude lipid extracts of biological sources are required for TLC-IR-MALDI-MS, omitting any laborious GSL downstream purification procedures. This strategy was successfully applied to the identification of cancer-associated GSLs in human hepatocellular and pancreatic tumors. Thus, the in situ TLC-IR-MALDI-MS of immunolabeled GSLs opens new doors by delivering specific structural information of trace quantities of GSLs with only a limited investment in sample preparation.

Localization of noncovalent complexes in MALDI-preparations by CLSM.

The unambiguous detection of noncovalent complexes (NCCs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is still a far cry from being routine. For protein NCCs such as their quaternary structure it has been reported that signals of the intact complex are only obtained for the first or at most the first few laser exposures of a given sample area. This observation was called the first-shot phenomenon. In the present study, this first-shot phenomenon has been investigated for the hexameric protein complex allophycocyanine (APC) by two independent methods, MALDI-MS with a (nearly) pH-neutral matrix 6-aza-2-thiothymine (6-ATT) and by imaging the fluorescence of the complex in APC-6-ATT preparations by confocal laser scan microscopy (CLSM). The intact APC heterohexamer loses its visible fluorescence upon dissociation into its subunits. Both methods consistently show that intact APC complexes are precipitated at the matrix crystal surface, but dissociate upon incorporation into the matrix crystals.

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

A novel method is presented for direct coupling of high-performance thin-layer chromatography (HPTLC) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of biomolecules. A first key feature is the use of a liquid matrix (glycerol), which provides a homogeneous wetting of the silica gel and a simple and fast MALDI preparation protocol. A second is the use of an Er:YAG infrared laser, which ablates layers of approximately 10-microm thickness of analyte-loaded silica gel and provides a soft desorption/ionization of even very labile analyte molecules. The orthogonal time-of-flight mass spectrometer employed in this study, finally provides a high accuracy of the mass determination, which is independent of any irregularity of the silica gel surface. The analytical potential of the method is demonstrated by the compositional mapping of a native GM3 (II(3)-alpha-Neu5Ac-LacCer) ganglioside mixture from cultured Chinese hamster ovary cells. The analysis is characterized by a high relative sensitivity, allowing the simultaneous detection of various major and minor GM3 species directly from individual HPTLC analyte bands. The lateral resolution of the direct HPTLC-MALDI-MS analysis is defined by the laser focus diameter of currently approximately 200 microm. This allows one to determine mobility profiles of individual species with a higher resolution than by reading off the chromatogram by optical absorption. The fluorescent dye primuline was, furthermore, successfully tested as a nondestructive, MALDI-compatible staining agent.

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.

Reverse Sanger sequencing of RNA by MALDI-TOF mass spectrometry after solid phase purification.

Several DNA/RNA sequencing strategies have been developed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In the reverse Sanger sequencing approach alpha-thiophosphate-containing NTPs are employed. Sequencing ladders are produced by the subsequent exonuclease cleavage, which is inhibited by the alpha-S-NTP at the 3' terminus. Here the reverse Sanger sequencing of RNA is described. The stability of RNA during the UV-MALDI process is higher relative to DNA, and RNA can be easily synthesized by transcription using bacteriophage RNA polymerase. alpha-S-rNTP was added to the reaction in a ratio of 1:3 to the native rNTPs and was incorporated statistically by the RNA polymerase. Four separate sequence ladders were produced, to avoid the problem of the only 1u mass difference between uridine and cytidine. However, it was shown that RNA transcription does not produce homogeneous transcripts. Therefore isolation of the full-length transcript is required to attain a non-ambiguous interpretation of cleavage spectra. This is achieved by the exclusive immobilization of the full-length transcript on a solid phase. The full-length transcripts were hybridized to magnetic beads, coated with short universal sequences, complementary to the in vitro RNA. After purification and isolation the RNA full-length transcript is cleaved by snake venom phosphodiesterase (SVP) and the obtained sequence ladder is analyzed by MALDI-MS.

Infrared laser post-ionization of large biomolecules from an IR-MALD(I) plume.

A two-infrared laser desorption/ionization method is described. A first laser, which was either an Er:YAG laser or an optical parametric oscillator (OPO), served for ablation/vaporization of small volumes of analyte/matrix sample at fluences below the ion detection threshold for direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A second IR-laser, whose beam intersected the expanding ablation plume at a variable distance and time delay, was used to generate biomolecular ions out of the matrix-assisted laser desorption (MALD) plume. Either one of the two above lasers or an Er:YSGG laser was used for post-ionization. Glycerol was used as IR-MALDI matrix, and mass spectra of peptides, proteins, as well as nucleic acids, some of which in excess of 10(5) u in molecular weight, were recorded with a time-of-flight mass spectrometer. A mass spectrum of cytochrome c from a water ice matrix is also presented. The MALD plume expansion was investigated by varying the position of the post-ionization laser beam above the glycerol sample surface and its delay time relative to the desorption laser. Comparison between the OPO (pulse duration, tau(L) = 6 ns) and the Er:YAG laser (tau(L) approximately 120 ns) as primary excitation laser demonstrates a significant effect of the laser pulse duration on the MALD process.

RNase T1 mediated base-specific cleavage and MALDI-TOF MS for high-throughput comparative sequence analysis.

Here we devise a new method for high-throughput comparative sequence analysis. The developed protocol comprises a homogeneous in vitro transcription/RNase cleavage system with the accuracy and data acquisition speed of matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS). In summary, the target region is PCR amplified using primers tagged with promoter sequences of T7 or SP6 RNA polymerase. Using RNase T1, the in vitro transcripts are base-specifically cleaved at every G-position. This reaction results in a characteristic pattern of fragment masses that is indicative of the original target sequence. To enable high-throughput analysis, samples are processed with automated liquid handling devices and nanoliter amounts are dispensed onto SpectroCHIP arrays for reliable and homogeneous MALDI preparation. This system enables rapid automated comparative sequence analysis for PCR products up to 1 kb in length. We demonstrate the feasibility of the devised method for analysis of single nucleotide polymorphisms (SNPs) and pathogen identification.

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.

Singlet-singlet annihilation in ultraviolet matrix-assisted laser desorption/ionization studied by fluorescence spectroscopy.

Laser-induced fluorescence spectroscopy was carried out on microcrystalline samples of three typical matrices under conditions of matrix-assisted laser desorption/ionization (MALDI). The emitted fluorescence intensity was determined as a function of incident laser fluence and a sublinear increase of the fluorescence intensity with laser fluence was found. A very good fit was obtained when the experimental fluorescence vs. fluence data were compared with a numerical model assuming that under typical MALDI fluence conditions a large fraction of molecules in the excited singlet state undergoes singlet-singlet annihilation. Throughout the fluence range relevant for MALDI, however, the experimental data could not be fit well to a model assuming resonant two-photon absorption as the process depopulating the singlet state. In a separate set of experiments, the singlet lifetimes of several typical crystalline MALDI matrices were determined and found to be considerably shorter than previously reported. While both singlet-singlet annihilation and resonant two-photon absorption have been discussed in the literature as candidates for pathways to primary matrix ion generation in MALDI, the data presented here suggest that singlet-singlet annihilation is the dominant mechanism for depopulating the singlet state in a matrix crystal excited at typical MALDI fluences.

Measurements of mean initial velocities of analyte and matrix ions in infrared matrix-assisted laser desorption ionization mass spectrometry.

The mean initial velocities of analyte ions ranging in molecular weight from 1000 Da to 150 kDa and desorbed with a pulsed Er:YAG laser from various solid-state and liquid IR MALDI matrices were measured along with those of the matrix ions. Experiments with UV MALDI were performed for comparison in addition for a 2,5-dihydroxybenzoic acid preparation. Two different measurement principles were employed, (1) a delayed extraction method, relying on the initial velocity-dependent increase of flight times with delay time between laser and HV ion extraction pulse, and (2) a field-free drift method in which the first region of a two-stage ion source was varied in length and the flight times compared. The two methods yielded somewhat different values for the mean initial ion velocities. Based on a detailed discussion of the measurement principles it is suggested that the actual initial velocities of IR MALDI ions lie between the limits set by the two methods. The influences of the analyte-to-matrix ratio, laser fluence, and laser wavelength on the initial ion velocities were also investigated. Significant differences between the desorption mechanisms for liquid and solid-state matrices were observed.