Stefan Berkenkamp

Performance of Infrared Matrix-assisted Laser Desorption/Ionization Mass Spectrometry with Lasers Emitting in the 3 μm Wavelength Range

The performance characteristics of two lasers emitting in the mid infrared, an Er-YAG (2.94 μm wavelength, 80–90 ns pulse width), and an Er-YSGG infrared laser (2.79 μm wavelength, 80 ns pulse width), in matrix-assisted laser desorption/ionization mass spectrometry (IR-MALDI-MS) of biological macromolecules, is reported. Glycerol and succinic acid were used as matrices. In IR-MALDI sample consumption per laser shot typically exceeds that of UV-MALDI by about two orders of magnitude. Using glycerol as matrix, the reproducibility of the ion signals from shot to shot is comparable to the best values achieved in UV-MALDI. The same holds true for the precision and accuracy of the mass determination. For succinic acid all these values are significantly worse, due to the strong sample heterogeneity as typically found in dried droplet preparations. Metastable fragmentation is comparable for UV- and IR-MALDI in the low mass range, but is significantly less for the IR in the mass range above ca. 20 kDa, leading to an improved mass resolution and an extended high mass limit for IR-MALDI.

Mechanisms of energy deposition in infrared matrix-assisted laser desorption/ionization mass spectrometry

Abstract The mechanisms of energy deposition in matrix-assisted laser desorption/ionization mass spectrometry with infrared lasers (IR-MALDI-MS) have been evaluated in a series of experiments. In a first part, the threshold fluences for the generation of IR-MALDI spectra were determined between 2.7 and 4.0 μm wavelength with an optical parametric oscillator as a tunable laser source for nine solid state and two liquid matrices of different chemical structure and compared to the infrared absorption spectra of the compounds. Preliminary spectra of IR-MALDI in the wavelength range of 1.45–1.75 μm are also presented using the overtone vibrations of a glycerol matrix. Matrices were chosen with regard to their IR-MALDI performance and to allow conclusions on the IR-absorption mechanisms. Whereas the wavelength dependence of the threshold fluence for non–hydrogen-bound C–H vibrations essentially follows the absorption spectrum of this functional group, strong discrepancies between the spectral dependence of threshold fluences and IR-absorption spectra were found for the vibrations of O–H and N–H groups around 3-μm wavelength that form strong intermolecular and intramolecular hydrogen bonds. In a second part, experiments are described that interrogate the two most probable mechanisms for the observed deviation of the threshold fluence behavior from the wavelength course of the IR-absorption spectra, that is, absorption by either free or weakened O–H and N–H stretching modes or by residual free water. All investigations were performed with glycerol and succinic acid as examples for common liquid and solid state matrices for IR-MALDI. For glycerol, a fluence-dependent, dynamic change in absorption during the laser pulse was revealed by laser transmission measurements on thin glycerol layers. This effect, characterized by a significant blue shift of the O–H stretch absorption, can be attributed to a weakening of intermolecular hydrogen bonds caused by the transient laser heating of the sample. Taking this effect into account, a good correspondence of the wavelength dependence of the threshold fluence with the infrared absorption under IR-MALDI conditions is derived for glycerol. For succinic acid, in contrast, the identification of the predominant absorption mechanism in the 3-μm wavelength range appears more difficult. A fluence-dependent absorption was not detected in laser transmission experiments with succinic acid single crystals. A change in analyte-to-matrix ratio, with the intention of inducing free absorbers near crystal defects, also did not influence the wavelength dependence of the threshold fluences. However, an influence of the surface-to-volume ratio on the wavelength-dependent threshold fluences was found by a comparison of three different preparation techniques for succinic acid, indicating a putative influence of weakly hydrogen-bound surface absorbers. In combination with the detailed analysis of the wavelength dependence of the threshold fluence given from the first part, a determination of the IR-MALDI process for succinic acid based on the absorption by weakly hydrogen-bound hydroxyl groups is suggested. No evidence for a significant contribution of residual free water absorption to the low-threshold fluences around 3-μm wavelength was found by monitoring a possible change in threshold fluence at the phase transition from water to ice and by reducing the analyte hydration and varying the water content in glycerol preparations. Also, in preparations with frozen hydrated proteins without organic matrices, the wavelength dependence of the threshold fluence did not reflect the spectral absorption of ice, supporting the assumption of a rather minor role of the absorption by residual water in IR-MALDI.

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.

Shiga Toxin Receptor Gb3Cer/CD77: Tumor-Association and Promising Therapeutic Target in Pancreas and Colon Cancer

Background Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas. Methodology/Principal Findings Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors. Conclusions/Significance Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.

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.

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 μm 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 μm, with an Er:YAG laser (λ = 2.94 μm) with two pulse durations of 100 and 185 ns, and an Er:YSGG laser (λ = 2.79 μm) 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.

Influence of the laser fluence in infrared matrix-assisted laser desorption/ionization with a 2.94 µm 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.

Direct coupling of high-performance thin-layer chromatography with UV spectroscopy and IR-MALDI orthogonal TOF MS for the analysis of cyanobacterial toxins.

Cyanobacteria are pathogenic prokaryotes and known for producing a high variety of cyclic hepatotoxic peptides in fresh and brackish water. Prominent members of these toxins are microcystin LR (MC LR) and nodularin (Nod), which are under suspicion to cause cancer. Various analytical methods have been reported for the detection of these cyclopeptides, and these are mainly based on liquid chromatography combined with mass spectrometric techniques. Here, we introduce a new approach based on the direct coupling of high-performance thin-layer chromatography (HPTLC) with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) using the liquid matrix glycerol. The analysis of the cyclopeptides involves the application of three complementary methods: (i) HPTLC separation of MC LR and Nod, (ii) their detection and quantification by UV spectroscopy at lambda = 232 nm, and (iii) direct identification of separated analytes on the HPTLC plate by IR-MALDI-o-TOF MS. Calibration curves exhibited a linear relationship of amount of analyte applied for HPTLC and UV absorption (R(2) > 0.99). The limits of detection were 5 ng for UV spectroscopy and 3 ng for mass spectrometric analysis of individual peptides. This novel protocol greatly improves the sensitive determination of toxins from pathogenic cyanobacteria in complex water samples. It was successfully applied to the detection and quantification of MC LR and Nod in a spiked, processed environmental water sample.

Comparative mass spectrometric analyses of Photofrin oligomers by fast atom bombardment mass spectrometry, UV and IR matrix-assisted laser desorption/ionization mass spectrometry, electrospray ionization mass spectrometry and laser desorption/jet-cooling photoionization mass spectrometry.

Photofrin (porfimer sodium) is a porphyrin derivative used in the treatment of a variety of cancers by photodynamic therapy. This oligomer complex and a variety of porphyrin monomers, dimers and trimers were analyzed with five different mass spectral ionization techniques: fast atom bombardment, UV and IR matrix-assisted laser desorption/ionization, electrospray ionization, and laser desorption/jet-cooling photoionization. All five approaches resulted in very similar oligomer distributions with an average oligomer length of 2.7 +/- 0.1 porphyrin units. In addition to the Photofrin analysis, this study provides a side-by-side comparison of the spectra for the five different mass spectrometric techniques.

Effect of Gas Pressure and Gas Type on the Fragmentation of Peptide and Oligosaccharide Ions Generated in an Elevated Pressure UV/ IR-MALDI Ion Source Coupled to an Orthogonal Time-of-Flight Mass Spectrometer

Matrix-assisted laser desorption ionization (MALDI) allows for the mass spectrometric (MS) analysis of thermally labile, non-volatile biomolecules. However, some residual analyte fragmentation typically accompanies the phase transition from the condensed to the gas phase and following plume expansion, even under optimized conditions. In-source decay (ISD) and post-source decay (PSD) MALDI MS are two techniques that make use of these phenomena and that can provide useful structural information by producing characteristic fragment ions of the analyte compounds. In orthogonal extracting time-of-flight mass spectrometry (o-TOF-MS), the pressure of the cooling gas in the ion source has a strong influence on the extent of analyte ion fragmentation. We investigated the effect of this parameter on peptide and oligosaccharide fragmentation by examining a range of pressures (from 0.05-1.8 mbar) in combination with seven different buffer gases (He, Ne, Ar, N(2), CO(2), CH(3), isobutane). Ions were generated by ultraviolet (UV) and/or by infrared (IR) MALDI. The influence of the ion extraction voltage on the analyte fragmentation also was investigated for a selected set of gas parameters. We observed that individual fragment ions exhibit characteristic fragment yield-pressure dependencies that can be classified into three groups. Type I ions resemble species that are also found in MALDI PSD MS analysis, while type II ions resemble typical ISD fragments. The yield-pressure relationship of type III ions suggests that these are the result of a combination of both processes. Comparing the yields of fragmentation for the different buffer gases reveals a correlation between their internal degrees of freedom and their collisional cooling efficiency. Changing the buffer gas pressure and/or extraction field provides an easy means to influence analyte ion fragmentation and to switch from the primary production of one type of fragment species to another. The method can therefore facilitate the structural characterization of MALDI-generated ions.