Alexander Pirkl

Small colony variants of Staphylococcus aureus reveal distinct protein profiles

Small‐colony variants (SCVs) of Staphylococcus aureus represent a slow‐growing subpopulation causing chronic and relapsing infections due to their physiological adaptation on an intracellular lifestyle. In this first proteomic study on physiological changes associated with a natural, clinically derived SCV, its proteomic profile was investigated in comparison to corresponding isogenic strains displaying normal (clinical wild‐type strain, complemented hemB mutant and spontaneous revertant of the clinical SCV) and SCV phenotypes (hemB mutant and gentamicin‐induced SCV). Applying an ultra‐high resolution chromatography and high mass accuracy MSE‐based label‐free relative and absolute protein quantification approach, the whole cytoplasmic proteome of this strain sextet was investigated in a growth phase‐controlled manner covering early‐exponential, late‐exponential and stationary phases. Of 1019 cytoplasmic proteins identified, 154 were found to be differently regulated between strains. All SCV phenotypes showed down‐regulation of the tricarboxylic acid (TCA) cycle‐related proteins and of a protein cluster involved in purine/pyrimidine and folate metabolism. In contrast to hemB mutant and gentamicin‐induced SCVs, the clinically derived SCVs showed no prominent up‐regulation of glycolytic proteins. The spontaneous switch into the normal phenotype resulted in up‐regulation of TCA cycle‐related parts, while oxidative stress‐related proteins were down‐regulated. However, the natural revertant from the clinical SCV retained also dominant protein features of the clinical SCV phenotype. In conclusion, physiological changes between normal and SCV S. aureus phenotypes are more complex than reflected by defined electron transport chain‐interrupting mutants and their complemented counterparts.

MALDI mass spectrometry imaging of bioactive lipids in mouse brain with a Synapt G2-S mass spectrometer operated at elevated pressure: improving the analytical sensitivity and the lateral resolution to ten micrometers.

Mass spectrometers from the Synapt-G1/G2 family (Waters) are widely employed for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). A lateral resolution of about 50 μm is typically achieved with these instruments, that is, however, below the often desired cellular resolution. Here, we show the first MALDI-MSI examples demonstrating a lateral resolution of about ten micrometers obtained with a Synapt G2-S HDMS mass spectrometer without oversampling. This improvement became possible by laser beam shaping using a 4:1 beam expander and a circular aperture for spatial mode filtering and by replacement of the default focusing lens. We used dithranol as an effective matrix for imaging of acidic lipids such as sulfatides, gangliosides, and phosphatidylinositols in the negative ion mode. At the same time, the matrix enables MS imaging of more basic lipids in the positive ion mode. Uniform matrix coatings with crystals having average dimensions between 0.5 and 3 μm were obtained upon spraying a chloroform/methanol matrix solution. Increasing the cooling gas pressure in the MALDI ion source after adding an additional gas line was furthermore found to increase the ion abundances of labile lipids such as gangliosides. The combined characteristics are demonstrated with the MALDI-MSI analysis of fine structures in coronal mouse brain slices.

Infrared Matrix-Assisted Laser Desorption/Ionization Orthogonal-Time-of-Flight Mass Spectrometry Employing a Cooling Stage and Water Ice As a Matrix

Although water ice has been utilized in the past as a matrix for infrared matrix-assisted laser desorption/ionization mass spectrometry (IR-MALDI-MS), it has not found a wider use due to limitations in the analytical performance and technical demands on the employment of the necessary cooling stage. Here, we developed a temperature-controlled sample stage for use with an orthogonal time-of-flight mass spectrometer (MALDI-o-TOF-MS). The stage utilizes a combination of liquid nitrogen cooling and counterheating with a Peltier element. It allows adjustment of the sample temperature between ~-120 °C and room temperature. To identify optimal irradiation conditions for IR-MALDI with the water ice matrix, we first investigated the influence of excitation wavelength, varied between 2.7 and 3.1 μm, and laser fluence on the signal intensities of molecular substance P ions. These data suggest the involvement of transient melting of the ice during the laser pulse and primary energy deposition into liquid water. As a consequence, the best analytical performance is obtained at a wavelength corresponding to the absorption maximum of liquid water of about 2.94 μm. The current data significantly surpass the previously reported analytical features. The particular softness of the method is, for example, exemplified by the analysis of noncovalently bound holo-myoglobin and of ribonuclease B. This is also the first report demonstrating the analysis of an IgG monoclonal antibody (MW ~ 150 kDa) from a water ice matrix. Untypical for MALDI-MS, high charge states of multiply protonated species were moreover observed for some of the investigated peptides and even for lacto-N-fucopentaose II oligosaccharides. Using water ice as matrix is of particular interest for MALDI MS profiling and imaging applications since matrix-free spectra are produced. The MS and tandem MS analysis of metabolites directly from frozen food samples is demonstrated with the example of a strawberry fruit.

Liquid AP‐UV‐MALDI Enables Stable Ion Yields of Multiply Charged Peptide and Protein Ions for Sensitive Analysis by Mass Spectrometry

In biological mass spectrometry (MS), two ionization techniques are predominantly employed for the analysis of larger biomolecules, such as polypeptides. These are nano-electrospray ionization [1, 2] (nanoESI) and matrix-assisted laser desorption/ionization [3, 4] (MALDI). Both techniques are considered to be “soft”, allowing the desorption and ionization of intact molecular analyte species and thus their successful mass-spectrometric analysis. One of the main differences between these two ionization techniques lies in their ability to produce multiply charged ions. MALDI typically generates singly charged peptide ions whereas nanoESI easily provides multiply charged ions, even for peptides as low as 1000 Da in mass. The production of highly charged ions is desirable as this allows the use of mass analyzers, such as ion traps (including orbitraps) and hybrid quadrupole instruments, which typically offer only a limited m/z range (< 2000–4000). It also enables more informative fragmentation spectra using techniques such as collisioninduced dissociation (CID) and electron capture/transfer dissociation (ECD/ETD) in combination with tandem MS (MS/MS). [5, 6] Thus, there is a clear advantage of using ESI in research areas where peptide sequencing, or in general, the structural elucidation of biomolecules by MS/MS is required. Nonetheless, MALDI with its higher tolerance to contaminants and additives, ease-of-operation, potential for highspeed and automated sample preparation and analysis as well as its MS imaging capabilities makes it an ionization technique that can cover bioanalytical areas for which ESI is less suitable. [7, 8] If these strengths could be combined with the analytical power of multiply charged ions, new instrumental configurations and large-scale proteomic analyses based on MALDI MS(/MS) would become feasible.

Analysis of Drosophila Lipids by Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometric Imaging

Drosophila melanogaster is a major model organism for numerous lipid-related diseases. While comprehensive lipidomic profiles have been generated for D. melanogaster, little information is available on the localization of individual lipid classes and species. Here, we show the use of matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) to profile lipids in D. melanogaster tissue sections. The preparation of intact cryosections from whole insects presents a challenge due to the brittle hydrophobic cuticle surrounding the body and heterogeneous tissue types beneath the cuticle. However, the introduction of a novel sucrose infiltration step and gelatin as an embedding media greatly improved the quality of tissue sections. We generated MS image profiles of six major lipid classes: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and triacylglycerides. In addition, signals corresponding to two male-specific sex pheromones were detected in the ejaculatory bulb, a specialized site of pheromone production. MSI performed with 35 μm lateral resolution provided high sensitivity detection of at least 92 different lipid species, based on exact mass. In contrast, MSI with 10 μm lateral resolution enabled the detection of 36 lipid species but allowed lipid profiling of individual organs. The ability to localize lipid classes in intact sections from whole Drosophila provides a powerful tool for characterizing the effects of diet, age, stress, and environment on lipid production and distribution.

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

BackgroundSuccessful 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.ResultsInfrared 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.ConclusionThe 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.

Analysis of Free Fatty Acids by Ultraviolet Laser Desorption Ionization Mass Spectrometry Using Insect Wings as Hydrophobic Sample Substrates

Physiologically relevant free fatty acids (FFAs) were analyzed by UV-laser desorption/ionization orthogonal extracting time-of-flight mass spectrometry (LDI-oTOF-MS). Dissected wings from Drosophila melanogaster fruit flies were used as the hydrophobic, laser energy strongly absorbing sample substrates. Using untreated substrates produces predominantly molecular [M + K](+) ions of the FFAs, whereas other alkali metal adducts can be generated by treating the wings with the corresponding alkali hydroxide before spotting of analyte. Limits of detection for the positive ion mode were determined for mixtures of isolated FFAs to values in the low 10 pmol range. Specific values depend on chain length and degree of unsaturation. R(2) coefficients for the analysis of saturated FFAs were found to be generally close to 0.98 over about 3 orders of magnitude if an internal standard (15:0 FFA) was added. Semiquantitative analyses of mixtures containing unsaturated FFAs are also possible but require more effort on the calibration strategy. Notably, both saturated and (poly-)unsaturated FFAs are detected sensitively in the presence of relatively high concentrations of other physiologically abundant lipids (phospholipids and triacyclglycerols). This simplifies screening of the FFA composition in crude tissue extracts. This feature is demonstrated by the analysis of a crude liver extract and that of fingermarks.

Progress in detection and structural characterization of glycosphingolipids in crude lipid extracts by enzymatic phospholipid disintegration combined with thin-layer chromatography immunodetection and IR-MALDI mass spectrometry.

In order to proceed in detection and structural analysis of glycosphingolipids (GSLs) in crude lipid extracts, which still remains a challenge in glycosphingolipidomics, we developed a strategy to structurally characterize neutral GSLs in total lipid extracts prepared from in vitro propagated human monocytic THP-1 cells, which were used as a model cell line. The procedure divides into (1) extraction of total lipids from cellular material, (2) enzymatical disintegration of phospholipids by treatment of the crude lipid extract with phospholipase C, (3) subsequent multiple thin-layer chromatography (TLC) overlay detection of individual GSLs with a mixture of various anti-GSL antibodies, and (4) structural analysis of immunostained GSLs directly on the TLC plate using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) in combination with collision-induced dissociation (CID). Whereas GSLs were mostly undetectable in untreated crude lipid extracts, pretreatment with phospholipase C resulted in clear-cut mass spectra. MS(1) and MS(2) analysis gave similar results when compared to those obtained with a highly purified neutral GSL preparation of THP-1 cells, which served as a control. We could demonstrate in this study the feasibility of simultaneous multiple immunodetection of individual neutral GSLs in one and the same TLC run and their structural characterization in crude lipid extracts after phospholipase C treatment, thereby avoiding laborious and long-lasting sample purification. This powerful combinatorial technique allows for efficient structural characterization of GSLs in small tissue samples and takes a step forward in the emerging field of glycosphingolipidomics.

Field-based ion generation from microscale emitters on natural and artificial objects for atmospheric pressure mass spectrometry

AbstractField-based ion generation is described for ambient mass spectrometry. The technique allows the analysis of endogenously expressed chemicals and exogenously applied compounds directly from the cuticle of live insects in real time. Cuticular hairs serve as electric field-enhancing structures and play a key role in ion generation. Artificial emitters such as graphite whiskers or sharp metal tips replicate this effect. FigureFruit fly in front of the ion trap entrance capillary (see file Online_abstract_figure.jpg).

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.