25 research outputs found
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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 âsoftâ, 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 (< 2000â4000). 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
Rapid metabolic profiling of Nicotiana tabacum defence responses against Phytophthora nicotianae using direct infrared laser desorption ionization mass spectrometry and principal component analysis
<p>Abstract</p> <p>Background</p> <p>Successful defence of tobacco plants against attack from the oomycete <it>Phytophthora nicotianae </it>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.</p> <p>Results</p> <p>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.</p> <p>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.</p> <p>Conclusion</p> <p>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.</p
Elucidating the molecular landscape of the stratum corneum
Characterization of the molecular structure of skin, especially the barrier layer, the stratum corneum, is a key research priority for generating understanding to improve diagnostics, aid pharmaceutical delivery, and prevent environmental damage. Our study uses the recently developed 3D OrbiSIMS technique to conduct in situ analysis of ex vivo human skin tissue and reveals the molecular chemistry of skin in unprecedented detail, as a result of the step change in high mass resolving power compared with previous studies. This characterization exposes the nonhomogeneity of the stratum corneum, both laterally and as a function of depth. Chemical variations relating to fundamental biological processes, such as the epidermal cholesterol sulfate cycle, are visualized using in situ analysis. We are able to resolve the debate around the chemical gradients present within the epidermis, for example, whether palmitic acid is of sebaceous origin or a true component of the stratum corneum. Through in situ depth analysis of cryogenically preserved samples, we are able to propose that it is actually a component of both surface sebum and the intrinsic lipid matrix. This approach also suggests similarity between the epidermis compounds found in human and porcine skin as a function of depth. Since porcine skin is a widely used model for permeation testing this result has clinical relevance. In addition to using this technique for endogenous species, we have used it to demonstrate the permeation of a commercially important antiaging peptide into the human stratum corneum. Due to its chemical similarity to native skin components and exceptionally low effective concentration, this information was previously unattainable
The 3D OrbiSIMSâlabel-free metabolic imaging with subcellular lateral resolution and high mass-resolving power
We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 ÎŒm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmittersâgamma-aminobutyric acid, dopamine and serotoninâwith high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone
Broadband spatio-temporal channel sounder for the 2.4 GHz ISM band
Issued as final repor
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 âŒâ120 °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 ÎŒm, 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
ÎŒm. 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-<i>N</i>-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
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 <i>Drosophila</i> Lipids by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Imaging
<i>Drosophila melanogaster</i> is a major model organism
for numerous lipid-related diseases. While comprehensive lipidomic
profiles have been generated for <i>D. melanogaster</i>,
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 <i>D. melanogaster</i> 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 ÎŒm lateral resolution provided high sensitivity detection
of at least 92 different lipid species, based on exact mass. In contrast,
MSI with 10 ÎŒm 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 <i>Drosophila</i> provides a powerful tool for characterizing the
effects of diet, age, stress, and environment on lipid production
and distribution
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]<sup>+</sup> 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. <i>R</i><sup>2</sup> 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-)Âunsaturated 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