Previous Mass Spectrometry Studies Research Articles

How Clavulanic Acid Inhibits Serine β-Lactamases.

Clavulanic acid is a medicinally important inhibitor of serine β-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LC‑MS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM-116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest.

Thermal desorption effects on fragment ion production from multi-photon ionized uridine and selected analogues.

Experiments on neutral gas-phase nucleosides are often complicated by thermal lability. Previous mass spectrometry studies of nucleosides have identified enhanced relative production of nucleobase ions (e.g. uracil+ from uridine) as a function of desorption temperature to be the critical indicator of thermal decomposition. On this basis, the present multi-photon ionization (MPI) experiments demonstrate that laser-based thermal desorption is effective for producing uridine, 5-methyluridine, and 2′-deoxyuridine targets without thermal decomposition. Our experiments also revealed one notable thermal dependence: the relative production of the sugar ion C5H9O4+ from intact uridine increased substantially with the desorption laser power and this only occurred at MPI wavelengths below 250 nm (full range studied 222–265 nm). We argue that this effect can only be rationalized plausibly in terms of changing populations of different isomers, tautomers, or conformers in the target as a function of the thermal desorption conditions. Furthermore, the wavelength threshold behavior of this thermally-sensitive MPI channel indicates a critical dependence on neutral excited state dynamics between the absorption of the first and second photons. The experimental results are complemented by density functional theory (DFT) optimizations of the lowest-energy structure of uridine and two further conformers distinguished by different orientations of the hydroxymethyl group on the sugar part of the molecule. The energies of the transitions states between these three conformers are low compared with the energy required for decomposition.

Differences in DNA methylation of insulin-like growth factor 2 and cadherin 13 in patients with preeclampsia.

In a previous mass spectrometry study of our research group, 25 proteins were found to be differentially expressed in cerebrospinal fluid of patients with preeclampsia compared to controls. The objective of the current study was to investigate DNA methylation of the genes encoding for the former mentioned proteins in an independent dataset. In a nested case-control study of the Rotterdam Periconceptional Cohort, placental tissue, umbilical cord white blood cells and human umbilical vein endothelial cells (HUVEC) were obtained of 13 patients with early-onset preeclampsia, 16 patients with late-onset preeclampsia and 83 normotensive controls (27 patients with fetal growth restriction, 20 patients with spontaneous preterm birth and 36 uncomplicated pregnancies). DNA methylation of 783CpGs in regions of 25 genes was measured. DNA methylation of selected candidate genes in early- and late-onset preeclampsia compared to fetal growth restriction, spontaneous preterm birth and uncomplicated controls. From the 783CpGs of the 25 selected genes, 15CpGs were differentially methylated between early-onset preeclampsia and spontaneous preterm birth (3.80 E-5≤p≤0.036). Four CpGs were differentially methylated between early-onset preeclampsia and fetal growth restriction (0.0002≤p≤0.037) and 13CpGs were differentially methylated between early onset preeclampsia and uncomplicated controls (0.0001≤p≤0.04). Differences in DNA methylation were found in placental tissue, umbilical cord white blood cells and HUVEC of patients with early onset preeclampsia compared to (un)complicated controls, but not in patients with late-onset preeclampsia. The genes showing the largest differential methylation encode insulin-like growth factor 2 binding protein and receptor and cadherin 13.

DFT perspective on the selectivity and mechanism of ligand-free Heck reaction involving allylic esters and arenediazonium salts

A density functional theory (DFT) study has been carried out with respect to the cationic and neutral mechanism of ligand-free Heck-Matsuda cross-coupling reaction involving allylic esters and arenediazonium salts. Full Heck-Matsuda (HM) catalytic cycles were explored in the presence of different possible combinations of ligands based on previous mass-spectrometry studies. DFT calculations were performed to examine the olefinic substrate influences on the regioselectivity of this ligand-free Heck reaction. A multi-step mechanism was revealed for the oxidative addition of palladium (0) center to carbon-nitrogen bond of arenediazonium cation. The cationic migratory insertion pathway is favored by a chelated coordination involving the palladium center and the allylic ester. In contrast, for neutral complexes, a bidentate coordination of acetate ligand is the favored pathway. Regarding the hydride elimination step, the resulting free energy profile is quite flat, revealing that the Heck product and the hydride complex are easily formed for both cationic and neutral cycles. The reductive elimination step assisted by acetate is a very exothermic process, going through small relative reaction barriers. These results constitute new insights to a better understanding of the selectivity and the mechanism of ligand-free Heck-Matsuda reactions.

Identification of lignin oligomers in Kraft lignin using ultra-high-performance liquid chromatography/high-resolution multiple-stage tandem mass spectrometry (UHPLC/HRMSn)

Kraft lignin is the main source of technically produced lignin. For the development of valuable products based on Kraft lignin, its molecular structure is important. However, the chemical composition of Kraft lignin is still not well known. So far, the analysis of Kraft lignin by mass spectrometry (MS) has been mainly focused on monomeric compounds. Previous MS studies on lignin oligomers (LOs) considered only synthesised LO standards and/or lignins produced by processes other than the Kraft process. Furthermore, published MS methods suffer from using high resolution only in the MS1 stage in multiple-stage tandem MS methods. A high resolution in all MSn stages would provide more detailed information about LO fragmentation pathways. Since lignin samples are complex mixtures of a large number of similar phenolic compounds, the selection of tentative LOs in the MS data is challenging. In this study, we present a method for non-targeted analysis of LOs in Kraft lignin using ultra-high-performance liquid chromatography/high-resolution multiple-stage tandem mass spectrometry (UHPLC/HRMSn). A pre-selection strategy for LOs has been established based on a data-dependent neutral loss MS3 method in combination with a principal component analysis-quadratic discriminant analysis classification model (PCA-QDA). The method was optimised using a design of experiments (DOE) approach. The developed approach improved the pre-selection of tentative LOs in complex mixtures. From 587 detected peaks, 36 peaks were identified as LOs.Graphical abstractᅟ

A New Approach to Evaluating Statistical Significance of Spectral Identifications

While nonlinear peptide natural products such as Vancomycin and Daptomycin are among the most effective antibiotics, the computational techniques for sequencing such peptides are still in their infancy. Previous methods for sequencing peptide natural products are based on Nuclear Magnetic Resonance spectroscopy and require large amounts (milligrams) of purified materials. Recently, development of mass spectrometry-based methods has enabled accurate sequencing of nonlinear peptide natural products using picograms of material, but the question of evaluating statistical significance of Peptide Spectrum Matches (PSM) for these peptides remains open. Moreover, it is unclear how to decide whether a given spectrum is produced by a linear, cyclic, or branch-cyclic peptide. Surprisingly, all previous mass spectrometry studies overlooked the fact that a very similar problem has been successfully addressed in particle physics in 1951. In this work, we develop a method for estimating statistical significance of PSMs defined by any peptide (including linear and nonlinear). This method enables us to identify whether a peptide is linear, cyclic, or branch-cyclic, an important step toward identification of peptide natural products.

Gel-based mass spectrometric and computational approaches to the mitochondrial proteome of Neurospora

We have used gel electrophoretic techniques including isoelectric focusing, blue native, acid–urea, 16-benzyldimethyl-n-hexadecylammonium chloride or SDS first dimensions and SDS Laemmli or tricine second dimensions to separate the proteins from highly-purified Neurospora mitochondria and sub-mitochondrial fractions (membrane, soluble, protein complexes and ribonucleoproteins). The products of 260 genes, many of them in multiple processed or modified forms, were identified by MALDI-TOF mass spectrometry. This work confirms the existence, expression, and mitochondrial localization of the products of 55 Neurospora genes previously annotated only as predicted or hypothetical, and of 101 genes not identified in previous mass spectrometry studies. Combined with previous mass spectrometry studies, and re-evaluation of genome annotations, we have compiled a curated list of 358 proteins identified in proteomic studies that are likely to be bona fide mitochondrial proteins plus 80 other identified proteins that may be mitochondrial. Literature data mining and computational predictions suggest that Neurospora mitochondria also contain another 299 proteins not yet identified in proteomics projects. Taken together, these data suggest that the products of at least 738 genes comprise the Neurospora mitochondrial proteome.

Characterization of White Spot Syndrome Virus Envelope Protein VP51A and Its Interaction with Viral Tegument Protein VP26

In this study, we characterize a novel white spot syndrome virus (WSSV) structural protein, VP51A (WSSV-TW open reading frame 294), identified from a previous mass spectrometry study. Temporal-transcription analysis showed that vp51A is expressed in the late stage of WSSV infection. Gene structure analysis showed that the transcription initiation site of vp51A was 135 bp upstream of the translation start codon. The poly(A) addition signal overlapped with the translation stop codon, TAA, and the poly(A) tail was 23 bp downstream of the TAA. Western blot analysis of viral protein fractions and immunoelectron microscopy both suggested that VP51A is a viral envelope protein. Western blotting of the total proteins extracted from WSSV virions detected a band that was close to the predicted 51-kDa mass, but the strongest signal was around 72 kDa. We concluded that this 72-kDa band was in fact the full-length VP51A protein. Membrane topology assays demonstrated that the VP51A 72-kDa protein is a type II transmembrane protein with a highly hydrophobic transmembrane domain on its N terminus and a C terminus that is exposed on the surface of the virion. Coimmunoprecipitation, colocalization, and yeast two-hybrid assays revealed that VP51A associated directly with VP26 and indirectly with VP28, with VP26 acting as a linker protein in the formation of a VP51A-VP26-VP28 complex.

Modulation of lysine acetylation-stimulated repressive activity by Erk2-mediated phosphorylation of RIP140 in adipocyte differentiation

Receptor-interacting protein 140 is a co-regulator for many transcription factors. Previous mass spectrometry studies showed that either phosphorylation or lysine acetylation of RIP140 directly enhanced its trans-repressive activity. In this study, we first identified p300 as a specific lysine acetyltransferase, and extracellular-signal-related kinase 2 (Erk2) as a specific kinase for threonine phosphorylation, of RIP140 in vivo. We further determined two specific acetylated lysine residues (Lys 158/Lys 287) and phosphorylated threonine residues (Thr 202/Thr 207) that were critical for its gene-repressive activity. We then delineated signal transduction from Erk2-mediated phosphorylation of RIP140 that enhanced its recruiting p300 for subsequent lysine acetylation, and demonstrated the kinetics of activation of this signal transduction pathway in differentiating adipocytes. Finally, the physiological significance of this cell signal transduction pathway was illustrated in rescuing experiments where the defect in fat accumulation of RIP140-null cultures was rescued by re-expressing the wild type RIP140 or its phospho-mimetic mutant, but not its acetylation deficient mutant. These results demonstrate the signal transduction pathway, initiated from Erk2 activation for specific threonine phosphorylation, followed by p300 recruitment for lysine acetylation, which ultimately enhances the gene-repressive activity of RIP140 and its functional role in fat accumulation in differentiated adipocytes.

Origin of ganglioside complex antibodies in Guillain–Barré syndrome

The origin of antibodies to ganglioside complexes, as new immunotargets for Guillain–Barré syndrome (GBS), is unknown. This was investigated in 21 GBS patients from which Campylobacter jejuni was isolated. Two of these patients had serum IgG to the GM1/GD1a complex and two other patients had IgG to the GQ1b/GD1a complex. These pairs of patients were clinically distinct. These antibodies all cross-reacted to lipo-oligosaccharides (LOS) from the autologous C. jejuni strain. Previous mass spectrometry studies on these LOS showed the presence of oligosaccharides with a similar structure, further supporting the hypothesis that in these patients LOS induced the ganglioside complex antibodies.

Augmentation of ultraviolet B radiation-induced tumor necrosis factor production by the epidermal platelet-activating factor receptor.

Ultraviolet B radiation (UVB) has been shown to damage human keratinocytes in part by inducing oxidative stress and cytokine production. Indeed, UVB-induced production of the pro-inflammatory and cytotoxic cytokine tumor necrosis factor alpha (TNF-alpha) has been implicated in the epidermal damage seen in response to acute solar radiation. Though the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and keratinocytes express PAF receptors linked to cytokine biosynthesis, it is not known whether PAF is involved in UVB-induced epidermal cell cytokine production. These studies examined the role of the PAF system in UVB-induced epidermal cell TNF-alpha biosynthesis using a novel model system created by retroviral-mediated transduction of the PAF receptor-negative human epidermal cell line KB with the human PAF receptor (PAF-R). Treatment of PAF-R-expressing KB cells with the metabolically stable PAF-R agonist carbamoyl-PAF resulted in increased TNF-alpha mRNA and protein, indicating that activation of the epidermal PAF-R was linked to TNF-alpha production. UVB irradiation of PAF-R-expressing KB cells resulted in significant increases in both TNF-alpha mRNA and protein in comparison to UVB-treated control KB cells. However, UVB treatment up-regulated cyclooxygenase-2 mRNA levels to the same extent in both PAF-R-expressing and control KB cells. Pretreatment with the antioxidant vitamin E or the PAF-R antagonists WEB 2086 and A-85783 inhibited UVB-induced TNF-alpha production in the PAF-R-positive but not control KB cells. These studies suggest that the epidermal PAF-R may be a pharmacological target for UVB in skin.