Olivér Ozohanics

GlycoMiner: a new software tool to elucidate glycopeptide composition

New computer software, GlycoMiner, has been developed to automatically identify tandem (MS/MS) spectra obtained in liquid chromatography/mass spectrometry (LC/MS) runs which correspond to N-glycopeptides. The program complements conventional proteomics analysis, and can be used in a high-throughput environment. The program interprets the spectra and determines the structure of the corresponding glycopeptides. GlycoMiner runs under Windows, can process spectra obtained on various instruments, and can be downloaded from our website (w3.chemres.hu/ms/glycominer). The algorithm works similarly to a human expert; evaluates the low mass oxonium ions; deduces oligosaccharide losses from the protonated molecule; and identifies the mass of the peptide residue. The program has been tested on tryptic digests of two glycopeptides: AGP (which has five different N-glycosylation sites) and transferrin (with two N-glycosylation sites). Results have been evaluated both manually and by GlycoMiner. Out of 3132 MS/MS spectra 338 were found to correspond to glycopeptides; identification by GlycoMiner showed a 0.1% false positive and 0.1% false negative rate. From these it was possible to identify 196 glycan structures manually; GlycoMiner correctly identified all of these, with no false positives. The rest were low quality spectra, not suitable for structure assignment.

Stimulation of reactive oxygen species generation by disease-causing mutations of lipoamide dehydrogenase

We investigated pathogenic mutations relevant in dihydrolipoamide dehydrogenase (LADH; gene: Dld) deficiency, a severe human disease, to elucidate how they alter reactive oxygen species (ROS) generation and associated biophysical characteristics of LADH. Twelve known disease-causing mutants of human LADH have been expressed and purified to homogeneity from E. coli. Detailed biophysical and biochemical characterization of the mutants has been performed applying circular dichroism (CD) spectroscopy, nano-spray mass spectrometry (MS), calibrated gel filtration and flavin adenine dinucleotide-content analysis. Functional analyses revealed that four of the pathogenic mutations significantly stimulated the ROS-generating activity of LADH and also increased its sensitivity to an acidic shift in pH. LADH activity was reduced by variable extents in the mutants exhibiting excessive ROS generation. It is remarkable that in the P453L mutant, enzyme activity was nearly completely lost with a ROS-forming activity becoming dominant, whereas the G194C mutation, common among Ashkenazi Jews, resulted in no alteration in LADH activity but a gain in the ROS-generating activity. There have been neither major conformational alterations nor monomerization of the functional homodimer of LADH associated with the higher ROS-generating capacity as measured by CD spectroscopy and size-exclusion chromatography combined with nano-spray MS, respectively. The excessive ROS generation of selected LADH mutants could be an important factor in the pathology and clinical presentation of human LADH deficiency and raises the possibility of an antioxidant therapy in the treatment of this condition.

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.

High-performance liquid chromatography coupled to mass spectrometry methodology for analyzing site-specific N-glycosylation patterns.

Analysis of protein glycosylation is a major challenge in biochemistry, here we present a nano-UHPLC-MS(MS) based methodology, which is suitable to determine site-specific N-glycosylation patterns. A few pmol glycoprotein is sufficient to determine glycosylation patterns (which opens the way for biomedical applications) and requires at least two separate chromatographic runs. One is using tandem mass spectrometry (for structure identification); the other single stage MS mode (for semi-quantitation). Analysis relies heavily on data processing. The previously developed GlycoMiner algorithm and software was used to identify glycopeptides in MS/MS spectra. We have developed a new algorithm and software (GlycoPattern), which evaluates single stage mass spectra, both in terms of glycopeptide identification (for minor glycoforms) and semi-quantitation. Identification of glycopeptide structures based on MS/MS analysis has a false positive rate of 1%. Minor glycoforms (when sensitivity is insufficient to obtain an MS/MS spectrum) can be identified in single stage MS using GlycoPattern; but in such a case the false positive rate is increased to 5%. Glycosylation is studied at the glycopeptide level (i.e. following proteolytic digestion). This way the sugar chains can be unequivocally assigned to a given glycosylation site (site-specific glycosylation pattern). Glycopeptide analysis has the further advantage that protein-specific glycosylation patterns can be identified in complex mixtures and not only in purified samples. This opens the way for medium high throughput analysis of glycosylation. Specific examples of site-specific glycosylation patterns of alpha-1-acid glycoprotein, haptoglobin and on a therapeutic monoclonal antibody, Infliximab are also discussed.

Structure and enzymatic mechanism of a moonlighting dUTPase

Genome integrity requires well controlled cellular pools of nucleotides. dUTPases are responsible for regulating cellular dUTP levels and providing dUMP for dTTP biosynthesis. In Staphylococcus, phage dUTPases are also suggested to be involved in a moonlighting function regulating the expression of pathogenicity-island genes. Staphylococcal phage trimeric dUTPase sequences include a specific insertion that is not found in other organisms. Here, a 2.1 Å resolution three-dimensional structure of a ϕ11 phage dUTPase trimer with complete localization of the phage-specific insert, which folds into a small β-pleated mini-domain reaching out from the dUTPase core surface, is presented. The insert mini-domains jointly coordinate a single Mg2+ ion per trimer at the entrance to the threefold inner channel. Structural results provide an explanation for the role of Asp95, which is suggested to have functional significance in the moonlighting activity, as the metal-ion-coordinating moiety potentially involved in correct positioning of the insert. Enzyme-kinetics studies of wild-type and mutant constructs show that the insert has no major role in dUTP binding or cleavage and provide a description of the elementary steps (fast binding of substrate and release of products). In conclusion, the structural and kinetic data allow insights into both the phage-specific characteristics and the generally conserved traits of ϕ11 phage dUTPase.

Digestion protocol for small protein amounts for nano-HPLC-MS(MS) analysis

A miniaturized tryptic digestion protocol for protein analysis has been developed, which works well for small amounts of proteins using small volume of reagents. The protocol starts from 10μL sample volume with total protein content in the low pmol or fmol range (alternatively expressed, in the low ng range). After adding various reagents the total volume of the tryptic digest will increase to 15μL only. This is especially advantageous for nano-HPLC-MS or MALDI analysis which requires (and allows) analysis of few μL aliquots only. Efficiency of the protocol was tested using nano-HLPC-MS(MS). The results show that the developed miniaturized digestion protocol performs at least as well, possibly even better, than conventional protocols using large sample amounts; and is far superior to digestion performed in larger volumes followed by solvent evaporation/resolvation. This is reflected both in signal intensities in MS and in the number of proteins identified by MS/MS.

Quantitative Comparison of Tandem Mass Spectra Obtained on Various Instruments

AbstractThe similarity between two tandem mass spectra, which were measured on different instruments, was compared quantitatively using the similarity index (SI), defined as the dot product of the square root of peak intensities in the respective spectra. This function was found to be useful for comparing energy-dependent tandem mass spectra obtained on various instruments. Spectral comparisons show the similarity index in a 2D “heat map”, indicating which collision energy combinations result in similar spectra, and how good this agreement is. The results and methodology can be used in the pharma industry to design experiments and equipment well suited for good reproducibility. We suggest that to get good long-term reproducibility, it is best to adjust the collision energy to yield a spectrum very similar to a reference spectrum. It is likely to yield better results than using the same tuning file, which, for example, does not take into account that contamination of the ion source due to extended use may influence instrument tuning. The methodology may be used to characterize energy dependence on various instrument types, to optimize instrumentation, and to study the influence or correlation between various experimental parameters. Graphical Abstractᅟ

Determination of energy metabolites in cancer cells by porous graphitic carbon liquid chromatography electrospray ionization mass spectrometry for the assessment of energy metabolism

A high performance liquid chromatography (HPLC) tandem mass spectrometric (MS/MS) method has been developed for the simultaneous determination of fifteen glucose, or acetate derived metabolites isolated from tumor cells. Glycolytic and tricarboxylic acid (TCA) cycle metabolites as well as acidic amino acids were separated on a HPLC porous graphitic carbon (PGC) column and simultaneously determined by means of triple quadrupole MS/MS using multiple reaction monitoring (MRM). Target compounds were eluted within 10 min with 8% v/v formic acid as an electronic modifier added to a 4:1 v/v methanol water mobile phase. The calibration is linear in the 1-100 μM concentration range for each analyte. The limit of detection ranges between 0.39 and 2.78 μM for the analytes concerned. To test the PGC-HPLC-MS/MS method in metabolomic studies, ZR-75.1 human mammary adenocarcinoma cells were labeled with U-(13)C glucose or 1-(13)C acetate. Applying the MRM mode, the incorporation of (13)C into metabolites, isolated from the tumor cells, and derived from glucose or acetate, could be properly identified.

Widespread alterations in the synaptic proteome of the adolescent cerebral cortex following prenatal immune activation in rats

An increasing number of studies have revealed associations between pre- and perinatal immune activation and the development of schizophrenia and autism spectrum disorders (ASDs). Accordingly, neuroimmune crosstalk has a considerably large impact on brain development during early ontogenesis. While a plethora of heterogeneous abnormalities have already been described in established maternal immune activation (MIA) rodent and primate animal models, which highly correlate to those found in human diseases, the underlying molecular background remains obscure. In the current study, we describe the long-term effects of MIA on the neocortical pre- and postsynaptic proteome of adolescent rat offspring in detail. Molecular differences were revealed in sub-synaptic fractions, which were first thoroughly characterized using independent methods. The widespread proteomic examination of cortical samples from offspring exposed to maternal lipopolysaccharide administration at embryonic day 13.5 was conducted via combinations of different gel-based proteomic techniques and tandem mass spectrometry. Our experimentally validated proteomic data revealed more pre- than postsynaptic protein level changes in the offspring. The results propose the relevance of altered synaptic vesicle recycling, cytoskeletal structure and energy metabolism in the presynaptic region in addition to alterations in vesicle trafficking, the cytoskeleton and signal transduction in the postsynaptic compartment in MIA offspring. Differing levels of the prominent signaling regulator molecule calcium/calmodulin-dependent protein kinase II in the postsynapse was validated and identified specifically in the prefrontal cortex. Finally, several potential common molecular regulators of these altered proteins, which are already known to be implicated in schizophrenia and ASD, were identified and assessed. In summary, unexpectedly widespread changes in the synaptic molecular machinery in MIA rats were demonstrated which might underlie the pathological cortical functions that are characteristic of schizophrenia and ASD.

Comparison of glycopeptide/glycoprotein enrichment techniques.

The analysis of post-translational modifications has become one of the mainstream topics in proteomics. Among these, glycosylation is among the most widespread, but its study is quite challenging. Analysis is mainly based on mass spectrometry, although alternatives are often used. Glycopeptide analysis is hindered by two main factors: First, mass spectrometric sensitivity of glycopeptides is only a fraction of that of peptides. The other relates to heterogeneity. In a protein there are often several glycosylation sites and, typically, various (often more than 10) different oligosaccharides are attached to each site. This spreads the already small glycopeptide signal intensity over several molecular species. Furthermore, to characterize glycosylation not only the most abundant, but also minor oligosaccharide variants attached to a given site need to be identified and (approximately) quantified. These difficulties often lead to severe problems relating to sensitivity and selectivity. In order to alleviate these issues, enrichment of glycopeptides and/or glycoproteins is often necessary. Enrichment can be accomplished either or both at the protein and at the peptide level. In the latter case the glycoprotein is first enzymatically digested using proteolytic enzymes, like trypsin. For the subsequent analysis of glycosylation, enzymatic digestion is (nearly) always needed. In practice, the alternatives are first enrichment, followed by digestion (corresponding to enrichment at the glycoprotein level), or first digestion then analysis (corresponding to enrichment at the peptide level). In the present work we have used both approaches. Digestion was performed by trypsin, according to an established methodology. There are many possibilities for enrichment; here we compare three alternatives useful for N-glycoproteins. All three are based on solid-phase extraction (SPE). These solution-based techniques are advantageous, as subsequent digestion and analysis (either using high-performance/ mass spectrometry (HPLC/MS) or matrix-assisted laser desorption/ionization (MALDI)) are far simpler than following gel-based methodologies, and they can be adapted for high throughput. We have used phenylboronic acid stationary phase – this binds to vicinal OH groups, i.e. most sugars and not much else. We have also used wheat germ agglutinin (WGA) lectin, which binds neuraminic acid (a sialic acid) units, a common constituent of all N-glycoproteins. There are several other lectins (like concanavalin A) which might be used for glycopeptide enrichment, but comparison of the specificity of various lectins is outside the scope of the present paper. Finally, we have used strong anion exchange which binds acidic groups, e.g. sialic acids,