Andreas Tholey

Quantitation of low molecular mass substrates and products of enzyme catalyzed reactions using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Relative peak-height ratios of products to substrates determined by MALDI-TOFMS allow the quantitative analysis of enzyme catalyzed reactions for screening purposes. Two examples were investigated: the first one was a lipase catalyzed reaction which produces 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (MET) using rac-alpha-phenylethylamine (PEA) as substrate. The second one was the pyruvate decarboxylase catalyzed formation of (1R)-1-hydroxy-1-phenyl-2-propanone (PAC) with benzaldehyde (BzA) as substrate. Here the corresponding oximes were analyzed after derivatization using hydroxylamine. The standard curves (r2 = 0.985 for MET, r2 = 0.991 for PAC) were linear over two orders of magnitude for MET and PAC concentrations. After optimization of the sample preparation an average relative standard deviation of 12.5% was obtained in both cases.

Metalloproteases meprin α and meprin β are C- and N-procollagen proteinases important for collagen assembly and tensile strength

Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a−/− and Mep1b−/− mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin β led to the generation of mature collagen molecules that spontaneously assembled into collagen fibrils. Thus, meprin α and meprin β are unique in their ability to process and release both C- and N-propeptides from type I procollagen in vitro and in vivo and contribute to the integrity of connective tissue in skin, with consequent implications for inherited connective tissue disorders.

Membrane-proximal domain of a disintegrin and metalloprotease-17 represents the putative molecular switch of its shedding activity operated by protein-disulfide isomerase.

A disintegrin and metalloprotease-17 (ADAM17) is a major sheddase responsible for the regulation of a wide range of biological processes, like cellular differentiation, regeneration, or cancer progression. Hitherto, the mechanism regulating the enzymatic activity of ADAM17 is poorly understood. Recently, protein-disulfide isomerase (PDI) was shown to interact with ADAM17 and to down-regulate its enzymatic activity. Here we demonstrate by NMR spectroscopy and tandem-mass spectrometry that PDI directly interacts with the membrane-proximal domain (MPD), a domain of ADAM17 involved in its dimerization and substrate recognition. PDI catalyzes an isomerization of disulfide bridges within the thioredoxin motif C600XXC603 of the MPD and results in a drastic structural change between an active open state and an inactive closed conformation. This conformational change of the MPD putatively acts as a molecular switch, facilitating a global reorientation of the extracellular domains in ADAM17 and regulating its shedding activity.

Tandem Mass Tag Protein Labeling for Top-Down Identification and Quantification

Top-down mass spectrometry holds tremendous potential for characterization and quantification of intact proteins. So far, however, very few studies have combined top-down proteomics with protein quantification. In view of the success of isobaric mass tags in quantitative bottom-up proteomics, we applied the tandem mass tag (TMT) technology to label intact proteins and examined the feasibility to directly quantify TMT-labeled proteins. A top-down platform encompassing separation via ion-pair reversed-phase liquid chromatography using monolithic stationary phases coupled online to an LTQ-Orbitrap Velos electron-transfer dissociation (ETD) mass spectrometer (MS) was established to simultaneously identify and quantify TMT-labeled proteins. The TMT-labeled proteins were found to be readily dissociated under high-energy collision dissociation (HCD) activation. The liberated reporter ions delivered expected ratios over a wide dynamic range independent of the protein charge state. Furthermore, protein sequence tags generated either by low-energy HCD or ETD activation along with the intact protein mass information allow for confident identification of small proteins below 35 kDa. We conclude that the approach presented in this pilot study paves the way for further developments and numerous applications for straightforward, accurate, and multiplexed quantitative analysis in protein chemistry and proteomics.

Highly accelerated feature detection in proteomics data sets using modern graphics processing units

MOTIVATION Mass spectrometry (MS) is one of the most important techniques for high-throughput analysis in proteomics research. Due to the large number of different proteins and their post-translationally modified variants, the amount of data generated by a single wet-lab MS experiment can easily exceed several gigabytes. Hence, the time necessary to analyze and interpret the measured data is often significantly larger than the time spent on sample preparation and the wet-lab experiment itself. Since the automated analysis of this data is hampered by noise and baseline artifacts, more sophisticated computational techniques are required to handle the recorded mass spectra. Obviously, there is a clear tradeoff between performance and quality of the analysis, which is currently one of the most challenging problems in computational proteomics. RESULTS Using modern graphics processing units (GPUs), we implemented a feature finding algorithm based on a hand-tailored adaptive wavelet transform that drastically reduces the computation time. A further speedup can be achieved exploiting the multi-core architecture of current computing devices, which leads to up to an approximately 200-fold speed-up in our computational experiments. In addition, we will demonstrate that several approximations necessary on the CPU to keep run times bearable, become obsolete on the GPU, yielding not only faster, but also improved results. AVAILABILITY An open source implementation of the CUDA-based algorithm is available via the software framework OpenMS (http://www.openms.de). SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Systems genomics evaluation of the SH-SY5Y neuroblastoma cell line as a model for Parkinson's disease

BackgroundThe human neuroblastoma cell line, SH-SY5Y, is a commonly used cell line in studies related to neurotoxicity, oxidative stress, and neurodegenerative diseases. Although this cell line is often used as a cellular model for Parkinson’s disease, the relevance of this cellular model in the context of Parkinson’s disease (PD) and other neurodegenerative diseases has not yet been systematically evaluated.ResultsWe have used a systems genomics approach to characterize the SH-SY5Y cell line using whole-genome sequencing to determine the genetic content of the cell line and used transcriptomics and proteomics data to determine molecular correlations. Further, we integrated genomic variants using a network analysis approach to evaluate the suitability of the SH-SY5Y cell line for perturbation experiments in the context of neurodegenerative diseases, including PD.ConclusionsThe systems genomics approach showed consistency across different biological levels (DNA, RNA and protein concentrations). Most of the genes belonging to the major Parkinson’s disease pathways and modules were intact in the SH-SY5Y genome. Specifically, each analysed gene related to PD has at least one intact copy in SH-SY5Y. The disease-specific network analysis approach ranked the genetic integrity of SH-SY5Y as higher for PD than for Alzheimer’s disease but lower than for Huntington’s disease and Amyotrophic Lateral Sclerosis for loss of function perturbation experiments.

A 2D reversed-phase × ion-pair reversed-phase HPLC-MALDI TOF/TOF-MS approach for shotgun proteome analysis

The separation of complex peptide mixtures in shotgun proteome analysis using a 2D separation scheme encompassing reversed-phase × ion-pair reversed-phase (IP-RP) liquid chromatography coupled online to electrospray ion trap mass spectrometry (MS) has been shown earlier to be superior in terms of separation efficiency and technical robustness compared to the classically used separation scheme encompassing strong cation exchange × IP-RP-chromatography in shotgun proteome analysis. In the present study, this novel separation scheme was coupled offline to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF-MS for the analysis of the same sample, a tryptic digest of the cytosolic proteome of the bacterium Corynebacterium glutamicum. Compared to the earlier study, the MALDI-based platform led to a significantly increased number of peptides (7,416 vs. 2,709) and proteins (1,208 vs. 468, without single peptide-based identifications), respectively. This represents the majority of all predicted cytosolic proteins in C. glutamicum. The high proteome coverage, as well as the large number of low-abundant proteins identified with this improved analytical platform, pave the way for new biological studies.

Characterization and Function of the First Antibiotic Isolated from a Vent Organism: The Extremophile Metazoan Alvinella pompejana

The emblematic hydrothermal worm Alvinella pompejana is one of the most thermo tolerant animal known on Earth. It relies on a symbiotic association offering a unique opportunity to discover biochemical adaptations that allow animals to thrive in such a hostile habitat. Here, by studying the Pompeii worm, we report on the discovery of the first antibiotic peptide from a deep-sea organism, namely alvinellacin. After purification and peptide sequencing, both the gene and the peptide tertiary structures were elucidated. As epibionts are not cultivated so far and because of lethal decompression effects upon Alvinella sampling, we developed shipboard biological assays to demonstrate that in addition to act in the first line of defense against microbial invasion, alvinellacin shapes and controls the worms epibiotic microflora. Our results provide insights into the nature of an abyssal antimicrobial peptide (AMP) and into the manner in which an extremophile eukaryote uses it to interact with the particular microbial community of the hydrothermal vent ecosystem. Unlike earlier studies done on hydrothermal vents that all focused on the microbial side of the symbiosis, our work gives a view of this interaction from the host side.

Zinc Ion-induced Domain Organization in Metallo-β-lactamases A FLEXIBLE “ZINC ARM” FOR RAPID METAL ION TRANSFER?

The reversible unfolding of metallo-β-lactamase from Chryseobacterium meningosepticum (BlaB) by guanidinium hydrochloride is best described by a three-state model including folded, intermediate, and unfolded states. The transformation of the folded apoenzyme into the intermediate state requires only very low denaturant concentrations, in contrast to the Zn2-enzyme. Similarly, circular dichroism spectra of both BlaB and metallo-β-lactamase from Bacillus cereus 569/H/9 (BcII) display distinct differences between metal-free and Zn2-enzymes, indicating that the zinc ions affect the folding of the proteins, giving a larger α-helix content. To identify the regions of the protein involved in this zinc ion-induced change, a hydrogen deuterium exchange study with matrix-assisted laser desorption ionization tandem time of flight mass spectrometry on metal-free and Zn1- and Zn2-BcII was carried out. The region spanning the metal binding metallo-β-lactamases (MBL) superfamily consensus sequence His-X-His-X-Asp motif and the loop connecting the N- and C-terminal domains of the protein undergoes a zinc ion-dependent structural change between intrinsically disordered and ordered states. The inherent flexibility even appears to allow for the formation of metal ion-bridged protein-protein complexes which may account for both electrospray ionization-mass spectroscopy results obtained upon variation of the zinc/protein ratio and stoichiometry-dependent variations of 199mHg-perturbed angular correlation of γ-rays spectroscopic data. We suggest that this flexible “zinc arm” motif, present in all the MBL subclasses, is disordered in metal-free MBLs and may be involved in metal ion acquisition from zinc-carrying molecules different from MBL in an “activation on demand” regulation of enzyme activity.

Proteomic study of human glioblastoma multiforme tissue employing complementary two-dimensional liquid chromatography- and mass spectrometry-based approaches.

An extensive data set comprising 2660 unique protein identifications was obtained for the proteome of a human brain tumor (glioblastoma multiforme) by combining the results of two complementary analytical strategies based on two-dimensional chromatography and mass spectrometry. A bottom-up method, performing peptide separation in both chromatographic dimensions was employed as well as a semi-top-down method, in which intact proteins were separated in the first and tryptic peptides in the second dimension. The identified proteins were assigned to their molecular functions and compared to previously identified proteins of glioblastoma multiforme (= astrocytoma WHO grade IV), lower WHO grade astrocytomas (grade II and III), and nontumor brain tissue. With the use of a subset of 104 identified membrane proteins, the properties of intact protein fractionation in the first dimension of the semi-top-down approach were elucidated in detail. The benefit of the semi-top-down approach was further demonstrated by the identification of a set of endogenous glioblastoma multiforme expressed proteins. These proteins correspond to recombinant antigens which were recently found to be reactive against autoantibodies in glioblastoma multiforme patients. The results indicate the usefulness of the semi-top-down approach for the investigation of immunogenic antigens in human tumor tissue samples.