Jeffrey N. Agar

Identification of a misfolded region in superoxide dismutase 1 that is exposed in amyotrophic lateral sclerosis.

Background: Misfolded SOD1 is associated with sporadic and familial ALS. Results: The epitope of C4F6, an antibody specific for misfolded SOD1, has been defined. Loops IV and VII in SOD1 modulate exposure of this epitope. Conclusion: Exposure of the C4F6 epitope correlates with heightened SOD1-mediated toxicity. Significance: Concealing the C4F6 epitope by stabilizing SOD1 loops IV and VII has therapeutic potential for ALS. Mutations and aberrant post-translational modifications within Cu,Zn-superoxide dismutase (SOD1) cause this otherwise protective enzyme to misfold, leading to amyotrophic lateral sclerosis (ALS). The C4F6 antibody selectively binds misfolded SOD1 in spinal cord tissues from postmortem human ALS cases, as well as from an ALS-SOD1 mouse model, suggesting that the C4F6 epitope reports on a pathogenic conformation that is common to misfolded SOD1 variants. To date, the residues and structural elements that comprise this epitope have not been elucidated. Using a chemical cross-linking and mass spectrometry approach, we identified the C4F6 epitope within several ALS-linked SOD1 variants, as well as an oxidized form of WT SOD1, supporting the notion that a similar misfolded conformation is shared among pathological SOD1 proteins. Exposure of the C4F6 epitope was modulated by the SOD1 electrostatic (loop VII) and zinc binding (loop IV) loops and correlated with SOD1-induced toxicity in a primary microglia activation assay. Site-directed mutagenesis revealed Asp92 and Asp96 as key residues within the C4F6 epitope required for the SOD1-C4F6 binding interaction. We propose that stabilizing the functional loops within SOD1 and/or obscuring the C4F6 epitope are viable therapeutic strategies for treating SOD1-mediated ALS.

The central nervous system transcriptome of the weakly electric brown ghost knifefish (Apteronotus leptorhynchus): de novo assembly, annotation, and proteomics validation

BackgroundThe brown ghost knifefish (Apteronotus leptorhynchus) is a weakly electric teleost fish of particular interest as a versatile model system for a variety of research areas in neuroscience and biology. The comprehensive information available on the neurophysiology and neuroanatomy of this organism has enabled significant advances in such areas as the study of the neural basis of behavior, the development of adult-born neurons in the central nervous system and their involvement in the regeneration of nervous tissue, as well as brain aging and senescence. Despite substantial scientific interest in this species, no genomic resources are currently available.ResultsHere, we report the de novo assembly and annotation of the A. leptorhynchus transcriptome. After evaluating several trimming and transcript reconstruction strategies, de novo assembly using Trinity uncovered 42,459 unique contigs containing at least a partial protein-coding sequence based on alignment to a reference set of known Actinopterygii sequences. As many as 11,847 of these contigs contained full or near-full length protein sequences, providing broad coverage of the proteome. A variety of non-coding RNA sequences were also identified and annotated, including conserved long intergenic non-coding RNA and other long non-coding RNA observed previously to be expressed in adult zebrafish (Danio rerio) brain, as well as a variety of miRNA, snRNA, and snoRNA. Shotgun proteomics confirmed translation of open reading frames from over 2,000 transcripts, including alternative splice variants. Assignment of tandem mass spectra was greatly improved by use of the assembly compared to databases of sequences from closely related organisms. The assembly and raw reads have been deposited at DDBJ/EMBL/GenBank under the accession number GBKR00000000. Tandem mass spectrometry data is available via ProteomeXchange with identifier PXD001285.ConclusionsPresented here is the first release of an annotated de novo transcriptome assembly from Apteronotus leptorhynchus, providing a broad overview of RNA expressed in central nervous system tissue. The assembly, which includes substantial coverage of a wide variety of both protein coding and non-coding transcripts, will allow the development of better tools to understand the mechanisms underlying unique characteristics of the knifefish model system, such as their tremendous regenerative capacity and negligible brain senescence.

Artifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation.

Bottom‐up MS studies typically employ a reduction and alkylation step that eliminates a class of PTM, S‐thiolation. Given that molecular oxygen can mediate S‐thiolation from reduced thiols, which are abundant in the reducing intracellular milieu, we investigated the possibility that some S‐thiolation modifications are artifacts of protein preparation. Cu/Zn‐superoxide dismutase (SOD1) was chosen for this case study as it has a reactive surface cysteine residue, which is readily cysteinylated in vitro. The ability of oxygen to generate S‐thiolation artifacts was tested by comparing purification of SOD1 from postmortem human cerebral cortex under aerobic and anaerobic conditions. S‐thiolation was ∼50% higher in aerobically processed preparations, consistent with oxygen‐dependent artifactual S‐thiolation. The ability of endogenous small molecule disulfides (e.g. cystine) to participate in artifactual S‐thiolation was tested by blocking reactive protein cysteine residues during anaerobic homogenization. A 50‐fold reduction in S‐thiolation occurred indicating that the majority of S‐thiolation observed aerobically was artifact. Tissue‐specific artifacts were explored by comparing brain‐ and blood‐derived protein, with remarkably more artifacts observed in brain‐derived SOD1. Given the potential for such artifacts, rules of thumb for sample preparation are provided. This study demonstrates that without taking extraordinary precaution, artifactual S‐thiolation of highly reactive, surface‐exposed, cysteine residues can result.

A rapid MALDI-TOF mass spectrometry workflow for Drosophila melanogaster differential neuropeptidomics

BackgroundNeuropeptides are a diverse category of signaling molecules in the nervous system regulating a variety of processes including food intake, social behavior, circadian rhythms, learning, and memory. Both the identification and functional characterization of specific neuropeptides are ongoing fields of research. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of nervous tissues from a variety of organisms allows direct detection and identification of neuropeptides. Here, we demonstrate an analysis workflow that allows for the detection of differences in specific neuropeptides amongst a variety of neuropeptides being simultaneously measured. For sample preparation, we describe a straight-forward and rapid (minutes) method where individual adult Drosophila melanogaster brains are analyzed. Using a MATLAB-based data analysis workflow, also compatible with MALDI-TOF mass spectra obtained from other sample preparations and instrumentation, we demonstrate how changes in neuropeptides levels can be detected with this method.ResultsOver fifty isotopically resolved ion signals in the peptide mass range are reproducibly observed across experiments. MALDI-TOF MS profile spectra were used to statistically identify distinct relative differences in organ-wide endogenous levels of detected neuropeptides between biological conditions. In particular, three distinct levels of a particular neuropeptide, pigment dispersing factor, were detected by comparing groups of preprocessed spectra obtained from individual brains across three different D. melanogaster strains, each of which express different amounts of this neuropeptide. Using the same sample preparation, MALDI-TOF/TOF tandem mass spectrometry confirmed that at least 14 ion signals observed across experiments are indeed neuropeptides. Among the identified neuropeptides were three products of the neuropeptide-like precursor 1 gene previously not identified in the literature.ConclusionsUsing MALDI-TOF MS and preprocessing/statistical analysis, changes in relative levels of a particular neuropeptide in D. melanogaster tissue can be statistically detected amongst a variety of neuropeptides. While the data analysis methods should be compatible with other sample preparations, the presented sample preparation method was sufficient to identify previously unconfirmed D. melanogaster neuropeptides.

QUDeX-MS: hydrogen/deuterium exchange calculation for mass spectra with resolved isotopic fine structure

BackgroundHydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is confounded by deuterium exchange-associated peaks overlapping with peaks of heavy, natural abundance isotopes, such as carbon-13. Recent studies demonstrated that high-performance mass spectrometers could resolve isotopic fine structure and eliminate this peak overlap, allowing direct detection and quantification of deuterium incorporation.ResultsHere, we present a graphical tool that allows for a rapid and automated estimation of deuterium incorporation from a spectrum with isotopic fine structure. Given a peptide sequence (or elemental formula) and charge state, the mass-to-charge ratios of deuterium-associated peaks of the specified ion is determined. Intensities of peaks in an experimental mass spectrum within bins corresponding to these values are used to determine the distribution of deuterium incorporated. A theoretical spectrum can then be calculated based on the estimated distribution of deuterium exchange to confirm interpretation of the spectrum. Deuterium incorporation can also be detected for ion signals without a priori specification of an elemental formula, permitting detection of exchange in complex samples of unidentified material such as natural organic matter. A tool is also incorporated into QUDeX-MS to help in assigning ion signals from peptides arising from enzymatic digestion of proteins. MATLAB-deployable and standalone versions are available for academic use at qudex-ms.sourceforge.net and agarlabs.com.ConclusionIsotopic fine structure HDX-MS offers the potential to increase sequence coverage of proteins being analyzed through mass accuracy and deconvolution of overlapping ion signals. As previously demonstrated, however, the data analysis workflow for HDX-MS data with resolved isotopic fine structure is distinct. QUDeX-MS we hope will aid in the adoption of isotopic fine structure HDX-MS by providing an intuitive workflow and interface for data analysis.

Secretion, isotopic labeling and deglycosylation of N-acylethanolamine acid amidase for biophysical studies

N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (Ntn) enzyme with a catalytic cysteine residue that has highest activity at acidic pH. The most prominent substrate hydrolyzed is palmitoylethanolamine (PEA), which regulates inflammation. Inhibitors of NAAA have been shown to increase endogenous levels of PEA, and are of interest as potential treatments for inflammatory disorders and other maladies. Currently, there are no X-ray or NMR structures of NAAA available to inform medicinal chemistry. Additionally, there are a limited number of enzyme structures available that are within the Ntn-hydrolase family, have a catalytic cysteine residue, and have a high sequence homology. For these reasons, we developed expression and purification methods for the production of enzyme samples amenable to structural characterization. Mammalian cells are necessary for post-translational processing, including signal sequence cleavage and glycosylation, that are required for a correctly folded zymogen before conversion to active, and mature enzyme. We have identified an expression construct, mammalian cell line, specific media and additives to express and secrete hNAAA zymogen and we further optimized propagation conditions and show this secretion method is suitable for isotopic labeling of the protein. We refined purification methods to achieve a high degree of protein purity potentially suited to crystallography. Glycosylated proteins can present challenges to biophysical methods. Therefore we deglycosylate the enzyme and show that the activity of the mature enzyme is not affected by deglycosylation.

Abstract 202: Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines

Introduction: Quantitative analytical systems have the ability to quantify the amount of constituents on a per entity basis. For example, flow cytometers use stoichiometric DNA stains to quantify the amount of DNA on a per cell basis, and allow discrimination of the cell cycle phases G0/G1, S-Phase, and G2/M. Quantitative imaging cytometers employ morphometric features (total DNA content, DNA maximum brightness, and nuclear area) to track the cell cycle during the mitotic phases. As cells enter mitosis, the condensation of the chromatin to form chromosomes increases the DNA density, while the nuclear area and volume decrease. Unfortunately, DNA dyes are harmful for live cells and can interfere with the results of toxicological assays. It is desirable to have a method for label-free long-term evaluation of the cell cycle in living cells. Methods: The Holomonitor®M4 (Phase Holographic Imaging, Lund, Sweden) is an incubator-adapted time-lapse imaging system that records interference patterns from a low power 635nm diode laser. Computer software is used to de-convolve the interference pattern at a plurality of heights to form 3-dimensional and 2-dimensional images of the sample thickness. These images are segmented and features (cellular area, optical thickness and volume, etc.) are calculated on a per cell basis. Positional information is recorded for each cell. Data can be presented as positional track charts, position vs. feature graphs, and feature vs. feature graphs. We obtained time-lapse videos using proprietary PHI software and also generated 4-dimensional plots (X-position, Y- position, optical thickness coded as the pixel intensity, and time) in ImageJ software. Results: In our studies of SKOV3 tumor cell lines, we found a set of cellular based features (cell volume, optical thickness, and cell area) that correlate with the DNA-based features we are accustomed to using in our traditional label-based systems. In the 4D plots, the history of each cell within the imaging area is presented for the entire evaluation time - typically 18-24 hours with 5 minute sampling increments. With a low optical thickness threshold, tracks of all of the cells are displayed. The image intensity threshold can be raised, so only mitotic events are displayed. Mitotic cells have a characteristic Y shaped tracks, appearing suddenly as the chromatin condenses, bifurcating as the daughter cells move away from each other, and then disappearing as the chromatin de-condenses. Cells blocked in mitosis present as persistent long tracks. The rapid degradation of apoptotic cells is often visible, while cells undergoing mitotic catastrophe suddenly disappear. Conclusions: Time-lapse label-free holographic imaging combining traditional analysis and novel display techniques is well suited for toxicological studies and visualization of drug effects on the cell cycle. Citation Format: Ed Luther, Jeffrey Agar, Mansoor Amiji. Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 202. doi:10.1158/1538-7445.AM2015-202

ProForma: A Standard Proteoform Notation

The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard.

Rapid discrimination of pediatric brain tumors by mass spectrometry imaging

PurposeMedulloblastoma, the most common primary pediatric malignant brain tumor, originates in the posterior fossa of the brain. Pineoblastoma, which originates within the pineal gland, is a rarer malignancy that also presents in the pediatric population. Medulloblastoma and pineoblastoma exhibit overlapping clinical features and have similar histopathological characteristics. Histopathological similarities confound rapid diagnoses of these two tumor types. We have conducted a pilot feasibility study analyzing the molecular profile of archived frozen human tumor specimens using mass spectrometry imaging (MSI) to identify potential biomarkers capable of classifying and distinguishing between medulloblastoma and pineoblastoma.MethodsWe performed matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry imaging on eight medulloblastoma biopsy specimens and three pineoblastoma biopsy specimens. Multivariate statistical analyses were performed on the MSI dataset to generate classifiers that distinguish the two tumor types. Lastly, the molecules that were discriminative of tumor type were queried against the Lipid Maps database and identified.ResultsIn this pilot study we show that medulloblastoma and pineoblastoma can be discriminated using molecular profiles determined by MSI. The highest-ranking discriminating classifiers of medulloblastoma and pineoblastoma were glycerophosphoglycerols and sphingolipids, respectively.ConclusionWe demonstrate proof-of-concept that medulloblastoma and pineoblastoma can be rapidly distinguished by using MSI lipid profiles. We identified biomarker candidates capable of distinguishing these two histopathologically similar tumor types. This work expands the current molecular knowledge of medulloblastoma and pineoblastoma by characterizing their lipidomic profiles, which may be useful for developing novel diagnostic, prognostic and therapeutic strategies.