Small Molecule LC-MS/MS Fragmentation Data Analysis and Application to Siderophore Identification

Rapid developments in tandem liquid chromatography-mass spectrometry (LC-MS/MS) have created wide interest in applications for the analysis of small molecule mixtures. MS/MS spectra can contain rich structural information, but because of the structural diversity of small molecules and different data acquisition methods, analysis algorithms and workflows frequently need to be tailored to individual research questions. This chapter shows how MATLAB can be used for LC-MS/MS-based structural characterization of small molecules. Starting with the import of raw data, ways for visualization and the creation of graphical user interfaces (GUIs) for individual applications are demonstrated. A selection of frequently used algorithms for pre-processing and data analysis is reviewed in context of their MATLAB implementation. The approaches are then tailored and applied to the analysis of iron-binding peptides (peptidic siderophores) by high-resolution LC-MS/MS. The method uses a database with siderophore structures to exploit prior knowledge about siderophore structural diversity for the interpretation of MS/MS spectra from known and new siderophores

An optical-IR jet in 3C133

We report the discovery of a new optical-IR synchrotron jet in the radio galaxy 3C133 from our HST/NICMOS snapshot survey. The jet and eastern hotspot are well resolved, and visible at both optical and IR wavelengths. The IR jet follows the morphology of the inner part of the radio jet, with three distinct knots identified with features in the radio. The radio-IR SED's of the knots are examined, along with those of two more distant hotspots at the eastern extreme of the radio feature. The detected emission appears to be synchrotron, with peaks in the NIR for all except one case, which exhibits a power-law spectrum throughout.Comment: ApJ accepted. 14 pages, 6 figure

Mutated in colorectal cancer (MCC) is a novel oncogene in B lymphocytes

BACKGROUND: Identification of novel genetic risk factors is imperative for a better understanding of B lymphomagenesis and for the development of novel therapeutic strategies. TRAF3, a critical regulator of B cell survival, was recently recognized as a tumor suppressor gene in B lymphocytes. The present study aimed to identify novel oncogenes involved in malignant transformation of TRAF3-deficient B cells. METHODS: We used microarray analysis to identify genes differentially expressed in TRAF3(−/−) mouse splenic B lymphomas. We employed lentiviral vector-mediated knockdown or overexpression to manipulate gene expression in human multiple myeloma (MM) cell lines. We analyzed cell apoptosis and proliferation using flow cytometry, and performed biochemical studies to investigate signaling mechanisms. To delineate protein-protein interactions, we applied affinity purification followed by mass spectrometry-based sequencing. RESULTS: We identified mutated in colorectal cancer (MCC) as a gene strikingly up-regulated in TRAF3-deficient mouse B lymphomas and human MM cell lines. Aberrant up-regulation of MCC also occurs in a variety of primary human B cell malignancies, including non-Hodgkin lymphoma (NHL) and MM. In contrast, MCC expression was not detected in normal or premalignant TRAF3(−/−) B cells even after treatment with B cell stimuli, suggesting that aberrant up-regulation of MCC is specifically associated with malignant transformation of B cells. In elucidating the functional roles of MCC in malignant B cells, we found that lentiviral shRNA vector-mediated knockdown of MCC induced apoptosis and inhibited proliferation in human MM cells. Experiments of knockdown and overexpression of MCC allowed us to identify several downstream targets of MCC in human MM cells, including phospho-ERK, c-Myc, p27, cyclin B1, Mcl-1, caspases 8 and 3. Furthermore, we identified 365 proteins (including 326 novel MCC-interactors) in the MCC interactome, among which PARP1 and PHB2 were two hubs of MCC signaling pathways in human MM cells. CONCLUSIONS: Our results indicate that in sharp contrast to its tumor suppressive role in colorectal cancer, MCC functions as an oncogene in B cells. Our findings suggest that MCC may serve as a diagnostic marker and therapeutic target in B cell malignancies, including NHL and MM. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13045-014-0056-6) contains supplementary material, which is available to authorized users

Accurate proteome-wide protein quantification from high-resolution 15N mass spectra

In quantitative mass spectrometry-based proteomics, the metabolic incorporation of a single source of 15N-labeled nitrogen has many advantages over using stable isotope-labeled amino acids. However, the lack of a robust computational framework for analyzing the resulting spectra has impeded wide use of this approach. We have addressed this challenge by introducing a new computational methodology for analyzing 15N spectra in which quantification is integrated with identification. Application of this method to an Escherichia coli growth transition reveals significant improvement in quantification accuracy over previous methods

Multiplexed single-cell proteomics using SCoPE2

Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis

Quantitative oculomotor assessment in hereditary ataxia: systematic review and consensus by the ataxia global initiative working group on digital-motor biomarkers

Oculomotor deficits are common in hereditary ataxia, but disproportionally neglected in clinical ataxia scales and as outcome measures for interventional trials. Quantitative assessment of oculomotor function has become increasingly available and thus applicable in multicenter trials and offers the opportunity to capture severity and progression of oculomotor impairment in a sensitive and reliable manner. In this consensus paper of the Ataxia Global Initiative Working Group On Digital Oculomotor Biomarkers, based on a systematic literature review, we propose harmonized methodology and measurement parameters for the quantitative assessment of oculomotor function in natural-history studies and clinical trials in hereditary ataxia. MEDLINE was searched for articles reporting on oculomotor/vestibular properties in ataxia patients and a study-tailored quality-assessment was performed. One-hundred-and-seventeen articles reporting on subjects with genetically confirmed (n=1134) or suspected hereditary ataxia (n=198), and degenerative ataxias with sporadic presentation (n=480) were included and subject to data extraction. Based on robust discrimination from controls, correlation with disease-severity, sensitivity to change, and feasibility in international multicenter settings as prerequisite for clinical trials, we prioritize a core-set of five eye-movement types: (i) pursuit eye movements, (ii) saccadic eye movements, (iii) fixation, (iv) eccentric gaze holding, and (v) rotational vestibulo-ocular reflex. We provide detailed guidelines for their acquisition, and recommendations on the quantitative parameters to extract. Limitations include low study quality, heterogeneity in patient populations, and lack of longitudinal studies. Standardization of quantitative oculomotor assessments will facilitate their implementation, interpretation, and validation in clinical trials, and ultimately advance our understanding of the evolution of oculomotor network dysfunction in hereditary ataxias

HST NIR Snapshot Survey of 3CR Radio Source Counterparts II: An Atlas and Inventory of the Host Galaxies, Mergers and Companions

We present the second part of an H-band (1.6 microns) atlas of z<0.3 3CR radio galaxies, using the Hubble Space Telescope Near Infrared Camera and Multi-Object Spectrometer (HST NICMOS2). We present new imaging for 21 recently acquired sources, and host galaxy modeling for the full sample of 101 (including 11 archival) -- an 87% completion rate. Two different modeling techniques are applied, following those adopted by the galaxy morphology and the quasar host galaxy communities. Results are compared, and found to be in excellent agreement, although the former breaks down in the case of strongly nucleated sources. Companion sources are tabulated, and the presence of mergers, tidal features, dust disks and jets are catalogued. The tables form a catalogue for those interested in the structural and morphological dust-free host galaxy properties of the 3CR sample, and for comparison with morphological studies of quiescent galaxies and quasar host galaxies. Host galaxy masses are estimated, and found to typically lie at around 2*10^11 solar masses. In general, the population is found to be consistent with the local population of quiescent elliptical galaxies, but with a longer tail to low Sersic index, mainly consisting of low-redshift (z<0.1) and low-radio-power (FR I) sources. A few unusually disky FR II host galaxies are picked out for further discussion. Nearby external sources are identified in the majority of our images, many of which we argue are likely to be companion galaxies or merger remnants. The reduced NICMOS data are now publicly available from our website (http://archive.stsci.edu/prepds/3cr/)Comment: ApJS, 177, 148: Final version; includes revised figures 1, 15b, and section 7.5 (and other minor changes from editing process. 65 pages, inc. 17 figure

Massage Therapy for Osteoarthritis of the Knee: A Randomized Dose-Finding Trial

In a previous trial of massage for osteoarthritis (OA) of the knee, we demonstrated feasibility, safety and possible efficacy, with benefits that persisted at least 8 weeks beyond treatment termination.We performed a RCT to identify the optimal dose of massage within an 8-week treatment regimen and to further examine durability of response. Participants were 125 adults with OA of the knee, randomized to one of four 8-week regimens of a standardized Swedish massage regimen (30 or 60 min weekly or biweekly) or to a Usual Care control. Outcomes included the Western Ontario and McMaster Universities Arthritis Index (WOMAC), visual analog pain scale, range of motion, and time to walk 50 feet, assessed at baseline, 8-, 16-, and 24-weeks.WOMAC Global scores improved significantly (24.0 points, 95% CI ranged from 15.3-32.7) in the 60-minute massage groups compared to Usual Care (6.3 points, 95% CI 0.1-12.8) at the primary endpoint of 8-weeks. WOMAC subscales of pain and functionality, as well as the visual analog pain scale also demonstrated significant improvements in the 60-minute doses compared to usual care. No significant differences were seen in range of motion at 8-weeks, and no significant effects were seen in any outcome measure at 24-weeks compared to usual care. A dose-response curve based on WOMAC Global scores shows increasing effect with greater total time of massage, but with a plateau at the 60-minute/week dose.Given the superior convenience of a once-weekly protocol, cost savings, and consistency with a typical real-world massage protocol, the 60-minute once weekly dose was determined to be optimal, establishing a standard for future trials.ClinicalTrials.gov NCT00970008