D. A. Schwartz

A proteomics approach to understanding protein ubiquitination.

There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.

An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets.

With the recent exponential increase in protein phosphorylation sites identified by mass spectrometry, a unique opportunity has arisen to understand the motifs surrounding such sites. Here we present an algorithm designed to extract motifs from large data sets of naturally occurring phosphorylation sites. The methodology relies on the intrinsic alignment of phospho-residues and the extraction of motifs through iterative comparison to a dynamic statistical background. Results show the identification of dozens of novel and known phosphorylation motifs from recently published serine, threonine and tyrosine phosphorylation studies. When applied to a linguistic data set to test the versatility of the approach, the algorithm successfully extracted hundreds of language motifs. This method, in addition to shedding light on the consensus sequences of identified and as yet unidentified kinases and modular protein domains, may also eventually be used as a tool to determine potential phosphorylation sites in proteins of interest.

Phosphoproteomic Analysis of the Developing Mouse Brain

Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.

Biological Sequence Motif Discovery Using motif‐x

The Web-based motif-x program provides a simple interface to extract statistically significant motifs from large data sets, such as MS/MS post-translational modification data and groups of proteins that share a common biological function. Users upload data files and download results using common Web browsers on essentially any Web-compatible computer. Once submitted, data analyses are performed rapidly on an associated high-speed computer cluster and they produce both syntactic and image-based motif results and statistics. The protocols presented demonstrate the use of motif-x in three common user scenarios.

Chandra Spectra of the Soft X-Ray Diffuse Background

We present an exploratory Chandra ACIS-S3 study of the diffuse component of the cosmic X-ray background (CXB) in the 0.3–7 keV band for four directions at high Galactic latitudes, with emphasis on details of the ACIS instrumental background modeling. Observations of the dark Moon are used to model the detector background. A comparison of the Moon data and the data obtained with ACIS stowed outside the focal area showed that the dark Moon does not emit significantly in our band. Point sources down to 3 � 10 � 16 ergs s � 1 cm � 2 in the 0.5–2 keV band are excluded in our two deepest observations. We estimate the contribution of fainter, undetected sources to be less than 20% of the remaining CXB flux in this band in all four pointings. In the 0.3–1 keV band, the diffuse signal varies strongly from field to field and contributes between 55% and 90% of the total CXB signal. It is dominated by emission lines that can be modeled by a kT ¼ 0:1 0:4 keV plasma. In particular, the two fields located away from bright Galactic features show a prominent line blend at E � 580 eV (O vii+O viii) and a possible line feature at E � 300 eV. The two pointings toward the North Polar Spur exhibit a brighter O blend and additional bright lines at 730–830 eV (Fe xvii). We measure the total 1–2 keV flux of 1:0 1:2 � 0:2 ðÞ �10 � 15 ergs s � 1 cm � 2 arcmin � 2 (mostly resolved) and the 2–7 keV flux of 4:0 4:5 � 1:5 ðÞ �10 � 15 ergs s � 1 cm � 2 arcmin � 2 .A tE > 2 keV, the diffuse emission is consistent with zero, to an accuracy limited by the short Moon exposure and systematic uncertainties of the S3 background. Assuming Galactic or local origin of the line emission, we put an upper limit of � 3 � 10 � 15 ergs s � 1 cm � 2 arcmin � 2 on the 0.3–1 keV extragalactic diffuse flux. Subject headings: intergalactic medium — ISM: general — methods: data analysis — X-rays: diffuse background — X-rays: ISM

DYX1C1 is required for axonemal dynein assembly and ciliary motility

DYX1C1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deleting exons 2–4 of Dyx1c1 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1 c.T2A start-codon mutation recovered from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also caused laterality and ciliary motility defects. In humans, we identified recessive loss-of-function DYX1C1 mutations in 12 individuals with PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA and IDA assembly factor DNAAF2 (KTU). Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4).

Large-Scale Phosphoprotein Analysis in Medicago truncatula Roots Provides Insight into in Vivo Kinase Activity in Legumes

Nitrogen fixation in legumes requires the development of root organs called nodules and their infection by symbiotic rhizobia. Over the last decade, Medicago truncatula has emerged as a major model plant for the analysis of plant-microbe symbioses and for addressing questions pertaining to legume biology. While the initiation of symbiosis and the development of nitrogen-fixing root nodules depend on the activation of a protein phosphorylation-mediated signal transduction cascade in response to symbiotic signals produced by the rhizobia, few sites of in vivo phosphorylation have previously been identified in M. truncatula. We have characterized sites of phosphorylation on proteins from M. truncatula roots, from both whole cell lysates and membrane-enriched fractions, using immobilized metal affinity chromatography and tandem mass spectrometry. Here, we report 3,457 unique phosphopeptides spanning 3,404 nonredundant sites of in vivo phosphorylation on 829 proteins in M. truncatula Jemalong A17 roots, identified using the complementary tandem mass spectrometry fragmentation methods electron transfer dissociation and collision-activated dissociation. With this being, to our knowledge, the first large-scale plant phosphoproteomic study to utilize electron transfer dissociation, analysis of the identified phosphorylation sites revealed phosphorylation motifs not previously observed in plants. Furthermore, several of the phosphorylation motifs, including LxKxxs and RxxSxxxs, have yet to be reported as kinase specificities for in vivo substrates in any species, to our knowledge. Multiple sites of phosphorylation were identified on several key proteins involved in initiating rhizobial symbiosis, including SICKLE, NUCLEOPORIN133, and INTERACTING PROTEIN OF DMI3. Finally, we used these data to create an open-access online database for M. truncatula phosphoproteomic data.

pLogo: a probabilistic approach to visualizing sequence motifs.

Methods for visualizing protein or nucleic acid motifs have traditionally relied upon residue frequencies to graphically scale character heights. We describe the pLogo, a motif visualization in which residue heights are scaled relative to their statistical significance. A pLogo generation tool is publicly available at http://plogo.uconn.edu/ and supports real-time conditional probability calculations and visualizations.

A CHANDRA SURVEY OF QUASAR JETS: FIRST RESULTS

We present results from Chandra X-ray imaging and spectroscopy of a flux-limited sample of flat-spectrum radio-emitting quasars with jetlike extended structure. Twelve of 20 quasar jets are detected in 5 ks ACIS-S exposures. The quasars without X-ray jets are not significantly different from those in the sample with detected jets except that the extended radio emission is generally fainter. New radio maps are combined with the X-ray images in order to elucidate the relation between radio and X-ray emission in spatially resolved structures. We find a variety of morphologies, including long straight jets and bends up to 90°. All X-ray jets are one-sided although the radio images used for source selection often show lobes opposite the X-ray jets. The FR II X-ray jets can all be interpreted as inverse Compton scattering of cosmic microwave background photons by electrons in large-scale relativistic jets although deeper observations are required to test this interpretation in detail. Applying this interpretation to the jets as a population, we find that the jets would be aligned to within 30° of the line of sight generally, assuming that the bulk Lorentz factor of the jets is 10.

Predicting Protein Post-translational Modifications Using Meta-analysis of Proteome Scale Data Sets

Protein post-translational modifications are an important biological regulatory mechanism, and the rate of their discovery using high throughput techniques is rapidly increasingly. To make use of this wealth of sequence data, we introduce a new general strategy designed to predict a variety of post-translational modifications in several organisms. We used the motif-x program to determine phosphorylation motifs in yeast, fly, mouse, and man and lysine acetylation motifs in man. These motifs were then scanned against proteomic sequence data using a newly developed tool called scan-x to globally predict other potential modification sites within these organisms. 10-fold cross-validation was used to determine the sensitivity and minimum specificity for each set of predictions, all of which showed improvement over other available tools for phosphoprediction. New motif discovery is a byproduct of this approach, and the phosphorylation motif analyses provide strong evidence of evolutionary conservation of both known and novel kinase motifs.