Liquid Chromatography Electron Capture Dissociation Tandem Mass Spectrometry (LC-ECD-MS/MS) versus Liquid Chromatography Collision-induced Dissociation Tandem Mass Spectrometry (LC-CID-MS/MS) for the Identification of Proteins

Electron capture dissociation (ECD) offers many advantages over the more traditional fragmentation techniques for the analysis of peptides and proteins, although the question remains: How suitable is ECD for incorporation within proteomic strategies for the identification of proteins? Here, we compare LC-ECD-MS/MS and LC-CID-MS/MS as techniques for the identification of proteins.Experiments were performed on a hybrid linear ion trap–Fourier transform ion cyclotron resonance mass spectrometer. Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin,lysozyme, cytochrome c, alcohol dehydrogenase, and β-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides. The results show that although protein coverage was lower for LC-ECDMS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags,providing greater confidence in protein assignment

Electron Capture Dissociation Mass Spectrometry of Tyrosine Nitrated Peptides

In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification.Here, we have investigated the electron capture dissociation (ECD) and collision-induced association (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains

Activated Ion Electron Capture Dissociation (AI ECD) of proteins: synchronization of infrared and electron irradiation with ion magnetron motion.

Here, we show that to perform activated ion electron capture dissociation (AI-ECD) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with a CO(2) laser, it is necessary to synchronize both infrared irradiation and electron capture dissociation with ion magnetron motion. This requirement is essential for instruments in which the infrared laser is angled off-axis, such as the Thermo Finnigan LTQ FT. Generally, the electron irradiation time required for proteins is much shorter (ms) than that required for peptides (tens of ms), and the modulation of ECD, AI ECD, and infrared multiphoton dissociation (IRMPD) with ion magnetron motion is more pronounced. We have optimized AI ECD for ubiquitin, cytochrome c, and myoglobin; however the results can be extended to other proteins. We demonstrate that pre-ECD and post-ECD activation are physically different and display different kinetics. We also demonstrate how, by use of appropriate AI ECD time sequences and normalization, the kinetics of protein gas-phase refolding can be deconvoluted from the diffusion of the ion cloud and measured on the time scale longer than the period of ion magnetron motion

Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex

Background: Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. Methodology/Principal Findings: We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Conclusions/Significance: Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells

Universal Rights and Wrongs

This paper argues for the important role of customers as a source of competitive advantage and firm growth, an issue which has been largely neglected in the resource-based view of the firm. It conceptualizes Penrose’s (1959) notion of an ‘inside track’ and illustrates how in-depth knowledge about established customers combines with joint problem-solving activities and the rapid assimilation of new and previously unexploited skills and resources. It is suggested that the inside track represents a distinct and perhaps underestimated way of generating rents and securing long-term growth. This also implies that the sources of sustainable competitive advantage in important respects can be sought in idiosyncratic interfirm relationships rather than within the firm itself

Regional Adoption of Business-to-Business Electronic Commerce in China: Role of E-Readiness

Adoption of B2B e-commerce is a powerful driver of economic success in developed and developing countries. However, adoption rates in developing countries lag far behind. This paper draws on the Perceived eReadiness Model and research on the influence of inter-organizational relationships and economic-cultural contexts to explain the importance of three factors—inter-organizational power dependence, cooperativeness, and regional economic-cultural differences—for achieving higher levels of Internet-based Electronic Data Interchange (EDI) in the developing country of China. We employ survey data to empirically test both the individual and joint influence of these factors. The findings suggest that beyond intra-organizational and external factors, managers and policy makers wanting to promote Internet-based EDI adoption in developing countries must also account for the inter-organizational relationships of firms and the economic and cultural circumstances of the regions in which they operate

Die "Roma-Frage" in Frankreich und Europa: Dekonstruktion eines Klischees

In der öffentlichen Wahrnehmung vieler europäischer Länder gelten Roma immer wieder als eine spezielle Bevölkerungsgruppe, die spezifische Probleme verursacht. Am Beispiel der Gruppe der "Roma-Migranten" in Frankreich fällt auf, dass ein Großteil der Schwierigkeiten, mit denen diese Einwanderergruppe konfrontiert ist, damit zusammenhängt, welche Wahrnehmungen und Klischees von "Roma" in Politik, Institutionen und Medien vorherrschen. Der Begriff "Roma-Migranten" ist noch relativ jung und umschreibt verschiedene familiäre Gruppen aus Zentraleuropa und vom Balkan, die infolge des Zusammenbruchs der kommunistischen Regime emigriert sind. In Frankreich umfasst diese Gruppe von Einwanderern 15.000 bis 20.000 Personen vorwiegend aus Rumänien. Sie leben zumeist in temporären, nicht genehmigten Siedlungen in den Vorstädten französischer Großstädte. Die prekären Lebensumstände sind allerdings nicht auf eine in irgendeiner Art ethnisch-kulturell zu begründenden Präferenz eines "fahrenden Volkes" zurückzuführen, sondern auf Mechanismen der Ausgrenzung, die politisch, juristisch und gesellschaftlich begründet sind

Ratings of age of acquisition of 299 words across 25 languages: Is there a cross-linguistic order of words?

We present a new set of subjective age-of-acquisition (AoA) ratings for 299 words (158 nouns, 141 verbs) in 25 languages from five language families (Afro-Asiatic: Semitic languages; Altaic: one Turkic language: Indo-European: Baltic, Celtic, Germanic, Hellenic, Slavic, and Romance languages; Niger-Congo: one Bantu language; Uralic: Finnic and Ugric languages). Adult native speakers reported the age at which they had learned each word. We present a comparison of the AoA ratings across all languages by contrasting them in pairs. This comparison shows a consistency in the orders of ratings across the 25 languages. The data were then analyzed (1) to ascertain how the demographic characteristics of the participants influenced AoA estimations and (2) to assess differences caused by the exact form of the target question (when did you learn vs. when do children learn this word); (3) to compare the ratings obtained in our study to those of previous studies; and (4) to assess the validity of our study by comparison with quasi-objective AoA norms derived from the MacArthur–Bates Communicative Development Inventories (MB-CDI). All 299 words were judged as being acquired early (mostly before the age of 6 years). AoA ratings were associated with the raters’ social or language status, but not with the raters’ age or education. Parents reported words as being learned earlier, and bilinguals reported learning them later. Estimations of the age at which children learn the words revealed significantly lower ratings of AoA. Finally, comparisons with previous AoA and MB-CDI norms support the validity of the present estimations. Our AoA ratings are available for research or other purposes