Henry W. Rohrs

An endogenous peptide positively selects and augments the activation and survival of peripheral CD4 + T cells

Although CD4+ and CD8+ T cells differ in their positively selecting signal strength, endogenous positively selecting ligands have only been identified for MHC class I-restricted T cell receptors (TCRs). Here we screened for ligands that can positively select MHC class II-restricted TCRs, using thymocytes from four I-Ek restricted TCR transgenic mice and a large panel of self peptides. One peptide, gp250, induced positive selection of AND CD4+ T cells, had no homology with the AND TCR agonist ligand, and was recognized with a high degree of specificity. gp250 acted as a co-agonist to initiate activation and enhance survival of peripheral AND CD4+ T cells. Thus, positively selecting ligands play critical roles in thymocyte development and in the activation and maintenance of peripheral T cells.Although CD4+ and CD8+ T cells differ in the strength of their positively selecting signal, endogenous positively selecting ligands have been identified only for major histocompatibility complex (MHC) class I–restricted T cell antigen receptors (TCRs). Here we screened for ligands able to positively select MHC class II–restricted TCRs using thymocytes from four I-Ek-restricted TCR-transgenic mice and a large panel of self peptides. One peptide, gp250, induced positive selection of AND CD4+ T cells, had no homology with the AND TCR agonist ligand and was recognized with a high degree of specificity. The gp250 peptide acted as a coagonist to initiate the activation and enhance the survival of peripheral AND CD4+ T cells. Thus, positively selecting ligands are critical in thymocyte development and in the activation and maintenance of peripheral T cells.

Top-down mass spectrometry: recent developments, applications and perspectives.

Top-down mass spectrometry is an emerging approach for the analysis of intact proteins. The term was coined as a contrast with the better-established, bottom-up strategy for analysis of peptide fragments derived from digestion, either enzymatically or chemically, of intact proteins. Although the term top-down originates from proteomics, it can also be applied to mass spectrometric analysis of intact large biomolecules that are constituents of protein assemblies or complexes. Traditionally, mass spectrometry has usually started with intact molecules, and in this regard, top-down approaches reflect the spirit of mass spectrometry. This article provides an overview of the methodologies in top-down mass spectrometry and then reviews applications covering protein posttranslational modifications, protein biophysics, DNAs/RNAs, and protein assemblies. Finally, challenges and future directions are discussed.

Isolation of the Magic-Size CdSe Nanoclusters [(CdSe)13(n-octylamine)13] and [(CdSe)13(oleylamine)13]†

The preparation, isolation, stoichiometric characterization, and dissolution of purified (CdSe)13 nanoclusters are described. We[1] and others[2] recently reported that (CdSe)13 nanoclusters were intermediates in the synthesis of CdSe quantum belts (nanoribbons). We now demonstrate that a lamellar intermediate phase[1] collected from the quantum-belt synthesis is [(CdSe)13(n-octylamine)13], the smallest, discrete, magic-size nanocluster of CdSe that has been observed.[3] Kinetic data show that free, soluble [(CdSe)13(oleylamine)13] nanoclusters are released from the insoluble [(CdSe)13(n-octylamine)13] upon ligand exchange.

Engineering nanorafts of calixarene polyphosphonates

The water-soluble calix[4]arene bearing p-substituted phosphonic acid groups is accessible in five steps in overall 62 % yield, with the hydrogen-bonding prowess of the acidic groups dominating its self-assembly processes. These include the formation of 3.0(3) nm and 20(2) nm nanorafts of the calixarene in water using spinning disc processing, stabilized by acetonitrile, and nanorafts in the gas phase (<or=20 molecules). The 20(2) nm particles transform into 3.0(3) nm particles prior to crystallization into a compact bilayer, whereas crystallization in the presence of large organic molecules gives an expanded bilayer interposed by layers of water molecules.

The Magic-Size Nanocluster (CdSe)34 as a Low-Temperature Nucleant for Cadmium Selenide Nanocrystals; Room-Temperature Growth of Crystalline Quantum Platelets

Reaction of Cd(OAc)2·2H2O and selenourea in primary-amine/secondary-amine cosolvent mixtures affords crystalline CdSe quantum platelets at room temperature. Their crystallinity is established by X-ray diffraction analysis (XRD), high-resolution transmission electron microscopy (TEM), and their sharp extinction and photoluminescence spectra. Reaction monitoring establishes the magic-size nanocluster (CdSe)34 to be a key intermediate in the growth process, which converts to CdSe quantum platelets by first-order kinetics with no induction period. The results are interpreted to indicate that the critical crystal-nucleus size for CdSe under these conditions is in the range of (CdSe)34 to (CdSe)68. The nanocluster is obtained in isolated form as [(CdSe)34(n-octylamine)16(di-n-pentylamine)2], which is proposed to function as crystal nuclei that may be stored in a bottle.

Charge carrier field emission determines the number of charges on native state proteins in electrospray ionization.

Although multiple charging in electrospray ionization (ESI) is essential to protein mass spectrometry, the underlying mechanism of multiple charging has not been explicated. Here, we present a new theory to describe ESI of native-state proteins and predict the number of excess charges on proteins in ESI. The theory proposes that proteins are ionized as charged residues in ESI, as they retain residual excess charges after solvent evaporation and do not desorb from charged ESI droplets. However, their charge state is not determined by the Rayleigh limit of a droplet of similar size to the protein; rather, their final charge state is determined by the electric field-induced emission of small charged solute ions and clusters from protein-containing ESI droplets. This theory predicts that the number of charges on a protein in ESI should be directly proportional to the square of the gas-phase protein diameter and to E*, the critical electric field strength at which ion emission from droplets occurs. This critical field strength is determined by the properties of the excess charge carriers (i.e., the solute) in droplets. Charge-state measurements of native-state proteins with molecular masses in the 5-76 kDa range in ammonium acetate and triethylammonium bicarbonate are in excellent agreement with theoretical predictions and strongly support the mechanism of protein ESI proposed here.

Revealing Novel Telomere Proteins Using in Vivo Cross-linking, Tandem Affinity Purification, and Label-free Quantitative LC-FTICR-MS

Telomeres are DNA-protein structures that protect chromosome ends from the actions of the DNA repair machinery. When telomeric integrity is compromised, genomic instability ensues. Considerable effort has focused on identification of telomere-binding proteins and elucidation of their functions. To date, protein identification has relied on classical immunoprecipitation and mass spectrometric approaches, primarily under conditions that favor isolation of proteins with strong or long lived interactions that are present at sufficient quantities to visualize by SDS-PAGE. To facilitate identification of low abundance and transiently associated telomere-binding proteins, we developed a novel approach that combines in vivo protein-protein cross-linking, tandem affinity purification, and stringent sequential endoprotease digestion. Peptides were identified by label-free comparative nano-LC-FTICR-MS. Here, we expressed an epitope-tagged telomere-binding protein and utilized a modified chromatin immunoprecipitation approach to cross-link associated proteins. The resulting immunoprecipitant contained telomeric DNA, establishing that this approach captures bona fide telomere binding complexes. To identify proteins present in the immunocaptured complexes, samples were reduced, alkylated, and digested with sequential endoprotease treatment. The resulting peptides were purified using a microscale porous graphite stationary phase and analyzed using nano-LC-FTICR-MS. Proteins enriched in cells expressing HA-FLAG-TIN2 were identified by label-free quantitative analysis of the FTICR mass spectra from different samples and ion trap tandem mass spectrometry followed by database searching. We identified all of the proteins that constitute the telomeric shelterin complex, thus validating the robustness of this approach. We also identified 62 novel telomere-binding proteins. These results demonstrate that DNA-bound protein complexes, including those present at low molar ratios, can be identified by this approach. The success of this approach will allow us to create a more complete understanding of telomere maintenance and have broad applicability.

A Single Peptide–MHC Complex Positively Selects a Diverse and Specific CD8 T Cell Repertoire

Goldilocks Immunology T cells are carefully calibrated in the thymus to react to invading pathogens and to ignore the self. This occurs through interactions between the T cell receptor and major histocompatibility complexes (MHCs) expressing self-peptides. A Goldilocks-like selection process is carried out whereby T cells that do not react or react too strongly to self-peptide MHCs are deleted, whereas those with interactions that are “just right” are allowed to survive. The result is T cells highly specific for a particular foreign peptide-MHC complex. Receipt of survival signals from “just-right” interactions (positive selection) and deletion of cells that are too reactive (negative selection) are spatially and temporally segregated in the thymus, and it is unclear at which stage T cells acquire their high degree of peptide-MHC specificity. By using mice expressing a single peptide-MHC complex, Wang et al. (p. 871) now show that this single complex is sufficient for selection of a CD8+ T cell repertoire with a broad range of specificity. Importantly, recognition of peptide MHC by these cells was highly specific, demonstrating that peptide-MHC specificity is acquired during positive selection in the thymus. Positive selection by a single peptide-MHC complex imparts exquisite specificity to developing T cells. Pathogen recognition by T cells is dependent on their exquisite specificity for self–major histocompatibility complex (MHC) molecules presenting a bound peptide. Although this specificity results from positive and negative selection of developing T cells in the thymus, the relative contribution of these two processes remains controversial. To address the relation between the selecting peptide-MHC complex and the specificity of mature T cells, we generated transgenic mice that express a single peptide–MHC class I complex. We demonstrate that positive selection of CD8 T cells in these mice results in an MHC-specific repertoire. Although selection on a single complex is peptide promiscuous, mature T cells are highly peptide specific. Thus, positive selection imparts MHC and peptide specificity on the peripheral CD8 T cell repertoire.

First Signature of Islet β-Cell-Derived Naturally Processed Peptides Selected by Diabetogenic Class II MHC Molecules

The diversity of Ags targeted by T cells in autoimmune diabetes is unknown. In this study, we identify and characterize a limited number of naturally processed peptides from pancreatic islet β-cells selected by diabetogenic I-Ag7 molecules of NOD mice. We used insulinomas transfected with the CIITA transactivator, which resulted in their expression of class II histocompatibility molecules and activation of diabetogenic CD4 T cells. Peptides bound to I-Ag7 were isolated and examined by mass spectrometry: some peptides derived from proteins present in secretory granules of endocrine cells, and a number were shared with cells of neuronal lineage. All proteins to which peptides were identified were expressed in β cells from normal islets. Peptides bound to I-Ag7 molecules contained the favorable binding motif characterized by acidic amino acids at the P9 position. The draining pancreatic lymph nodes of prediabetic NOD mice contained CD4 T cells that recognized three different natural peptides. Furthermore, four different peptides elicited CD4 T cells, substantiating the presence of such self-reactive T cells. The overall strategy of identifying natural peptides from islet β-cells opens up new avenues to evaluate the repertoire of self-reactive T cells and its role in onset of diabetes.

Bilayer, nanoimprint lithography

Nanoimprint lithography has been shown to be a viable means of patterning polymer films in the sub-100 nm range. In this work, we demonstrate the use of a bilayer resist to facilitate the metal liftoff step in imprinter fabrication. The bilayer resist technology exhibits more uniform patterns and fewer missing features than similar metal nanoparticle arrays fabricated with single layer resist. The bilayer resist relies upon the differential solubility between poly(methyl methacrylate) and poly(methyl methacrylate methacrylic acid copolymer). Evidence is presented that shows the technique has a resolution of better than 10 nm.