Stacked Josephson junction SQUID

Operation of a Superconducting Quantum Interference Device (SQUID) made of stacked Josephson junctions is analyzed numerically for a variety of junction parameters. Due to a magnetic coupling of junctions in the stack, such a SQUID has certain advantages as compared to an uncoupled multi-junction SQUID. Namely, metastability of current-flux modulation can be reduced and a voltage-flux modulation can be improved if junctions in the stack are phase-locked. Optimum operation of the SQUID is expected for moderately long, strongly coupled stacked Josephson junctions. A possibility of making a stacked Josephson junction SQUID based on intrinsic Josephson junctions in high-Tc superconductor is discussed.Comment: 4 pages, 3 figures, presented at SQUID-2001 (Stenungsbaden September 2001

How to Dig Deeper? Improved Enrichment Methods for Mucin Core-1 Type Glycopeptides

Two different workflows were tested in order to develop methods that provide deeper insight into the secreted O-glycoproteome. Bovine serum samples were subjected to lectin affinity-chromatography both at the protein- and peptide-level in order to selectively isolate glycopeptides with the most common, mucin core-1 sugar. This enrichment step was implemented with either protein-level mixed-bed ion-exchange chromatography or with peptide-level electrostatic repulsion hydrophilic interaction chromatography. Both methods led to at least 65% of the identified products being glycopeptides, in comparison to similar to 25% without the additional chromatography steps [Darula, Z., and Medzihradszky, K. F. (2009) Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum. Mol. Cell. Proteomics 8, 2515-2526]. In order to improve not only the isolation but also the characterization of the glycopeptides exoglycosidases were used to eliminate carbohydrate extensions from the directly peptide-bound GalNAc units. Consequent tandem MS analysis of the mixtures using higher-energy collision-dissociation and electron-transfer dissociation led to the identification of 124 glycosylation sites in 51 proteins. While the electron-transfer dissociation data provided the bulk of the information for both modified sequence and modification site assignment, the higher-energy collision-dissociation data frequently yielded confirmation of the peptide identity, and revealed the presence of some core-2 or core-3 oligosaccharides. More than two-thirds of the sites as well as the proteins have never been reported modified. Molecular & Cellular Proteomics 11: 10.1074/mcp.O111.016774, 1-10, 2012

Noise and conversion properties of Y-Ba-Cu-O Josephson mixers at operating temperatures above 20 K

We have measured the noise performance and conversion efficiency of Y-Ba-Cu-O bicrystal Josephson mixers at operating temperatures between 20 and 60 K and at operating frequencies around 90 GHz. A double-sideband mixer noise temperature of about 1600 K and a conversion efficiency of -10 dB at 20 K operating temperature has been measured using the Y-factor method. The absorbed local oscillator power was in the range of 10 nW. The dependence of the mixer performance on the normalized frequency Omega and the fluctuation parameter Gamma has been studied. In accordance with the resistively shunted junction model, the experimental data show the presence of excess noise. The temperature dependence of the mixer noise temperature can be explained by the variation of the linewidth of the Josephson oscillations with the operating temperature. (C) 2000 American Institute of Physics. [S0003-6951(00)00113-3]

Carbamidomethylation Side Reactions May Lead to Glycan Misassignments in Glycopeptide Analysis

Iodoacetamide is perhaps the most widely used reagent for the alkylation of free sulfhydryls in proteomic experiments. Here, we report that both incomplete derivatization of Cys side chains and overalkylation of the peptides may lead to the misassignment of glycoforms when LC-MS/MS with electron-transfer dissociation (ETD) alone is used for the structural characterization of glycopeptides. Accurate mass measurements do not help, because the elemental compositions of the misidentified and correct modifications are identical. Incorporation of "higher-energy C-trap dissociation" (HCD), i.e., beam-type collision-induced dissociation data into the database searches with ETD data may prove decisive in most cases. However, the carbamidomethylation of Met residues leads to sulfonium ether formation, and the resulting fixed positive charge triggers a characteristic fragmentation, that eliminates the normal Y, fragment from the HCD spectra of N-linked glycopeptides, producing an abundant Y-1-48 Da ion instead (the nominal mass difference is given relative to the unmodified amino acid sequence), that easily can be mistaken for the side chain loss from Met sulfoxide. In such cases, good quality ETD data may indicate the discrepancy, and will also display abundant fragments due to CH3-S-CH2CONH2 elimination from the charge-reduced precursor ions. Our observations also draw attention to the underreported interference of different unanticipated covalent modifications

Módszerfejlesztés fehérje-glikoziláció analízisére = Method development for protein glycosylation analysis

Az extracelluláris glikoziláció tanulmányozása némiképp elhanyagolt kutatási terület. Ennek az egyik oka az elképesztő heterogenitás: egy adott pozíció hol módosított, hol nem, és számtalan különböző cukor-szerkezetet viselhet, így a glikopeptidek többnyire szubsztöchiometrikus mennyiségben fordulnak elő. Ráadásul a poszt-transzlációs módosítások vizsgálatára általában használatos tömegspektrometria is nehezebben boldogul a glikopeptidekkel. Mi szérum-fehérjék Ser és Thr oldalláncát módosító gyakori és egyszerű cukrok vizsgálatára fókuszáltunk. Marhaszérummal dolgoztunk. Egy dúsítási eljárást dolgoztunk ki egy cukorkötő-fehérje (lektin) segítségével. A glikopeptid-elegyet egy új MS/MS technika: elektron-transzfer disszociáció (ETD) segítségével analizáltuk. Ennek sikeréhez az adatbázis-lekereső szoftvert is optimalizálni kellett az ETD adatokhoz. Kutatásunk során kb. 40 új glikozilációs helyet azonosítottunk. Ennyit eddig senkinek nem sikerült egyetlen kísérlet-sorozatból. Sejten belül is előfordul O-glikoziláció, egyetlen GlcNAc kerül a Ser/Thr oldalláncokra, regulációs és jelátviteli szerepe van. Bár biológiai szempontból nagyon eltér az extracelluláris rokonságtól, hasonló analitikai kihívást jelent. Erre a módosításra is kidolgoztunk egy dúsítási eljárást. | Studying extracellular glycosylation is a somewhat neglected research area. Partly because the incredible heterogeneity glycoproteins feature both in site occupancy and in the number of different sugar structures modifying the same site. Thus, glycopeptides almost always are present in substoichiometric quantities. In addition, these modifications are a bit difficult to tackle with mass spectrometry that is generally used for the analysis of post-translational modifications. We focused on some simple and frequently occurring sugars modifying the side-chains of Ser and Thr residues of serum proteins. We worked with bovine serum. We developed an enrichment method using a carbohydrate-binding protein (lectin). We characterized the glycopeptide mixtures utilizing a novel MS/MS technique, electron-transfer dissociation (ETD). For this purpose the softwer used for database searching also had to be optimized. We identified ~40 novel glycosylation sites, more than anybody ever assigned in a single study. O-glycosylation occurs within the cell too: a single GlcNAc is deposited on Ser/Thr side chains. It fulfills a regulatory, signaling function. Though biologically very distant from its extracellular relatives, it represents a similar analytical challenge. We developed an enrichment method for this modification too