We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, 18O, specifically into the N-glycosylation site. The 18O-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.

A proteome of a model organism, Caenorhabditis elegans, was analyzed by an integrated liquid chromatography (LC)-based protein identification system, which was constructed by microscale two-dimensional liquid chromatography (2DLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. Soluble and insoluble protein fractions were prepared from a mixed growth phase culture of the worm C. elegans, digested with trypsin, and fractionated separately on the 2DLC system. The separated peptides were directly analyzed by on-line ESI-MS/MS in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database, wormpep 66, for protein identification. The total number of proteins of the composite proteome identified in this method was 1,616, including 110 secreted/targeted proteins and 242 transmembrane proteins. The codon adaptation indices of the identified proteins suggested that the system could identify proteins of relatively low abundance, which are difficult to identify by conventional 2D-gel electrophoresis (GE) followed by an offline mass spectrometric analysis such as peptide mass fingerprinting. Among the approximately 5,400 peptides assigned in this study, many peptides with post-translational modifications, such as N-terminal acetylation and phosphorylation, were detected. This expression profile of C. elegans, containing 571 hypothetical gene products, will serve as the basic data of a major proteome set expressed in the worm.

The authors propose and demonstrate the fabrication of InN∕GaN multiple quantum well (MQW) consisting of 1 ML and fractional monolayer InN well insertion in GaN matrix under In-polarity growth regime. Since the critical thickness of InN epitaxy on GaN is about 1 ML and the growth temperature for 1 ML InN insertion can be remarkably higher, the proposed MQW structure can avoid/reduce generation of misfit dislocation, resulting in higher quality MQW-structure nature in principle than former InN-based MQWs. The proposed InN∕GaN MQWs are potentially applicable to room temperature operating excitonic devices working in short-wavelength visible colors.

It has been suggested that γδ T cells are involved in certain autoimmune disorders. To establish reference data for clinical studies to explore the role of γδ T cells in autoimmune bone marrow failure syndrome, we examined the γδ T-cell repertoire in 120 healthy Japanese individuals by flow cytometry. The average numbers of T lymphocytes in blood were as follows: 1,084 ± 369 (SD) αβ T cells, 68 ± 44 γδ T cells, 16 ± 12 Vδ1 T cells, and 43 ± 36 Vδ2 T cells (/μl). Absolute numbers of γδ T cells decreased with aging (R = -0.378, P < 0.001). The decrease of γδ T cells was the result of reduction of Vδ2, but not of Vδ1, T cells. Numbers of Vδ2 T cells were significantly higher in male than in female donors (P = 0.007). The Vδ2 T cells but not Vδ1 T cells showed a rapid reduction in cell numbers on mitogen stimulation, which was accompanied by modest down-regulation of Bcl-2 protein expression. These results indicate that age and gender have a major impact on γδ T-cell repertoire in Japanese donors, as well as European and American donors. The age-related decrease of Vδ2 T cells may be explained by their susceptibility to activation-induced cell death.

Structural and optical characterization of InGaN/GaN multiple quantum wells grown by molecular beam epitaxyThe authors propose and demonstrate fine structure novel InN/GaN multiple quantum wells ͑MQWs͒ consisting of ultimately thin InN wells around 1 ML inserted in a GaN matrix grown under In-polarity growth regime by molecular beam epitaxy. Since the critical thickness of InN epitaxy on the c-plane GaN is about 1 ML and also the growth temperature for 1 ML InN insertion can be remarkably higher than conventional one, the proposed MQW structure can avoid new generation of misfit dislocation at the heterointerface, in principle, and results in high quality MQW structure due to the effects of enhanced surface migration at higher temperatures. It is shown that demonstrated 1 ML InN/GaN MQW structures indicate surprisingly higher structural quality/ properties than those former-reported InN-based heterostructures. Self-ordering mechanism arising from immiscibility nature in between InN and GaN will also contribute for depositing sharp and atomically flat InN well. The proposed MQW structure has physically and practically important meanings leading to room temperature operating GaN-based excitonic devices and also efficient photonic devices working in short wavelength visible colors.

We developed a multiplexed two-dimensional separation system based on reversed phase (RP)--strong cation exchange (SCX) chromatography as a front-end device for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization (nanoESI) mass spectrometry. Tryptic peptide mixtures were fractionated on a reversed-phase HPLC column, and each fraction was loaded onto multiplexed SCX microcolumns. Because this second chromatography was carried out in parallel, the analysis time is independent of the fraction number in the first RP-HPLC separation. The resultant samples were desalted/concentrated and eluted onto a MALDI plate with matrix-containing elution solutions in parallel, or eluted with optimized solutions for nanoESI and loaded onto nanoESI sprayers by an automated instrument. The soluble portion of HCT116 lysate was digested and fractionated using a 48-plexed chromatography system. Approximately 1000 unique peaks were detected in MALDI-MS with 3000 MS/MS spectra, while 724 peptides with ultrahigh peptide mass accuracy (sub-ppm error) were identified in nanoESI-FTICR mass spectrometry with five integrated selected ion monitoring scans. Since MS measurement with this off-line LC-LC approach is not restricted by continuous LC elution, it is expected to be useful especially in cases where repeated analysis with different scan modes or long-term data acquisition is required.

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