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      • Using size exclusion chromatography-RPLC and RPLC-CIEF as two-dimensional separation strategies for protein profiling

        Simpson, David C.,Ahn, Seonghee,Pasa-Tolic, Ljiljana,Bogdanov, Bogdan,Mottaz, Heather M.,Vilkov, Andrey N.,Anderson, Gordon A.,Lipton, Mary S.,Smith, Richard D. WILEY-VCH Verlag 2006 Electrophoresis Vol.27 No.13

        <P>Bottom-up proteomics (analyzing peptides that result from protein digestion) has demonstrated capability for broad proteome coverage and good throughput. However, due to incomplete sequence coverage, this approach is not ideally suited to the study of modified proteins. The modification complement of a protein can best be elucidated by analyzing the intact protein. 2-DE, typically coupled with the analysis of peptides that result from in-gel digestion, is the most frequently applied protein separation technique in MS-based proteomics. As an alternative, numerous column-based liquid phase techniques, which are generally more amenable to automation, are being investigated. In this work, the combination of size-exclusion chromatography (SEC) fractionation with RPLC-Fourier-transform ion cyclotron resonance (FTICR)-MS is compared with the combination of RPLC fractionation with CIEF-FTICR-MS for the analysis of the Shewanella oneidensis proteome. SEC-RPLC-FTICR-MS allowed the detection of 297 proteins, as opposed to 166 using RPLC-CIEF-FTICR-MS, indicating that approaches based on LC-MS provide better coverage. However, there were significant differences in the sets of proteins detected and both approaches provide a basis for accurately quantifying changes in protein and modified protein abundances.</P>

      • SCISCIESCOPUS

        Overview of the HUPO Plasma Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database

        Omenn, Gilbert ,S.,States, David,J.,Adamski, Marcin,Blackwell, Thomas ,W.,Menon, Rajasree,Hermjakob, Henning,Apweiler, Rolf,Haab, Brian ,B.,Simpson, Richard ,J.,Eddes, J WILEY-VCH 2005 PROTEOMICS -WEINHEIM- Vol.5 No.13

        <P>HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anti-coagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics.med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay-based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs.</P><P>We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches.</P><P>This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan. These PPP results on complexity, dynamic range, incomplete sampling, false-positive matches, and integration of diverse datasets for plasma and serum proteins lay a foundation for development and validation of circulating protein biomarkers in health and disease.</P>

      • KCI우수등재

        진공표준의 국제비교 연구

        홍승수(Seung-Soo Hong),신용현(Yong-Hyeon Shin),임종연(Jong-Yeon Lim),이상균(Sang-Kyun Lee),정광화(Kwang-Hwa Chung),David Simpson,Fiona Redgrave,Michael Perkin,Bernard Waller,M. Hirata$ 한국진공학회(ASCT) 1997 Applied Science and Convergence Technology Vol.6 No.4

        Capacitance Diaphragm Gauge(CDG)를 전달표준기로 사용하여 한국과 영국의 저진공표준의 국제 비교를 수행한 결과 양국 표준기에서 교정한 CDG의 압력비의 차이는 0.05% 이었다. 또한 두 개의 Spinning Rotor Gauge(SRG)를 이용하여 한국과 일본의 고진공표준의 국제비교를 수행한 결과 양국 표준기에서 교정한 SRG의 accommodation coefficient(σ)의 차이가 0.4% 이내이었다. CDG와 SRG를 이용한 한국과 영국의 저진공표준기 및 한국과 일본의 고진공표준기의 국제비교 결과 CDG 및 SRG 자체의 고유오차범위 이내에서 결과가 일치함을 확인할 수 있었다. The international intercomparison of low vacuum standards between Korea and United Kingdom was performed using a Capacitance Diaphragm Gauge (CDG) as a transfer standards. The resultant difference in pressure of the CDG was 0.05%. Similialy, the difference in accommodation coefficient of a spinning rotor gauge (SRG) between Japan and Korea was verified within 0.4% for high vacuum standards. The intercomparisons of low and high vaccum standards of Korea, United Kingdom, and Japan using a CDG and SRG show that the vacuum standards agree within the inherent uncertainty limits.

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