负责人 英语怎么说1 Title: Relative protein quantification by isobaric SILAC with immonium ion splitting (ISIS)
Mara Colzani1, Frédéric Schütz2, Alexandra Potts1, Patrice Waridel1 and Manfredo Quadroni1
1 Center for Integrative Genomics, University of Lausanne, Quartier Sorge, 1015 Lausanne-Dorigny, Switzerland
2 Bioinformatics Core Facility, Swiss Institute of Bioinformatics, Quartier Sorge, 1015 Lausanne, Switzerland
Correspondence:
Mara Colzani
Telephone: +41 21 692 56 76
nceFax: +41 21 692 57 05
吸血鬼日记第四季14
E-mail: lzani@unil.ch
Manfredo Quadronieverymomentofmylife
Telephone: +41 21 692 56 76
Fax: +41 21 692 57 05principal
E-mail: manfredo.quadroni@unil.ch
MCP Papers in Press. Published on December 28, 2007 as Manuscript M700440-MCP200 Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc. by on April 17, ponline
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Running Title: Isobaric SILAC with immonium ion splitting (ISIS)
by on April 17, 2008
雅虎通英文版
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Abbreviations
SILAC
stable isotope labelling with amino acids in cell cultures mmu
milli-mass units PTM
post translational modifications iTRAQ isobaric tags for relative and absolute quantitation
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道歉的英文Summary
Metabolic labelling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical Stable Isotope Labelling with Amino acids in Cell cultures (SILAC) employs pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated and their intensities ud for mass spectrometry-bad relative protein quantification.
We prent an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labelled either with 13C on the carbonyl (C1) carbon or 15N on backbone nitrogen. Labelled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra, but generate upon fragmentation distinct immonium ions parated by 1 u. When labelled protein samples are mixed, the intensities of the immonium
macron
ions can be ud for the relative quantification of the parent proteins. We validated the labelling of cellular proteins with valine, isoleucine and leucine, with coverage of 97% of all tryptic peptides. We improved the nsitivity for the detection of the quantification ions on a pulsing instrument by employing a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally, the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with tho obtained by a classical 2D-DIGE
analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labelling schemes like iTRAQ. We discuss advantages and disadvantages of ISIS technique as well as possible ways to improve it. by on April 17, ponline
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Introduction
Methods for quantitative proteomics bad on stable isotope labelling have become in the last decade very powerful tools to investigate cellular process [1, 2, 3]. The main applications of such methods have been the analysis of changes in protein expression [4] as well as the elucidation of networks of molecular (protein-protein [5] or DNA-protein) interactions or the study of post translational modifications (PTM’s) [6, 7]. Isotope labelling methods can be classified in two broad class. Tho bad on residue-specific chemical derivatization with labelled reagents have the great advantage of offering greater flexibility in the choice of the chemistry and are universally applicable. However they can suffer from the complexity of the steps involved and from the risk of side-reactions. Metabolic labelling approaches, on the other hand, are only possible for organisms w
ho cells can be cultured in strictly controlled conditions. They offer however the advantage that no additional labelling steps are involved and
that the proteins maintain all their native properties. Such techniques are especially attractive when complex biochemical purifications are necessary to obtain the samples to be studied, since extracts of cells can be mixed at the very beginning of the procedure, thereby eliminating artefacts due to slight variations in the purification conditions for the different samples.
配方英语Many chemical labelling schemes have been devid. In turn, they can be subdivided in two groups bad on the approaches needed for quantification. Most techniques rely on measuring the intensity of light/heavy parent ions in MS survey scans to establish intensity ratios. One technical issue with such an approach is the need to establish an unambiguous correlation (bad on m/z and retention times in liquid chromatography) between tandem MS spectra ud for by on April 17, ponline
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agenda
identification and their precursor peaks in MS survey scans. ICAT [8] and a multitude of other labelling schemes [9, 10, 11, 12] belong to this class.
The cond group is formed by techniques that exploit quantitative information embedded in tandem MS spectra rather than in survey scans [13, 14]. Earlier examples of this principle ud protea-mediated 16O /18O labelling [15, 16, 17] . Such approaches require however co-fragmentation of the precursors and this necessitates very clo or identical mass, such as in the iTRAQ scheme. The latter is a special ca that employs isobaric tagging reagents which fragment to give, for every peptide, diagnostic low mass ions ud for quantification. Compared with methods with distinct mass precursors, the techniques prent the advantages that no precursor mass splitting results in higher signal intensity and data handling is easier since only MS/MS data are needed, thus eliminating the need to integrate peaks along chromatographic runs. Appropriate design of the reagent has allowed to vary reporter ion mass and this has been ud to implement 4- and 8-fold multiplexing workflows.
By contrast, classical SILAC [18] is a metabolic labelling method similar, from the point of view of the quantification procedure, to non-isobaric precursor methods. In order to achieve accurate quantification, it needs high resolution mass data with low noi levels, together with a powerful soft
ware able to correlate MS/MS identifications with full scan MS data and extract integrated ion intensities for the precursors and cognate, but often unidentified labelled analogues.
We propo an alternative SILAC scheme that combines some uful features of iTRAQ-like workflows with the convenience of SILAC for studying samples from cultured cells. The technique is bad on the incorporation of isobaric labelled amino acids which generate distinct residue immonium ion fragments. We show that reliable quantification is possible purely on
MS/MS spectra, using very simple data extraction tools. by on April 17, ponline
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