Genomics

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63

Identification of targets of the protein disulfide reductase thioredoxin


ABSTRACT: Identification of targets of the protein disulfide reductase thioredoxin using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and thiol specific differential labeling with isotope-coded affinity tags (ICAT). Reduction of specific target disulfides is quantified by measuring ratios of cysteine residues labeled with the ?heavy? (13C) and ?light? (12C) ICAT reagents in peptides derived from tryptic digests of Trx-treated and non-treated samples. Keywords: protein, LC-MS/MS, ICAT Overall design: Biological Material: Dissected embryos from seeds from the malting barley (Hordeum vulgare) cultivar Barke (2002 harvest) germinated for 48 h as previously described (Bønsager et al., 2007). Sample preparation: Sample A. Barley embryo protein extract, treated with iodoacetamide (IAM) during extraction, was incubated ± thioredoxin and after 1 h incubation at RT, IAM was again added (final concentration 10 mM) to irreversibly block cysteines reduced by thioredoxin. Excess IAM was removed and remaining cysteine residues were completely reduced by tris(2-carboxyethyl)phosphine (TCEP) under denaturing conditions and ICAT-labeled (+Trx, ICATL; -Trx, ICATH), essentially as recommended by the manufacturer (Applied Biosystems). The ICATL and ICATH labeled samples were mixed 1:1 and subjected to trypsin digestion followed by isolated of ICAT labeled peptides by avidin affinity chromatography and LC-MS/MS analysis on an Agilent 1100 nanoflow HPLC (Agilent, Palo Alto CA) coupled to a QSTAR quadrupole time-of-flight mass spectrometer (Applied Biosystems). Data were acquired in information dependent mode with a cycle of a survey mass spectrum followed by tandem mass spectra of the three most intense multiply charged ions (1 s each) after which the selected ions were placed on an exclusion list for 45 s. Sample B (control). Same as for sample A except IAM addition after thioredoxin incubation was omitted. Data processing and peptide quantification: The raw data .wiff files were processed with the Mascot script for Analyst (QS 2.0) version 1.6b20 (Matrix Science, London, UK). The charge state was determined from the survey spectrum with a default charge setting of multiply charged precursors. The MS/MS data was centroided and de-isotoped without MS/MS averaging to generate the Mascot Generic Format (MGF) peak list. Spectra with less than 10 peaks were rejected. Database searches of the centroided data were performed using the Mascot search engine (Matrix Science) against the Viridiplantae (Green Plants) subset of Swiss-Prot 54.6, nrNCBI (downloaded on 20071206) and the Barley EST database (barley gene index [HvGI] release 9.0). The following search criteria were used: MS mass tolerance 0.2 Da; MS/MS tolerance 0.2 Da; no trypsin miss-cleavage allowed. Variable modifications: ICATH (cysteine), ICATL (cysteine), carbamidomethyl (cysteine) and oxidation (methionine). Tentative consensus sequences (TC sequences) identified in HvGI release 9.0 were blasted (http://www.ncbi.nlm.nih.gov/blast) against the nrNCBI database. Quantification of ICAT labeled peptide pairs was performed using the software MS quant version 1.4.3a12 (http://www.msquant.sourceforge.net). Spectra were manually validated and peptides with a sequence tag of at least three amino acids and a Mascot score of 20 were accepted. References: Bønsager BC, Finnie C, Roepstorff P, Svensson B (2007) Spatio-temporal changes in germination and radical elongation of barley seeds tracked by proteome analysis of dissected embryo, aleurone layer, and endosperm tissues. Proteomics 7:4528-4540.

INSTRUMENT(S): Identification of targets of the protein disulfide reductase thioredoxin

SUBMITTER: Per Hagglund  

PROVIDER: GSE11650 | GEO | 2008-11-20

SECONDARY ACCESSION(S): PRJNA106161

REPOSITORIES: GEO

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Publications

Identification of thioredoxin disulfide targets using a quantitative proteomics approach based on isotope-coded affinity tags.

Hägglund Per P   Bunkenborg Jakob J   Maeda Kenji K   Svensson Birte B  

Journal of proteome research 20081201 12


Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific diffe  ...[more]

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