Same as for Sample (Trx-treated) except IAM addition after thioredoxin incubation was omitted.
Growth protocol
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).
Extracted molecule
protein
Extraction protocol
na
Label
na
Label protocol
na
Hybridization protocol
na
Scan protocol
na
Description
Sample preparation: Barley embryo protein extract, treated with iodoacetamide (IAM) during extraction, was incubated ± thioredoxin for 1 h at RT. 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. 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.
Data processing
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.
Identification of targets of the protein disulfide reductase thioredoxin
Data table header descriptions
ID_REF
peptide sequence
VALUE
The average ICATH/ICATL ratio as calculated in the Msquant software version 1.4.3a12 (http://www.msquant.sourceforge.net)
Score
Mascot peptide score
MCR (Mass charge ratio) [Th]
The experimentally determined mass/charge ratio
Charge
The peptide charge
Measured mass [Da]
The peptide mass calculated based on column I and J
Mascot calculated mass [Da]
The theoretical mass of the peptide as calculated by the mascot search engine
Uncalibrated mass relative error [ppm]
The error between the measured mass and the theoretical mass
Calibrated mass relative error [ppm]
The error betweeen the measured mass and the theoretical mass after calibration in the software Msquant version 1.4.3a12 (http://www.msquant.sourceforge.net)
