Abstract
Protein expression profiles in yeast cells, in response to salinity stress, were determined using the cleavable isotope-coded affinity tag (cICAT) labeling strategy. The analysis included separation of the mixed protein samples by SDS-PAGE, followed by excision of the entire gel lane, and division of the lane into 14 gel regions. Regions were subjected to in-gel digestion, biotin affinity chromatography, and analysis by nano-scale microcapillary liquid chromatography coupled to tandem mass spectrometry. The novel (13)C-labeled ICAT reagents have identical elution profiles for labeled peptide pairs and broadly spread the distribution of labeled peptides during reversed-phase chromatography. A total of 560 proteins were identified and quantified, with 51 displaying more than 2-fold expression differences. In addition to some known proteins involved in salt stress, four RNA-binding proteins were found to be up-regulated by high salinity, suggesting that selective RNA export from the nucleus is important for the salt-stress response. Some proteins involved in amino acid synthesis, which have been observed to be up-regulated by amino acid starvation, were also found to increase their abundance on salt stress. These results indicate that salt stress and amino acid starvation cause overlapping cellular responses and are likely to be physiologically linked.
Highlights
Protein expression profiles in yeast cells, in response to salinity stress, were determined using the cleavable isotope-coded affinity tag labeling strategy
The isotope-coded affinity tags (ICAT) reagent consists of three components: (i) a reactive group that reacts with the free thiol functionality of cysteine residues; (ii) a linker in which stable isotopes have been incorporated; and (iii) a biotin tag that makes possible affinity isolation and detection of peptides labeled with either the heavy or light versions of the ICAT reagent
This study presents the use of cleavable (c)ICAT reagents for the large-scale quantitative profiling of protein expression after salinity stress
Summary
Preparation of Proteins for ICAT Labeling—Yeast cells (BJ5459, MATa ura trp lys 801 leu pep4::HIS3 prb11.6R canl) were grown in yeast extract/peptone/dextrose medium at 30 °C to an OD600 of approximately 0.8. The combined supernatants were concentrated to approximately 100 l using a SpeedVac. Affinity Purification and Cleavage of ICAT-labeled Peptides—To affinity-purify the ICAT-labeled peptides, 30 l of 25% acetonitrile, 5 mM KH2PO4, and 350 mM KCl (pH 2.7) and 500 l of Affinity Load Buffer (pH 7.2) (Applied Biosystems) were added to the tryptic digests prior to loading the ICAT-labeled peptides onto an avidin cartridge (Applied Biosystems) following the manufacture’s protocol. The eluate from the avidin cartridge was completely dried in a SpeedVac. Identification and Quantitation of Proteins by Mass Spectrometry— The dried ICAT-labeled peptides were dissolved in a solution containing 5% acetonitrile, 0.4% acetic acid, 0.005% heptafluorobutyric acid, and 5% formic acid and pressure-loaded onto a fused silica microcapillary (100-m ID ϫ 10 cm) reversed-phase column packed, in the laboratory, with Magic C18 beads (Michrom BioResources, Auburn, CA). The data were searched against the yeast protein database followed by manual interpretation of all MS/MS spectra from proteins with changes greater than 2-fold
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