Abstract
Cadmium (Cd(2+)) is one of well-known toxic heavy metal ions. To gain a global understanding how Cd(2+) affects cells at the molecular level, we systematically studied the cellular response of the fission yeast Schizosaccharomyces pombe to Cd(2+) using our integrated proteomic strategy of amino acid-coded mass tagging (AACT) and liquid chromatography-tandem mass spectrometry. Our proteome-wide investigation unequivocally identified 1133 S. pombe proteins. Of which, the AACT-based quantitative analysis revealed 106 up-regulated and 55 down-regulated proteins on the Cd(2+) exposure. The most prevalent functional class in the up-regulated proteins, approximately 28% of our profile, was the proteins involved in protein biosynthesis, showing a time-dependent biphasic expression pattern characteristic with rapid initial induction and later repression. Most significantly, 27 proteins functionally classified as cell rescue and defense were up-regulated for oxygen and radical detoxification, heat shock response, and other stress response. Furthermore, the large precursor sequence coverage of our AACT approach allowed us to unequivocally identify and quantitate different isozymes for glutathione S-transferase, which have close similarity in their amino acid sequence. Our quantitative dataset also showed that 80% of the up-regulated proteins found in the S. pombe response were different from those in the Saccharomyces cerevisiae response. The function of some of the key identifications was validated through biochemical assays. It is very interesting that the induction of cysteine synthase expression was not observed in our study, although it has been proven as a critical enzyme to supply free cysteines for the enhancing synthesis of Cd(2+)-sequestering molecules such as glutathione and phytochelatins in plants and some yeasts. Our quantitative proteomic result instead suggested that, as an alternative mechanism for the detoxification of Cd(2+), S. pombe produced significantly higher level of inorganic sulfide to immobilize cellular Cd(2+) as a form of CdS nanocrystallites capped with glutathione and/or phytochelatins.
Highlights
Cadmium (Cd2؉) is one of well-known toxic heavy metal ions
Large-scale Identification and Quantification for the Proteins Sensitive to Cd2ϩ Exposure—The integrated strategy of amino acid-coded mass tagging (AACT) and LC-mass spectrometry (MS)/MS was used to elucidate the cellular responses to Cd2ϩ in the fission yeast S. pombe at the proteomic level (Scheme 1)
Our experiments discovered that all proteins functionally classified as oxygen and radical detoxification were significantly up-regulated, including both S. pombe ortholog proteins of the above proteins identified in S. cerevisiae and catalase, glutaredoxin, superoxide dismutase [Cu-Zn], and thioredoxin reductase (Table II)
Summary
Reagents and Yeast Strain—The deuterium-enriched amino acid precursor, L-leucine-5,5,5-d3-98% (Leu-d3), was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). Because we used Leu-d3 in cell culture for amino acid-specific mass tagging, those peptides containing leucine were further analyzed for the quantification of differentially expressed proteins. This process can validate the database search results For those Leu-d3-containing peptides satisfying both criteria, the quantitative results for their corresponding proteins were obtained by examining the peptide mass map of certain precursor ions on chromatographic profiles, and the induction ratios of proteins were obtained both by measuring the mono-isotopic peak intensity of unlabeled (light) and Leu-d3-labeled (heavy) peptides and by comparing the integral peak areas of the pairs. A total of 250 l of the supernatant was used for assaying the synthesized cysteine by a ninhydrin method [36]
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