Abstract：
:Reversible thiol oxidation of cysteine residues occurs in many intracellular catalytic and signaling processes. Here we describe an optimized protocol, which can be completed in ∼5 d, to unambiguously identify specific cysteine residues that are transiently and reversibly oxidized by comparing two complex biological samples obtained from yeast cell cultures at the proteome level. After 'freezing' the in vivo thiol stage of cysteine residues by medium acidification, we first block reduced thiols in extracts with iodoacetamide (IAM), and then we sequentially reduce and label reversible oxidized thiols with the biotin-based heavy or light IAM derivatives, which are known as isotope-coded affinity tag (ICAT) reagents, so that the two samples can be compared at once after combination of the labeled extracts, trypsin digestion, streptavidin-affinity purification of peptides containing oxidized cysteines, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. For the same protein extracts, before cysteine-containing peptide enrichment, individual relative protein concentrations are obtained by stable-isotope dimethyl labeling.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
García-Santamarina S,Boronat S,Domènech A,Ayté J,Molina H,Hidalgo Edoi
10.1038/nprot.2014.065subject
Has Abstractpub_date
2014-05-01 00:00:00pages
1131-45issue
5eissn
1754-2189issn
1750-2799pii
nprot.2014.065journal_volume
9pub_type
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