Журнал протеомики и биоинформатики
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Development of Deuterated-Leucine Labeling with Immunoprecipitation to Analyze Cellular Protein Complex

Shufang Liang, Xuejiao Xu, Haojie Lu and Pengyuan Yang

The deuterated-leucine (Leu-d 3 ) labeling is one kind of stable isotope labeling by amino acids in cell culture (SILAC), which has been widely used to compar e and quantify pr otein r elative expr ession. W e expanded an integrated immunoprecipitation (IP) coupled with SILAC approach (SILAC-IP) to differentiate the specific bind- ing par tners associated with a bait pr otein in two populations of cells. By this SILAC-IP strategy , the identified specific-binding proteins were quantified by tracking pairs of Leu-d 3 labeled and unlabeled peptides from the mass spectra, which could differentiate specific-binding proteins from nonspecific partners in high confidence. W e applied SILAC-IP method to dif fer entiate specific-binding pr oteins associated with 14-3-3â in human hepato- cellular carcinoma cell line QGY7703 between those in the liver cell line QSG7701. The proteins including HSP86, SKB1hs, GADPH and MEP50 were identified to associate with 14-3-3â in QGY7703 with high binding level than in QSG7701 cells.