TAILIEUCHUNG - Proteomic Applications in Biology Part 12

Tham khảo tài liệu 'proteomic applications in biology part 12', khoa học tự nhiên, công nghệ sinh học phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | 176 Proteomic Applications in Biology highlight the clear separation between the known and predicted Golgi- and ER-localized protein clusters. Significantly a number of cell wall biosynthetic enzymes were identified including a number of glycosyltransferases. This confirmed LOPIT as a valid method for discriminating between Golgi- and ER-localized proteins from Arabidopsis crude membrane fractions Dunkley et al. 2004 . Further development of the LOPIT technique replaced ICAT with isotope tagging of Arabidopsis membrane peptide fractions for both relative and absolute protein quantitation iTRAQ Dunkley et al. 2006 Fig. 4 . The iTRAQ method is a progression of ICAT by labeling the free primary amines of peptides with four different iTRAQ reporter tags 114 115 116 and 117 m z . They are detectable by MS MS which allows for simultaneous quantification analysis of up to four peptide samples Wiese et al. 2007 . Arabidopsis membrane peptide fractions were differentially tagged with the four iTRAQ reporters fractionated and analyzed by MudPIT and Q-TOF MS. The addition of SCX to RP LC-MS MS provided superior peptide separation and identification resulting in 689 Arabidopsis protein identifications. Multivariate analysis of iTRAQ-labeled MS MS data revealed 89 proteins in the Golgi density gradient cluster. This more extensive analysis further validated the approach as further cell wall biosynthetic enzymes such as glycosyltransferases and sugar interconverting enzymes were identified as well as transporters V-ATPase components and a variety of proteins with likely Golgi functions Dunkley et al. 2006 . This was a significant improvement on the initial LOPIT set of ten Arabidopsis Golgi-localized proteins by ICAT and LC-MS MS Dunkley et al. 2004 . To test its robustness in other biological system LOPIT was used to investigate the subcellular distribution of proteins from Drosophila embryos. A total of 329 Drosophila proteins were identified and localized to three subcellular

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