新合成蛋白标记蛋白质组.pptx

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1、AHA第1页/共22页BONCAT(bioorthogonal noncanonical amino acid tagging)azidohomoalanine(AHA,1)David Tirrells groupSubstrates are newly synthesized proteinsBONCAT is based on introduction of AHAAHA is acepted by tRNA synthetaseAHA incorporation is unbiasedincorporation of AHA enables chemoselective tagging

2、with an alkyne affinity tag第2页/共22页Growth rate and viability of cell cultured in AHAmethionine auxotrophic E.coli strain M9+19aa+MetOD=1.0,washM9+19aa+MetM9+19aa+AHAcell viability,Growth curve第3页/共22页Growth rate and viability of cell cultured in AHA第4页/共22页Number of colony forming units per OD600 pe

3、r ml of culture over time 第5页/共22页Growth of E.coli strain MTD123 on a range of concentrations of AHA 第6页/共22页Growth rate,protein synthesis and viability in the present of AHA and methinonine is the same during the first 30 min.After prolonged labeling growth arrest does occur第7页/共22页Quantitation of

4、newly synthesized proteins induced by heat shockM9+19aa+MetM9+19aa+MetABAB37oC44oC114115116117iTRAQ reporter1:1:1:1Protein extraction,digestionOn diagonal fraction were discarded,off diagonal fraction were pooledICAT cation exchange cartridgeFirst run on RP-HPLCSecond run on RP-HPLCTCEP treatment O/

5、NA labeling time of 15 minsAHAAHA37oC第8页/共22页The principle of the COFRADIC approach第9页/共22页Chromatographic separation of enriched labeled peptides isolated by COFRADIC.第10页/共22页Extracted ion chromatogram of a peptide identified in two forms diaminobutyrate(DAB)residue Homoserine(HS)residue 第11页/共22页

6、Tandem-MS spectra of a peptide observed in two forms.第12页/共22页Proposed TCEP-induced reaction scheme 第13页/共22页第14页/共22页第15页/共22页Venn diagrams of proteins identified in this and other studies.第16页/共22页第17页/共22页第18页/共22页Relative abundance of newly synthesized proteins.66 proteins 292 proteins 1.5-2186

7、proteins 1.5第19页/共22页COFRADIC has several advantagesit utilizes standard chromatographic techniques and chemicals to achieve separation of labeled from unlabeled peptides.it provides chromatographic fractionation as well as enrichment of labeled peptides,thereby facilitating mass spectrometric identification.it provides a simple and robust approach to enrichment of labeled peptides.第20页/共22页Thank you第21页/共22页感谢您的观看！第22页/共22页