新合成蛋白标记蛋白质组.pptxVIP

AHASubstrates are newly synthesized proteinsBONCAT is based on introduction of AHAAHA is acepted by tRNA synthetaseAHA incorporation is unbiasedincorporation of AHA enables chemoselective tagging with an alkyne affinity tagazidohomoalanine (AHA, 1) David Tirrell’s groupBONCAT (bioorthogonal noncanonical amino acid tagging)methionine auxotrophic E. coli strain M9+19aa+MetOD=1.0, washM9+19aa+MetM9+19aa+AHAGrowth rate and viability of cell cultured in AHAcell viability,Growth curveGrowth rate and viability of cell cultured in AHANumber of colony forming units per OD600 per ml of culture over time Growth of E. coli strain MTD123 on a range of concentrations of AHA 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 occur114115116117ABABQuantitation of newly synthesized proteins induced by heat shockM9+19aa+Met37oCM9+19aa+MetAHAAHAA labeling time of 15 mins37oC44oCProtein extraction, digestioniTRAQ reporter1:1:1:1ICAT cation exchange cartridgeFirst run on RP-HPLCTCEP treatment O/NSecond run on RP-HPLCOn diagonal fraction were discarded, off diagonal fraction were pooledThe principle of the COFRADIC approachChromatographic separation of enriched labeled peptides isolated by COFRADIC. Extracted ion chromatogram of a peptide identified in two forms diaminobutyrate (DAB) residue Homoserine (HS) residue Tandem-MS spectra of a peptide observed in two forms. Proposed TCEP-induced reaction scheme Venn diagram’s of proteins identified in this and other studies. Relative abundance of newly synthesized proteins. 66 proteins 292 proteins ~1.5-2186 proteins 1.5COFRADIC 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 an