文档介绍：会计学
1
新合成蛋白标记蛋白质组
BONCAT (bioorthogonal noncanonical amino acid tagging)
azidohomoalanine (AHA, 1)
David Tirrell’s group
Substrates are newly synthesized proteins
BONCAT is based on introduction of AHA
AHA is acepted by tRNA synthetase
AHA incorporation is unbiased
incorporation of AHA enables chemoselective tagging with an alkyne affinity tag
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Growth rate and viability of cell cultured in AHA
methionine auxotrophic E. coli strain
M9+19aa+Met
OD=, wash
M9+19aa+Met
M9+19aa+AHA
cell viability,
Growth curve
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Growth rate and viability of cell cultured in AHA
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Number of colony forming units per OD600 per ml of culture over time
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Growth of E. coli strain MTD123 on a range of concentrations of AHA
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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
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Quantitation of newly synthesized proteins induced by heat shock
M9+19aa+Met
M9+19aa+Met
A
B
A
B
37oC
44oC
114
115
116
117
iTRAQ reporter
1:1:1:1
Protein extraction, digestion
On diagonal fraction were discarded, off diagonal fraction were pooled
ICAT cation exchange cartridge
First run on RP-HPLC
Second run on RP-HPLC
TCEP treatment O/N
A labeling time of 15 mins
AHA
AHA
37oC
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The principle of the COFRADIC approach
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Chromatographic separation of enriched labeled peptides isolated by COFRADIC.
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