AHA
BONCAT (bioorthogonal noncanonical amino acid tagging)
azidohomoalanine (AHA, 1)
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
Relative abundance of newly synthesized proteins.
66 proteins >2 92 proteins ~1.5-2 186 proteins <1.5
COFRADIC has several advantages
it utilizes standard chromatographic techniques and chemicals
David Tirrell’s group
Growth rate and viability of cell cultured in AHA
methionine auxotrophic E. coli strain
M9+19aa+Met OD=1.0, wash
M9+19aa+Met M9+19aa+AHA
Quantitation of newly synthesized proteins induced by heat shock
M9+19aa+Met 37oC M9+19aa+Met
A labeling time of 15 mins
ห้องสมุดไป่ตู้AHA A 37oC B
Protein extraction, digestion
iTRAQ reporter
114
115
AHA A 44oC B
116
117
1:1:1:1 ICAT cation exchange cartridge
First run on RP-HPLC TCEP treatment O/N Second run on RP-HPLC On diagonal fraction were discarded, off diagonal fraction were pooled
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.
it provides a simple and robust approach to enrichment of
labeled peptides.
Thank you
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
cell viability, Growth curve
Growth rate and viability of cell cultured in AHA
Number 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
The principle of the COFRADIC approach
Chromatographic separation of enriched labeled peptides isolated by COFRADIC.
Extracted ion chromatogram of a peptide identified in two forms
to achieve separation of labeled from unlabeled peptides.
it provides chromatographic fractionation as well as
enrichment of labeled peptides, thereby facilitating mass spectrometric identification.