The TMT is based on the reaction of the Primary amine at the N-terminus and the lysine with the NHS-ester group of the isotopically labeled tag. Once successfully tagged the peptide mixture is subject to MS analysis were the TMT-peptide displays a label-specific, low mass reporter ion during MS2.
The NHS-based TMT strategy requires all peptides to be freely accessible within a lysate to obtain efficient tagging. TMT can be apply for intact proteins as well, but applying it to intact cell in vivo was new to me.
The authors investigated the labelling efficienicy among different cancer cell lines and stated that in vivo TMT labeling requires an additional enrichment step to achive decent labeling efficiencies which are still lower compared to tagging on the peptide level (roughly 50% of identified peptides were tagged with invivo TMT after enrichment). The enrichment was done using an anti-TMT antibody to pull down all labeled peptides.
Compared to TMT labelling on the peptide level which appeared to be 100% of all identified peptides in vivo labelling with subsequent
Tandem Mass Tag (TMT) is a common technique for quantification of peptides at the MS2 level.
The TMT is based on the reaction of the Primary amine at the N-terminus and the lysine with the NHS-ester group of the isotopically labeled tag. Once successfully tagged the peptide mixture is subject to MS analysis were the TMT-peptide displays a label-specific, low mass reporter ion during MS2.
The NHS-based TMT strategy requires all peptides to be freely accessible within a lysate to obtain efficient tagging. TMT can be apply for intact proteins as well, but applying it to intact cell in vivo was new to me.
showed rather minor efficiency. However reproducibility was definitely given. Surprisely, the in vivo labelling strategy had no specificity to the location of the proteins. Specifically, a bias towards surface protein has not been revealed.
Tandem Mass Tag (TMT) is a common technique for quantification of peptides at the MS2 level.
The TMT is based on the reaction of the Primary amine at the N-terminus and the lysine with the NHS-ester group of the isotopically labeled tag. Once successfully tagged the peptide mixture is subject to MS analysis were the TMT-peptide displays a label-specific, low mass reporter ion during MS2.
The NHS-based TMT strategy requires all peptides to be freely accessible within a lysate to obtain efficient tagging. TMT can be apply for intact proteins as well, but applying it to intact cell in vivo was new to me.
The authors investigated the labelling efficienicy among different cancer cell lines and stated that in vivo TMT labeling requires an additional enrichment step to achive decent labeling efficiencies which are still lower compared to tagging on the peptide level (roughly 50% of identified peptides were tagged with invivo TMT after enrichment). The enrichment was done using an anti-TMT antibody to pull down all labeled peptides.
Compared to TMT labelling on the peptide level which appeared to be 100% of all identified peptides in vivo labelling with subsequent immunoprecipation showed rather minor efficiency. However reproducibility was definitely given. Surprisely, the in vivo labelling strategy had no specificity to the location of the proteins. Specifically, a bias towards surface protein has not been revealed.
showed rather minor efficiency. However reproducibility was definitely given. Surprisely, the in vivo labelling strategy had no specificity to the location of the proteins. Specifically, a bias towards surface protein has not been revealed.
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