%0 Journal Article
%T Targeted LC-MS quantification of intact TAT-fusion therapeutics: a case study.
%A Zhao X
%A Sun J
%A Sun L
%A Chen J
%A Cheng Y
%A Duan X
%J Bioanalysis
%V 7
%N 8
%D 2015
%M 25633639
%F 2.695
%R 10.4155/bio.15.17
%X BACKGROUND: While HIV-1 TAT peptide-conjugation shows great promise on improving intracellular delivery of biotherapeutics in vitro and in vivo, quantification of TAT-fusion therapeutics in biological matrices represents a daunting challenge.
METHODS: A sensitive MS approach for accurate quantification of intact TAT-fusion protein/polypeptide in plasma was developed. i) A semi-automated 96-well ion-exchange solid phase extraction was developed; ii) a rapid LC separation on C4 was devised; iii) a TAT-fusion analog was constructed as internal standard.
RESULTS: We reported that low percentage of supercharging reagents enabled a significant sensitivity improvement of MS for intact TAT-fusion protein/polypeptide analysis. We showed a proof of concept by successfully developing a sensitive LC/MRM-MS method for quantifying GAP161, a TAT-conjugating RasGAP mimics, in rat plasma.
CONCLUSIONS: This work represents the first quantification of TAT-fusion therapeutics in biological samples by an LC-MS based method.