Targeted LC-MS Quantification of Intact Tat-Fusion Therapeutics: A Case Study

Abstract

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. Materials & 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. Conclusion: This work represents the first quantification of TAT-fusion therapeutics in biological samples by an LC-MS based method.

Keywords::

• intact protein analysis
• supercharging reagents
• targeted MS quantification
• TAT-fusion therapeutics

Supplementary Data

Financial & competing interests disclosure

This work was supported by the National Natural Science Foundation of China (Grant No. 81302739 to X Duan) and a research foundation from Beijing Bureau of Human Resources and Social Security (to X Duan). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 81302739 to X Duan) and a research foundation from Beijing Bureau of Human Resources and Social Security (to X Duan). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.