Zhao Xiaoping, Sun Jianan, Sun Lu, Chen Jianhua, Cheng Yuanguo, Duan Xiaotao
State Key Laboratory of Pathogen & Biosecurity, Beijing, China.
Bioanalysis. 2015;7(8):981-90. doi: 10.4155/bio.15.17. Epub 2015 Jan 29.
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.
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.
This work represents the first quantification of TAT-fusion therapeutics in biological samples by an LC-MS based method.
虽然HIV-1 TAT肽缀合在体外和体内改善生物治疗药物的细胞内递送方面显示出巨大潜力，但在生物基质中对TAT融合治疗药物进行定量是一项艰巨的挑战。
开发了一种灵敏的质谱方法，用于准确定量血浆中完整的TAT融合蛋白/多肽。i）开发了一种半自动96孔离子交换固相萃取方法；ii）设计了一种在C4上的快速液相色谱分离方法；iii）构建了一种TAT融合类似物作为内标。
我们报道，低百分比的超电荷试剂能够显著提高质谱对完整TAT融合蛋白/多肽分析的灵敏度。我们通过成功开发一种灵敏的液相色谱/多反应监测-质谱方法来定量大鼠血浆中的GAP161（一种TAT缀合的RasGAP模拟物），证明了这一概念。
这项工作代表了首次通过基于液相色谱-质谱的方法对生物样品中的TAT融合治疗药物进行定量。
