title: 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.
  
  
  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.
authors: State Key Laboratory of Pathogen & Biosecurity, Beijing, China.
authors: Xiaoping
authors: X
authors: Zhao
authors: Jianan
authors: J
authors: Sun
authors: Lu
authors: L
authors: Sun
authors: Jianhua
authors: J
authors: Chen
authors: Yuanguo
authors: Y
authors: Cheng
authors: Xiaotao
authors: X
authors: Duan
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D006801:Humans; D008297:Male; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D052616:Solid Phase Extraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D020703:ras GTPase-Activating Proteins / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000032:analysis*
chemical_list: D011498:Protein Precursors; D020703:ras GTPase-Activating Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C050502:gonadotropin releasing hormone associated peptide; D007987:Gonadotropin-Releasing Hormone
keywords: TAT-fusion therapeutics; intact protein analysis; supercharging reagents; targeted MS quantification