title: Development of a highly sensitive liquid chromatography/tandem mass spectrometry method to quantify total and free levels of a target protein, interferon-gamma-inducible protein-10, at picomolar levels in human serum.
abstract: BACKGROUND
  Liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays are increasingly being used for absolute quantitation of proteins due to high specificity and low cost. However, the major challenge for the LC/MS method is insufficient sensitivity. This paper details the strategies developed to maximize the sensitivity from aspects of chromatography, mass spectrometry, and sample preparation to achieve a highly sensitive LC/MS method.
  
  
  METHODS
  The method is based on the LC/MS/MS measurement of a surrogate peptide generated from trypsin digestion of interferon-gamma-inducible protein-10 (IP-10). The sample preparation strategy involved selectively extracting IP-10 and removing high-abundance serum proteins through acidified protein precipitation (PPT). It was revealed in this work that these high-abundance serum proteins, if not separated from the protein of interest, could cause significant ionization saturation and high background noise in selected reaction monitoring (SRM), leading to a 100-fold higher lower limit of quantification (LLOQ).
  
  
  RESULTS
  Our method demonstrated that the acidified PPT could be optimized to selectively extract the protein of interest with full recovery of 97% to 103%, while the high-abundance serum proteins could be effectively removed with minimal matrix effect of 90% to 93%. For the first time, a highly sensitive LC/MS method with a LLOQ of 31.62 pM for the quantitation of IP-10 has been achieved, which is a 100-fold improvement over the generic method.
  
  
  CONCLUSIONS
  The described method offers excellent sensitivity with advantages of being antibody reagent independent and leads to significant cost and time savings. It has been successfully employed to determine both total and free IP-10 levels in human serum samples. This method development strategy may also be applied to other small proteins.
authors.affiliation: Research and Development, Bristol-Myers Squibb, Princeton, NJ, 08543, USA.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Zhang
authors.forename: Qing
authors.initials: Q
authors.lastname: Xiao
authors.forename: Baomin
authors.initials: B
authors.lastname: Xin
authors.forename: Wendy
authors.initials: W
authors.lastname: Trigona
authors.forename: Adrienne A
authors.initials: AA
authors.lastname: Tymiak
authors.forename: Ashok R
authors.initials: AR
authors.lastname: Dongre
authors.forename: Timothy V
authors.initials: TV
authors.lastname: Olah
mesh_terms: D001798:Blood Proteins / Q000737:chemistry; D054357:Chemokine CXCL10 / Q000097:blood* / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D005561:Formates; D006801:Humans; D016018:Least-Squares Analysis; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin
chemical_list: D001798:Blood Proteins; C114753:CXCL10 protein, human; D054357:Chemokine CXCL10; D005561:Formates; C030544:formic acid; D014357:Trypsin