title: An integrated quantification method to increase the precision, robustness, and resolution of protein measurement in human plasma samples.
abstract: BACKGROUND
  Current quantification methods for mass spectrometry (MS)-based proteomics either do not provide sufficient control of variability or are difficult to implement for routine clinical testing.
  
  
  RESULTS
  We present here an integrated quantification (InteQuan) method that better controls pre-analytical and analytical variability than the popular quantification method using stable isotope-labeled standard peptides (SISQuan). We quantified 16 lung cancer biomarker candidates in human plasma samples in three assessment studies, using immunoaffinity depletion coupled with multiple reaction monitoring (MRM) MS. InteQuan outperformed SISQuan in precision in all three studies and tolerated a two-fold difference in sample loading. The three studies lasted over six months and encountered major changes in experimental settings. Nevertheless, plasma proteins in low ng/ml to low μg/ml concentrations were measured with a median technical coefficient of variation (CV) of 11.9% using InteQuan. The corresponding median CV using SISQuan was 15.3% after linear fitting. Furthermore, InteQuan surpassed SISQuan in measuring biological difference among clinical samples and in distinguishing benign versus cancer plasma samples.
  
  
  CONCLUSIONS
  We demonstrated that InteQuan is a simple yet robust quantification method for MS-based quantitative proteomics, especially for applications in biomarker research and in routine clinical testing.
authors.affiliation: Integrated Diagnostics, 219 Terry Avenue North, Suite 100, 98109 Seattle, WA USA.
authors.forename: Xiao-Jun
authors.initials: XJ
authors.lastname: Li
authors.affiliation: Integrated Diagnostics, 219 Terry Avenue North, Suite 100, 98109 Seattle, WA USA.
authors.forename: Lik Wee
authors.initials: LW
authors.lastname: Lee
authors.affiliation: Integrated Diagnostics, 219 Terry Avenue North, Suite 100, 98109 Seattle, WA USA.
authors.forename: Clive
authors.initials: C
authors.lastname: Hayward
authors.affiliation: Integrated Diagnostics, 219 Terry Avenue North, Suite 100, 98109 Seattle, WA USA ; Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., M4-A830, 98109 Seattle, WA USA.
authors.forename: Mi-Youn
authors.initials: MY
authors.lastname: Brusniak
authors.affiliation: Integrated Diagnostics, 219 Terry Avenue North, Suite 100, 98109 Seattle, WA USA.
authors.forename: Pui-Yee
authors.initials: PY
authors.lastname: Fong
authors.affiliation: Integrated Diagnostics, 219 Terry Avenue North, Suite 100, 98109 Seattle, WA USA ; DuPont Industrial Biosciences, 925 Page Mill Road, Palo, 94304 Alto, CA USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: McLean
authors.affiliation: Integrated Diagnostics, 219 Terry Avenue North, Suite 100, 98109 Seattle, WA USA.
authors.forename: JoAnne
authors.initials: J
authors.lastname: Mulligan
authors.affiliation: Integrated Diagnostics, 219 Terry Avenue North, Suite 100, 98109 Seattle, WA USA.
authors.forename: Douglas
authors.initials: D
authors.lastname: Spicer
authors.affiliation: Integrated Diagnostics, 219 Terry Avenue North, Suite 100, 98109 Seattle, WA USA.
authors.forename: Kenneth C
authors.initials: KC
authors.lastname: Fang
authors.affiliation: Integrated Diagnostics, 219 Terry Avenue North, Suite 100, 98109 Seattle, WA USA.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Hunsucker
authors.affiliation: Integrated Diagnostics, 219 Terry Avenue North, Suite 100, 98109 Seattle, WA USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Kearney
keywords: Bioinformatics; Clinical proteomics; Immunoaffinity depletion; Mass spectrometry; Multiple reaction monitoring; Plasma or serum analysis; Quantitative proteomics
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