title: A highly sensitive targeted mass spectrometric assay for quantification of AGR2 protein in human urine and serum.
abstract: Anterior gradient 2 (AGR2) is a secreted, cancer-associated protein in many types of epithelial cancer cells. We developed a highly sensitive targeted mass spectrometric assay for quantification of AGR2 in urine and serum. Digested peptides from clinical samples were processed by PRISM (high pressure and high resolution separations coupled with intelligent selection and multiplexing), which incorporates high pH reversed-phase liquid chromatography (LC) separations to fractionate and select target fractions for follow-on LC-selected reaction monitoring (LC-SRM) analyses. The PRISM-SRM assay for AGR2 showed a reproducibility of <10% CV and limit of quantification (LOQ) values of ∼130 pg/mL in serum and ∼10 pg per 100 μg of total protein mass in urine, respectively. A good correlation (R(2) = 0.91) was observed for the measurable AGR2 concentrations in urine between SRM and enzyme-linked immunosorbent assay (ELISA). On the basis of an initial cohort of 37 subjects, urinary AGR2/PSA concentration ratios showed a significant difference (P = 0.026) between noncancer and cancer. Large clinical cohort studies are needed for the validation of AGR2 as a useful diagnostic biomarker for prostate cancer. Our work validated the approach of identifying candidate secreted protein biomarkers through genomics and measurement by targeted proteomics, especially for proteins where no immunoassays are available.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory , Richland, Washington 99352.
authors.forename: Tujin
authors.initials: T
authors.lastname: Shi
authors.forename: Yuqian
authors.initials: Y
authors.lastname: Gao
authors.forename: Sue Ing
authors.initials: SI
authors.lastname: Quek
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Fillmore
authors.forename: Carrie D
authors.initials: CD
authors.lastname: Nicora
authors.forename: Dian
authors.initials: D
authors.lastname: Su
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.forename: Jacob
authors.initials: J
authors.lastname: Kagan
authors.forename: Sudhir
authors.initials: S
authors.lastname: Srivastava
authors.forename: Karin D
authors.initials: KD
authors.lastname: Rodland
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Chan
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Alvin Y
authors.initials: AY
authors.lastname: Liu
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
mesh_terms: D000595:Amino Acid Sequence; D002138:Calibration; D056148:Chromatography, Reverse-Phase; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009088:Mucoproteins; D015513:Oncogene Proteins; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: C502296:AGR2 protein, human; D009088:Mucoproteins; D015513:Oncogene Proteins; D011506:Proteins