title: Antibody-free, targeted mass-spectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serum.
abstract: Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)-based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano-liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50-100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Tujin
authors.initials: T
authors.lastname: Shi
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Fillmore
authors.forename: Xuefei
authors.initials: X
authors.lastname: Sun
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.forename: Athena A
authors.initials: AA
authors.lastname: Schepmoes
authors.forename: Mahmud
authors.initials: M
authors.lastname: Hossain
authors.forename: Fang
authors.initials: F
authors.lastname: Xie
authors.forename: Si
authors.initials: S
authors.lastname: Wu
authors.forename: Jong-Seo
authors.initials: JS
authors.lastname: Kim
authors.forename: Nathan
authors.initials: N
authors.lastname: Jones
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Moore
authors.forename: Ljiljana
authors.initials: L
authors.lastname: Pasa-Tolić
authors.forename: Jacob
authors.initials: J
authors.lastname: Kagan
authors.forename: Karin D
authors.initials: KD
authors.lastname: Rodland
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.forename: Keqi
authors.initials: K
authors.lastname: Tang
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D015415:Biomarkers / Q000378:metabolism; D001798:Blood Proteins / Q000032:analysis; D002138:Calibration; D002256:Carbonic Anhydrases / Q000097:blood; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D017430:Prostate-Specific Antigen / Q000097:blood; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D049490:Systems Biology / Q000379:methods
chemical_list: D000906:Antibodies; D015415:Biomarkers; D001798:Blood Proteins; D011506:Proteins; D017430:Prostate-Specific Antigen; D002256:Carbonic Anhydrases