title: Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion.
abstract: We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM), for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ∼12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successfully quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
authors.forename: Tujin
authors.initials: T
authors.lastname: Shi
authors.forename: Xuefei
authors.initials: X
authors.lastname: Sun
authors.forename: Yuqian
authors.initials: Y
authors.lastname: Gao
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Fillmore
authors.forename: Athena A
authors.initials: AA
authors.lastname: Schepmoes
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.forename: Jintang
authors.initials: J
authors.lastname: He
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Moore
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: Alvin Y
authors.initials: AY
authors.lastname: Liu
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Keqi
authors.initials: K
authors.lastname: Tang
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
mesh_terms: D015415:Biomarkers / Q000097:blood*; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D013058:Mass Spectrometry; D017430:Prostate-Specific Antigen / Q000097:blood*; D011471:Prostatic Neoplasms / Q000097:blood* / Q000473:pathology; D040901:Proteomics / Q000379:methods
chemical_list: D015415:Biomarkers; D001798:Blood Proteins; D017430:Prostate-Specific Antigen