title: Multi-peptide nLC-PC-IDMS-SRM-based assay for the quantification of biomarkers in the chicken ovarian cancer model.
abstract: A novel form of ovomacroglobulin/ovostatin (OVOS2) predicted from EST data was previously identified in the chicken ovarian cancer model using a mass spectrometry-based shotgun label-free proteomics strategy. The quantitative label-free data from plasma showed a significant increase over time with the spontaneous onset and progression of ovarian cancer making it a potential protein biomarker for further study. Two other proteins of interest identified from this initial study included vitellogenin-1 (Vit-1), a lipid-transport protein tied to egg production, and transthyretin (TTR), a retinol binding transport protein currently used in the clinical management of ovarian cancer. A multiplexed protein cleavage isotope dilution mass spectrometry (PC-IDMS) assay was developed to quantify OVOS2, Vit-1, and TTR by selected reaction monitoring (SRM). A total of 6 stable isotope labeled (SIL) peptide standards were used in the assay with three tryptic peptides from OVOS2, one for Vit-1, and two for TTR. The assay was developed for use with un-depleted raw plasma combined with the filter assisted sample preparation (FASP) method and its use was also demonstrated for matched ovary tissue samples. The PC-IDMS data for the two TTR peptides did not correlate with each other with more than a 10-fold difference in concentration for all 5 time points measured. The PC-IDMS data from the longitudinal plasma samples correlated well for OVOS2 and Vit-1 whereas TTR was inconclusive. Interestingly, the absolute amount for one of the OVOS2 SIL peptides was 2-fold less compared with the other two SIL peptides. These data illustrate the successes and challenges of qualifying quantitative levels of proteins from an in-gel digestion sample preparation followed by LC-MS/MS (GeLC) label-free discovery-based approach to a targeted SRM-based quantitative assay in plasma and tissues.
authors.affiliation: W.M. Keck FT-ICR Mass Spectrometry Laboratory and Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Genna L
authors.initials: GL
authors.lastname: Andrews Kingon
authors.forename: James N
authors.initials: JN
authors.lastname: Petitte
authors.forename: David C
authors.initials: DC
authors.lastname: Muddiman
authors.forename: Adam M
authors.initials: AM
authors.lastname: Hawkridge
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D002138:Calibration; D002247:Carbon Isotopes; D002645:Chickens; D004195:Disease Models, Animal; D005260:Female; D006801:Humans; D007201:Indicator Dilution Techniques; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010051:Ovarian Neoplasms / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis*; D011228:Prealbumin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards; D014819:Vitellogenins / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D000511:alpha-Macroglobulins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011228:Prealbumin; D014819:Vitellogenins; D000511:alpha-Macroglobulins; C036828:ovomacroglobulin