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.