Measuring the intra-individual variability of the plasma proteome in the chicken model of spontaneous ovarian adenocarcinoma

Abstract

The domestic chicken (Gallus domesticus) has emerged as a powerful experimental model for studying the onset and progression of spontaneous epithelial ovarian cancer (EOC) with a disease prevalence that can exceed 35% between 2-7 years of age. A novel experimental strategy for biomarker discovery is reported herein that combines the chicken model of EOC, longitudinal plasma sample collection with matched tissues, advanced mass spectrometry-based proteomics, and concepts derived from the index of individuality. Blood was drawn from 148 age-matched chickens starting at 2.5 years of age every three months for one-year. At the conclusion of the one-year sample collection period, the 73 birds that remained alive were euthanized, necropsied, and tissues were collected. Pathological assessment of resected tissues from these 73 birds confirmed that 5 birds (6.8%) developed EOC. A proteomics workflow including in-gel digestion, nanoLC coupled to high performance mass spectrometry, and label-free (spectral counting) was used to measure the biological intra-individual variability (CVW) of the chicken plasma proteome. Longitudinal plasma sample sets from two birds within the 73-bird biorepository were selected for this study; one bird was considered “healthy” and the second bird developed late-stage EOC. A total of 116 proteins from un-depleted plasma were identified with 80 proteins shared among all sample sets. Within- and between-run analytical variability (CVA) of the label-free proteomics workflow was measured using a single plasma sample analyzed multiple times. Ovomacroglobulin (ovostatin) was found to increase over a 6 month period in the late-stage EOC bird providing an initial candidate protein for further investigation.

Multi-peptide nLC-PC-IDMS-SRM-based Assay for the quantification of biomarkers in the chicken ovarian cancer model

“…These aspects combined with the ability to control genetic, environmental, and sample collection variables make the chicken a powerful experimental system for MS-based strategies at the global and targeted level. We recently explored the intra-individual and analytical variability of the plasma proteome 28 and N-linked glycome 29 from healthy and EOC birds to identify candidate biomarkers. In this study, we developed a protein cleavage (PC) isotope dilution mass spectrometry (IDMS) selected reaction monitoring (SRM) assay to validate observations in the plasma proteomics study.…”

Towards an Animal Model of Ovarian Cancer: Cataloging Chicken Blood Proteins Using Combinatorial Peptide Ligand Libraries Coupled with Shotgun Proteomic Analysis for Translational Research

“…In the end, Huang et al (2006) were only able to identify 30 protein IDs. In another report, a total of 116 proteins were identified from undepleted chicken plasma using a 1DGE-LC-MS/MS protocol (Hawkridge et al, 2010). In comparison, the number of protein IDs obtained in this study is far greater.…”

“…Chickens are considered a valid model to study ovarian cancer because they have high rates of developing spontaneous ovarian tumors (Wilson, 1953;Fredrickson, 1987). Other authors have searched for potential cancer biomarkers in chicken plasma as an alternative to human samples (Huang et al, 2006;Barua et al, 2009;Hawkridge et al, 2010). Among the 264 proteins identified in this study, several proteins are related to ovarian cancer pathogenesis or are potential diagnostic biomarkers.…”

The PeptideAtlas of the Domestic Laying Hen

“…The peptides CSGEPFQAITSDDSG R from H9L385 and CSGEPFQAITSDDSG H IYAFR from Q90WR3 (polymorphic site noted) were each observed in several hundred quality spectra across experiments, many with nearly full y-ion series, lending credence to the existence of the non-genomic protein sequence. Disaggregation of the samples to examine individual organisms from a published study 24 reveals that specific chickens can present with either one or both of the sequences. Chickens #602, 612, 630, 639, and 666 exhibit peptides from both sequences, while #600, 601, and 650 exhibit only the unique peptide from H9L385 and #620 exhibits the form from Q90WR3, implying both hetero- and homozygosity of the polymorphism occur in the population.…”

“…Birds were managed in accordance with the Institute for Laboratory Animal Research Guide with all of the husbandry practices being approved by and under the oversight of North Carolina State University Institutional Animal Care and Use Committee (IACUC). Plasma and tissue from the initial cohort of B-strain hens were obtained in a previous study, 24 with samples collected from a second cohort of 300 C-strain hens similarly. In brief, ~2mL of blood was collected from each bird every four months beginning at an age of 26 weeks.…”