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 and 7 years of age. An 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 (Harris, Clin Chem 20: 1535-1542, 1974). Blood was drawn from 148 age-matched chickens starting at 2.5 years of age every 3 months for 1 year. At the conclusion of the 1 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 five birds (6.8%) developed EOC. A proteomics workflow including in-gel digestion, nanoLC coupled to high-performance mass spectrometry, and label-free (spectral counting) quantification was used to measure the biological intra-individual variability (CV(W)) 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. Analytical variability (CV(A)) of the label-free proteomics workflow was measured using a single plasma sample analyzed five times and was found to be ≥CV(W) in both birds for 16 proteins (20%) and in either bird for 25 proteins (31%). Ovomacroglobulin (ovostatin) was found to increase (p < 0.001) over a 6 month period in the late-stage EOC bird providing an initial candidate protein for further investigation.

Figure 1

Hematoxylin and eosin stained tissue sections for the Healthy bird (602B) with a fully functional ovary at 4× (A) and 20× (B) and a late stage EOC bird (650B) at 4× (C) and 20× (D). The dashed boxes in (A) and (C) indicate the magnified regions in images (B) and (D). Black arrows indicate immature follicles for the Healthy bird (A). Black arrow indicates an oocyte within an immature follicle and the blue arrow indicates the ovarian surface epithelial (OSE) layer (B). Neoplastic cells invading the ovarian stroma (C). Dilated cystic glands and glandular structures of variable-size indicated by yellow arrows (D). Scale bars at 4× in (A) and (C) = 1 mm and at 20× in (B) and (D) = 100 μm.

Figure 2

One-dimensional gels of longitudinal plasma samples from Healthy (red), EOC (green), and Analytical (blue) birds with corresponding time points/replicates and total protein (μg) loaded onto each lane. The total numbers of proteins identified from each gel lane, in each bird, and for the entire study are provided directly below the 1D-gels. (A) Venn diagram showing the identified protein overlap between the Healthy, EOC, and Analytical sample sets.(B) The number of proteins in the Healthy (red), EOC (green), and Analytical (blue) plotted as function of fraction of proteins identified per lane.(C) The number of proteins identified plotted as a function of fraction of proteins identified per lane.(D) Note: ^{a, b, c} denote the source of the numbers between Figure 1A and 1B, 1C, and 1D.

Figure 3

The coefficient of variation (%) at each time point (CV_{Individual Timepoints}) for the 80 proteins identified in all 15 gel lanes plotted as a function of the average spectral counts (Average SpC). A histogram plotting the number of proteins (Frequency) relative to the CV_{Individual Timepoints} in 10% bin widths adjoins each plot. Healthy (A), EOC (B), and Analytical (C) birds.

Figure 4

Quantitative SpC data for the Healthy (red) and EOC (green) birds plotted as a function of sampling time point are shown for serum albumin (A), vitellogenin-1 (B), and transthyretin (C). The solid lines terminate at the average SpC (n=3) for each time point.

Figure 5

Quantitative SpC data for the Healthy (red) and EOC (green) birds plotted as a function of sampling time point for ovomacroglobulin. The solid lines terminate at the average SpC (n=3) for each time point. The p-value (<0.001) is the probability that the that the SpC differences between time points for the EOC bird are insignificant (ANOVA).