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Abstract INTRODUCTION:
Ascites, the pathologic accumulation of fluid in the peritoneal cavity, is common at diagnosis, initial debulking surgery, and with recurrence in high-grade serous ovarian carcinoma (HGSOC). Greater ascites volumes are associated with shorter progression-free survival and overall survival. Since ascites reaccumulates frequently, is readily available in large volumes for sampling, and reflects the peritoneal microenvironment, ascites is a valuable matrix for proteomic biomarker discovery.
The majority of the ascites protein mass is comprised of few proteins, and sample preparation methods using enrichment or depletion are required to obtain adequate depth of mass spectrometry-based proteome coverage. Nanotrap® Protein Enrichment Affinity Kits (PEAK) (Ceres Nanosciences) contain magnetic hydrogel Nanotrap particles (A, B, C) with complementary chemistries to enhance the depth of proteome coverage with low abundant protein enrichment and high abundant protein exclusion. Our previous studies with the Nanotrap particles in ascites fluid demonstrated particles A, AC, and ABC are the optimal single or combination Nanotrap particles for the proteomic analysis of ascites.1-2 The impact of incorporating Nanotrap particle technology on the differential protein abundance of ascites fluid is not well-described.
OBJECTIVE(S):
Our goal was to evaluate the Nanotrap hydrogel technology’s capacity to quantify differential protein abundance in the ascites fluid of HGSOC patients. Differential relative protein abundance was evaluated using a spike-in study based on recovery and measurement bias of recombinant non-human proteins spiked into ascites fluid at a range of known quantities. In addition, the fold-change in rank of endogenous ascites fluid proteins was evaluated in the Nanotrap particle-enriched samples vs. the non-enriched (Neat) samples to determine how Nanotrap-based enrichment alters the proteome landscape.
METHODS:
Ten patient-derived HGSOC ascites fluid specimens were obtained from an ovarian cancer biorepository at the University of Minnesota and pooled. A feasibility pilot study was conducted with Enolase (S. cerevisiae) spiked into ascites fluid at 1:10 and 1:100 ratios with and without enrichment with Nanotrap particle B, followed by reduction, alkylation, and overnight digestion with trypsin at 37°C. Enolase-spiked neat ascites was processed in parallel. Peptides were separated using a Vanquish microflow LC system coupled to a Q Exactive Plus mass spectrometer operated in parallel reaction monitoring data acquisition mode with n=2 peptides/protein selected as targets. The sum of the peak areas for the top two most abundant peptides represented the protein abundance. Data analysis was performed using Skyline. Global proteomic data were acquired using a data-dependent acquisition (DDA) method. Data analysis was conducted using Proteome Discoverer with Sequest. Proteins identified in ≥2 of 3 replicates of the Neat and Nanotrap-enriched samples (n=120 proteins) were ranked by their intensity-based abundance. Fold-change calculations of the protein rankings for each Nanotrap particle alone and in combination vs. Neat were determined. A fold-change <1.0 indicated protein enrichment with the Nanotrap particle(s), a fold-change >1.0 indicated protein depletion with the Nanotrap particle(s), and a fold-change ≈1 indicated no change in relative abundance ranking vs. Neat.
RESULTS:
Analysis of the intensity-based relative abundance of the endogenous ascites proteins indicated a mean of 43.5% of proteins were enriched and 55.3% were depleted by the Nanotrap particles, with 1.2% demonstrating no change in relative abundance ranking vs. Neat. Particle C yielded the lowest correlation between the ranking of the proteins in the Neat vs. enriched samples (r=0.3090). The widest fold-change range (0.025-117.5) was obtained from the proteins enriched with particles A+C, followed by the combination of particles A+B+C (0.077-110). Fold-change ranges following enrichment with individual particles A (0.116-69.5) and B (0.105-71.0) were similar. Serotransferrin and Hemopexin were the proteins with the greatest magnitude of depletion across all Nanotrap particles, with the exception of particle C, which was the most efficient at depleting Albumin. Apolipoprotein B, Apolipoprotein C-III, and Ceruloplasmin were the proteins with the greatest degree of enrichment with particles A, B, and C, respectively. This selective protein depletion and enrichment reflects the particle chemistries. Particles A and B maintain a net electronegative surface charge, enabling selective capture of proteins through electrostatic and hydrophobic interaction mechanisms. Particle C possesses a net electropositive surface charge, enabling strong electrostatic interaction and preferable capture of negatively charged proteins and biomolecules.
Preliminary results from the spike-in study indicate an overall under-recovery of the recombinant Enolase spiked into the ascites samples prior to enrichment with Nanotrap particle B compared to the recovery of Enolase spiked into the Neat ascites samples without subsequent enrichment. Among the enriched samples, the lowest bias was -22.9% compared to 5.4% for the Neat samples, which likely reflects the function of the Nanotrap particles in selectively altering the composition of the proteome. Ongoing studies include an expanded evaluation of the quantitative measurement accuracy of a broader panel of recombinant proteins spiked into ascites ± Nanotrap particle enrichment.
DISCUSSION:
The incorporation of ascites fluid as a proteomic biomarker in HGSOC patient care could impact diagnosis, prediction, prognosis, and disease monitoring. Overall, the Nanotrap particles exhibit an 0.8:1 balance between the enrichment of low abundance proteins and the depletion of high abundance proteins to enable the balanced interrogation of the ascites fluid proteome. |
= Discovery stage. (55.92%, 2026)
= Translation stage. (23.22%, 2026)
= Clinically available. (20.85%, 2026)
