Investigation of the ovarian and prostate cancer peptidome for candidate early detection markers using a novel nanoparticle biomarker capture technology

Abstract

Current efforts to identify protein biomarkers of disease use mainly mass spectrometry (MS) to analyze tissue and blood specimens. The low-molecular-weight "peptidome" is an attractive information archive because of the facile nature by which the low-molecular-weight information freely crosses the endothelial cell barrier of the vasculature, which provides opportunity to measure disease microenvironment-associated protein analytes secreted or shed into the extracellular interstitium and from there into the circulation. However, identifying useful protein biomarkers (peptidomic or not) which could be useful to detect early detection/monitoring of disease, toxicity, doping, or drug abuse has been severely hampered because even the most sophisticated, high-resolution MS technologies have lower sensitivities than those of the immunoassays technologies now routinely used in clinical practice. Identification of novel low abundance biomarkers that are indicative of early-stage events that likely exist in the sub-nanogram per milliliter concentration range of known markers, such as prostate-specific antigen, cannot be readily detected by current MS technologies. We have developed a new nanoparticle technology that can, in one step, capture, concentrate, and separate the peptidome from high-abundance blood proteins. Herein, we describe an initial pilot study whereby the peptidome content of ovarian and prostate cancer patients is investigated with this method. Differentially abundant candidate peptidome biomarkers that appear to be specific for early-stage ovarian and prostate cancer have been identified and reveal the potential utility for this new methodology.

Figures

Fig. 1

SDS-PAGE analysis of human serum sample pre- and post-incubation with NIPAm/AAc nanoparticles. Lane 1 Serum from healthy donor before incubation with particles. Lane 2 Supernatant obtained after centrifugation of the mix of incubation containing serum and nanoparticles. Lane 3 Eluate from nanoparticles: the samples are enriched in low-molecular-weight proteins, while albumin is mostly excluded

Fig. 2

Comparison of the LC-MS chromatography of serum and elution from nanoparticle. The abundant base peaks are labeled with their retention time. The peak intensities of albumin tryptic peptides such as AAFTECCQAADK (retention time 21.86 min), FQNALLVR (retention time 27.03 min), LVNEVTEFAK (retention time 28.42 min), KVPQVSTPTLVEVSR (retention time 28.90 min), RPCFSALEVDETYVPK (retention time 31.01 min), and AVMDDFAAFVEK (33.37 min) are high in the serum sample and are significantly low in the elution from nanoparticle sample

Fig. 3

Verification of nanoparticle capture workflow. The chemokines CCL12 and CCL28 were spiked, as internal standard process controls, in 1:10 and 10:1 ratios in ovarian cancer case and benign control sera, respectively (top), while in pre- and post-prostatectomy sera in the ratios of 10:1 and 1:10 (bottom). Each vertical bar in the histogram represents one serum sample from a cancer patient (red) or a control subject (blue), and the bar height corresponds to the peptide ion abundance measured

Fig. 4

Nanoparticle capture–mass spectrometry workflow. Sera from patients and controls are incubated with NIPAm/AAc particles, eluates from particles are analyzed by HPLC-LTQ-Orbitrap mass spectrometry, and the data analysis is performed using BioSieve and Scaffold software

Fig. 5

Venn diagram visualization of group 1 (CaP-specific proteins; increased spectral counts in pre- vs. post-prostatectomy serum) not found upregulated in OC; group 3 (OC-specific proteins; increased spectral counts in OC vs. benign control) not found upregulated in CaP; and group 2 commonly upregulated proteins in both CaP and OC

Fig. 6

MS/MS spectra for peptide analytes with statistically significant elevations. The precursor b and y ion masses are shown, as well as the matched amino acid sequence (top right for each panel) for MS/MS spectra corresponding to a a CaP-specific peptide from vitronectin precursor and b an OC-specific peptide from leucine-rich alpha 2 glycoprotein 1