Quantitative proteomic analysis of a genetically induced prostate inflammation mouse model via custom 4-plex DiLeu isobaric labeling

Abstract

Inflammation is involved in many prostate pathologies including infection, benign prostatic hyperplasia, and prostate cancer. Preclinical models are critical to our understanding of disease mechanisms, yet few models are genetically tractable. Here, we present a comparative quantitative proteomic analysis of urine from mice with and without prostate-specific inflammation induced by conditional prostate epithelial IL-1β expression. Relative quantification and sample multiplexing was achieved using custom 4-plex N,N-dimethyl leucine (DiLeu) isobaric tags and nanoflow ultrahigh-performance liquid chromatography coupled to high-resolution tandem mass spectrometry. Each set of 4-plex DiLeu reagents allows four urine samples to be analyzed simultaneously, providing high-throughput and accurate quantification of urinary proteins. Proteins involved in the acute phase response, including haptoglobin, inter-α-trypsin inhibitor, and α₁-antitrypsin 1-1, were differentially represented in the urine of mice with prostate inflammation. Mass spectrometry-based quantitative urinary proteomics represents a promising bioanalytical strategy for biomarker discovery and the elucidation of molecular mechanisms in urological research.

Keywords: benign prostatic hyperplasia; inflammation; interleukin-1β; lower urinary tract symptoms, mass spectrometry; urine proteomics.

Fig. 1.

Overall workflow of quantitative urinary proteomics analysis using 4-plex N,N-dimethyl leucine (DiLeu) labeling. Data analysis was conducted using Proteome Discoverer software followed by offline analysis. MWCO, molecular weight cutoff; LC-MS/MS, liquid chromotography tandem mass spectroscopy.

Fig. 2.

Histological analysis of IL-1β-mediated inflammation in induced mouse prostate inflammation-IL-1-driven (IMPI-1) prostate lobes at 6.5 wk after doxycycline administration. A and B: ventral IMPI-1 prostate from an animal maintained on normal drinking water. C and D: ventral IMPI-1 prostate from an animal maintained continuously on doxycycline-supplemented drinking water (2 mg/ml). Note the presence of pockets of inflammatory cells in the stroma and epithelium. Original magnifications: ×10 for A and C and ×25 for B and D.

Fig. 3.

Volcano plot of mouse urine proteins; -log₁₀ P value (paired Student's t-test) versus log₂ of the prostatic IL-1β induced-to-baseline ratio.

Fig. 4.

Example MS/MS fragmentation of N,N-dimethyl leucine (DiLeu) labeled urinary peptide representing major urinary protein 3. The b- and y-product ions represent the backbone fragmentation of the peptide for identification. The intensities of reporter ions are used for relative quantification.

Fig. 5.

Partial least-squares discriminant analysis score plot of all identified and quantified mouse prostate inflammation urinary proteins. Each data point represents a biological replicate at baseline or induced, averaged across technical replicates.

Fig. 6.

PANTHER overrepresentation test: 261 total mouse prostate inflammation urinary proteins; binomial test with Bonferroni correction, α = 0.05 for induced to baseline versus the program-generated reference mouse proteome. Gray shading corresponds to Fig. 7. GO, Gene Ontology.

Fig. 7.

PANTHER overrepresentation test: 261 total mouse prostate inflammation urinary proteins; binomial test with Bonferroni correction, α = 0.05 for induced to baseline versus the program-generated reference mouse proteome. Categories were manually assigned from Fig. 6.