A Proteomics Signature of Mild Hypospadias: A Pilot Study

Abstract

Background and Objective: Mild hypospadias is a birth congenital condition characterized by the relocation of the male urethral meatus from its typical anatomical position near the tip of the glans penis, to a lower ventral position up to the brim of the glans corona, which can also be accompanied by foreskin ventral deficiency. For the most part, a limited number of cases have known etiology. We have followed a high-throughput proteomics approach to study the proteome in mild hypospadias patients. Methods: Foreskin samples from patients with mild hypospadias were collected during urethroplasty, while control samples were collected during elective circumcision (n = 5/group). A high-throughput, quantitative proteomics approach based on multiplexed peptide stable isotope labeling (SIL) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to ascertain protein abundance changes in hypospadias patients when compared to control samples. Results: A total of 4,815 proteins were quantitated (2,522 with at least two unique peptides). One hundred and thirty-three proteins from patients with mild hypospadias showed significant abundance changes with respect to control samples, where 38 proteins were increased, and 95 proteins were decreased. Unbiased functional biological analysis revealed that both mitochondrial energy production and apoptotic signaling pathways were enriched in mild hypospadias. Conclusions: This first comprehensive proteomics characterization of mild hypospadias shows molecular changes associated with essential cellular processes related to energy production and apoptosis. Further evaluation of the proteome may expand the search of novel candidates in the etiology of mild hypospadias and could also lead to the identification of biomarkers for this congenital urogenital condition.

Keywords: etiology; hypospadias; liquid chromatography-tandem mass spectrometry (LC-MS/MS); proteomics; quantitative proteomics.

Figure 1

Proteomics study design. Foreskin samples were collected during scheduled urethroplasty from patients with mild hypospadias (n = 5) or from individuals submitted to a circumcision procedure (control group, n = 5). One-hundred milligrams of foreskin samples from each patient were homogenized and proteins extracted from tissue. A total of 100 μg of protein was processed per sample. Protein extracts were digested with trypsin using the FASP technology. Subsequently, peptide samples were labeled using a 10-plex TMT kit. Each sample was labeled with a specific TMT mass tag. After labeling, samples were pooled and separated into five fractions using high pH reversed-phase peptide fractionation. Peptides were resuspended in 0.1% formic acid and analyzed by LC-MS/MS. Upon fragmentation, the specific reporter ion released from each of the 10 mass tags shows in the low-mass region of the MS/MS spectrum. The intensity of this reporter ion was used to measure relative peptide abundance levels among samples, from which relative protein abundance between control and mild hypospadias cases was inferred (see text for details).

Figure 2

Mild hypospadias protein profile differs from controls. Zq values from a total of 2,522 proteins identified with two or more peptides were subjected to PCA and PLS-DA. (A) Score plot for PC1 (29.2% variance explained) vs. PC2 (18.1% variance explained). (B) Score plot for PLS1 (17.5% variance explained) vs. PLS2 (21.9% variance explained). The ellipses illustrate 95% confidence region for these groups. n = 5/group.

Figure 3

Proteins found downregulated in the proteome from mild hypospadias patients. Ninety-five proteins were found decreased between control and mild hypospadias samples. Heatmaps depict protein abundance changes in terms of Zq across samples (C1-C5 for control and H1-H5 for mild hypospadias samples). The magnitude of abundance change [increased (red) or decreased (blue)] is shown according to the color scale on the right.

Figure 4

Proteins found upregulated in the proteome from mild hypospadias patients. Thirty-eight proteins were found increased between control and mild hypospadias samples. Heatmaps depict protein abundance changes in terms of Zq across samples (C1-C5 for control and H1-H5 for mild hypospadias samples). The magnitude of abundance change [increased (red) or decreased (blue)] is shown according to the color scale on the right.

Figure 5

Validation of cytochrome C by ELISA. Cytochrome C was found in significantly lower concentrations in mild hypospadias samples (median = 8.86 ng/ml) in comparison to controls (median = 12.56 ng/ml) samples in agreement with proteomics results. Dashed line represents the median value. Dotted lines represent quartiles, top: 75% percentile and bottom: 25% percentile. ** = p < 0.05. Controls, n = 10; Mild hypospadias, n = 8.