Relative Quantitation of Proteins in Expressed Prostatic Secretion with a Stable Isotope Labeled Secretome Standard

Expressed prostatic secretion (EPS) is a proximal fluid directly derived from the prostate and, in the case of prostate cancer (PCa), is hypothesized to contain a repertoire of cancer-relevant proteins. Quantitative analysis of the EPS proteome may enable identification of proteins with utility for PCa diagnosis and prognosis. The present investigation demonstrates selective quantitation of proteins in EPS samples from PCa patients using a stable isotope labeled proteome standard (SILAP) generated through the selective harvest of the “secretome” from the PC3 prostate cancer cell line grown in stable isotope labeled cell culture medium. This stable isotope labeled secretome was digested with trypsin and equivalently added to each EPS digest, after which the resultant mixtures were analyzed by liquid chromatography–tandem mass spectrometry for peptide identification and quantification. Relative quantification of endogenous EPS peptides was accomplished by comparison of reconstructed mass chromatograms to those of the chemically identical SILAP peptides. A total of 86 proteins were quantified from 263 peptides in all of the EPS samples, 38 of which were found to be relevant to PCa. This work demonstrates the feasibility of using a SILAP secretome standard to simultaneously quantify many PCa-relevant proteins in EPS samples

HO-1, TACO-1, and HIF-1α expression in Elesclomol-treated melanoma cells.

<p>(<b>A</b>) Whole-cell (WC), (<b>B</b>) mitochondrial and WC, and (<b>C</b>) nuclear (Nu) and cytoplasmic (Cy) lysates, prepared from WM1158 metastatic melanoma cells following treatment with increasing doses of Elesclomol (ELM). Controls were WM1158 melanoma cells that received the drug vehicle DMSO, or no treatment (no tx). The blots were probed with antibody to HO-1, TACO-1, HIF-1α, or α-tubulin, which served as loading control. LDH5 was used as a cytoplasmic protein control.</p

Basal OCR in relation to the ECAR in short-term cultures of human melanocytes, and primary and metastatic melanoma cell lines.

<p>Depicted are HEMs (red symbol), primary (green symbol) and metastatic melanoma cell lines (blue symbol), and two Vemurafenib-resistant melanoma cell lines (dark blue symbol). The two melanoma cell lines derived from tumors of a same patient are depicted by open circles.</p

Phase-contrast analysis of Elesclomol-treated melanoma cells.

<p>Phase-contrast images of a pigmented (WM852) and an amelanotic (C32) melanoma cell line treated for 12 hr with drug vehicle (DMSO), or a low (20 nM), or high dose (500 nM) of Elesclomol (ELM). (Images were captured at 20× magnification).</p

Selection of Elesclomol-resistant cells.

<p>(<b>A</b>) Following 60 days of every second day treatment of WM983-B cells with Elesclomol, a bioenergetics analysis was performed to measure the effects of continuous Elesclomol treatment upon OXPHOS and glycolysis. The arrow points to increased baseline ECAR in response to Elesclomol treatment for 60 days. (<b>B</b>) Measurement of steady-state ATP levels in WM983-B cells treated for 60 days with Elesclomol or only PBS.</p

Proteins from each of the Top 3 most significantly dysregulated pathways identified by SILAC and subsequent IPA-Tox analysis of WM1158 cells treated with Elesclomol (E) or the drug vehicle DMSO (V).

<p>u - proteins identified by unique peptides; c - proteins identified by common peptides.</p

Analysis of ρ0 melanoma cells.

<p>(<b>A</b>) Equal amounts of DNA isolated from each cell line (parental; ρ0) were analyzed by qPCR for a small (0.22 kb) mitochondrial sequence (primer set 14,620/14,841), a large (8.9 kb) mitochondrial sequence (primer set 5,999/14,841), and a 12.2 kb DNA polymerase β primer set serving as a positive control for nuclear gene expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040690#pone.0040690-Qian1" target="_blank">[25]</a>. qPCR samples not containing DNA or primer sets served as negative controls. (<b>B</b>) Pharmacologic profile of OCR and ECAR of parental (solid circles) and ρ0 (open circles) WM1158 melanoma cell lines as determined by the Seahorse X24 analyzer. (<b>C</b>) Three<b>-</b>day MTT proliferation analysis of WM1158, WM983-B, and WM852 ρ0 and parental cells (no ethidium bromide, EtBr).</p

Bioenergetics analysis of melanoma cells.

<p>(<b>A</b>) Seahorse XF24 Flux analysis of Lu1205 and WM983-B metastatic melanoma cells treated for 2 hr with increasing doses of Elesclomol salt (ELM) (20, 60, 100, or 200 nM). After baseline OCR and ECAR determination, the cells were treated with oligomycin (O), FCCP (F), rotenone (R), or 2-deoxyglycose (2DG). Melanoma cells sensitive to Elesclomol, which had low reserve capacity and could not upregulate oxygen consumption in response to FCCP, are indicated by arrows. (<b>B</b>) Seahorse XF24 analysis of WM1158 cells and Vemurafenib-resistant melanoma cell lines (TPF10-741; TPF11-43) treated for 2 hr with 200 nM of Elesclomol salt (ELM) or only PBS. (C) Analysis of mitochondrial membrane potential in WM983-A and WM983-B using TMRM fluorescence following increasing doses of Elesclomol salt (20, 60, 100, 200, 500 nM) in the presence or absence of copper (5 µM).</p

Top 5 most significantly dysregulated pathways identified by SILAC and subsequent IPA-Tox analysis.

<p>WM1158 cells treated for 4 hr with Elesclomol (500 nM) or only DMSO.</p