Immuno–MS Based Targeted Proteomics: Highly Specific, Sensitive, and Reproducible Human Chorionic Gonadotropin Determination for Clinical Diagnostics and Doping Analysis

The human chorionic gonadotropin (hCG) proteins constitute a diverse group of molecules that displays biomarker value in pregnancy detection and cancer diagnostics, as well as in doping analysis. For the quantification of hCGβ and qualitative differentiation between other hCG variants in a selective, sensitive, and reproducible manner, the targeted proteomics approach based on mass spectrometric (MS) selected reaction monitoring (SRM) detection was exploited. By optimizing immunoaffinity extraction using monoclonal antibodies coated to magnetic beads, access was granted for the MS to the low-abundance target proteins, ensuring proper sensitivity with limits of detection (LODs) of 2 and 5 IU/L, respectively, for urine and serum samples. Validation according to key elements and recommendations defined by the European Medicines Agency in <i>Guideline on Validation of Bioanalytical Methods</i> was performed. For both matrixes this demonstrated good within-day precision results (within 20% for the lowest concentration, and within 15% for the medium and high concentration), good accuracy results (within 15% for all concentrations), and proper linearity, >0.997 for serum and of 0.999 for urine, in the concentration range up to 5000 IU/L. The method’s application in clinical diagnostics was tested on samples from a pregnant woman and from patients previously diagnosed with testicular cancer. For doping analysis, samples from one man having received injection of the hCG-containing pharmaceutical Pregnyl were analyzed. The method proved to be quantitatively accurate with indisputable identification specificity, reducing risks of false positive and false negative results. The successfully validated method advocates thus for more extended use of MS in routine analysis

Multiplexing Determination of Small Cell Lung Cancer Biomarkers and Their Isovariants in Serum by Immunocapture LC-MS/MS

A multiplex method for the determination of the small cell lung cancer (SCLC) markers progastrin releasing peptide (ProGRP) and neuron specific enolase (NSE) is presented, which involves coextraction by immunoaffinity (IA) beads and codetermination by selected reaction monitoring (SRM). The performance was compared with two IA SRM methods which were recently validated for individual marker determination. The multiplexing method reduces sample volume, handling time per sample, and reagent consumption and shows good linearity, recovery, quantitative measurements, and sensitivity with lower limit of detection (LLOD) values of 7.2 pM (=90 pg/mL) and 4.5 pM (=210 pg/mL) and lower limit of quantitation (LLOQ) values of 24 pM (=300 pg/mL) and 15 pM (=700 pg/mL), for total ProGRP and γ-NSE, respectively. The novel aspect of this approach is the multiplexing of ProGRP and NSE with the additional ability to perform fingerprinting by the selective determination of ProGRP isoform 1, ProGRP isoform 3, and total ProGRP, as well as the α- and the γ-subunit of NSE isoenzymes. Six serum samples from patients with SCLC were analyzed to demonstrate the methods feasibility to simultaneously differ between and individually quantify ProGRP, NSE, and their isoform and isoenzyme variants, respectively. Both the presence of and variation between all the isoforms and isoenzymes, as well as covarying results with the conventional immunometric assays for total ProGRP and γ-NSE, were seen in the analyses of patient serum samples

DHT upregulates AR expression and increases PSA secretion in LNCaP cells.

<p>(<b>A</b>) DHT upregulates AR expression in LNCaP cells in both time-dependent and concentration-dependent manners (left panel); there are also higher levels of PSA in the DHT treated cells revealed by the ELISA method (right panel). (<b>B</b>) Immunocytochemistry shows nuclear positivity of AR in LNCaP cells and its expression is upregulated by DHT treatment; but AR expression is negative in the PC-3 cells with/without DHT treatment (bar scale: 50 µm).</p

Stem-like phenotype analyses by flow cytometry.

<p>(A–C) CD44, CD24 and CD90 expressions were analyzed by flow cytometry in LNCaP and PC-3 cells cultivated with/without DHT in 10 nM for 48 hours. CD44 was induced by DHT treatment in both cell lines (A). No statistically significant difference of CD24 expression level was shown in both cell lines (B). CD90 expression was significantly increased by DHT stimulation in both cell lines (C). (* means <i>P</i><0.05).</p

Clinical and pathologic characteristics for 117 patients with malignant prostate cancer.

<p>PSA, prostatic specific antigen.</p

DHT induces cell growth and clonogenicity in prostate cancer cell lines.

<p>(A) Cell growth curves show statistically significant difference in LNCaP cells with/without DHT treatment, but not in PC-3 cells (* means <i>P</i><0.05). (B) Representative photographs of colony formation in both cell lines demonstrate that more colonies were formed by 1 nM DHT treatment and even more colonies were obtained by 10 nM DHT treatment (bar scale: 50 mm). (C) Histograms of colony formation efficiency show statistically higher efficiencies in the cells treated with low concentration of DHT (<i>P</i><0.01), and even higher efficiencies in the cells by high concentration of DHT (<i>P</i><0.001). (D) Representative photographs for sphere formation for both cell lines in the cells with/without DHT treatment (bar scale: 50 µm). (E) Histograms for sphere formation efficiency show higher efficiencies in the cells added with 1 nM DHT (<i>P</i><0.05), and even higher efficiencies in the cells stimulated with 10 nM DHT (<i>P</i><0.01).</p

Immunohistochemistry of SHBG expression in prostate cancer tissues.

<p>(A) Positive and negative control of SHBG in liver tissues. (B) Weak SHBG positivity is shown in a benign prostate tumor and strong SHBG immunoreactivity is revealed in a malignant Gleason score 8 cancer tissue. (C) Representative immunohistochemical images of different scores of prostate cancer samples are shown. Bar scale in all of the images is 150 µm.</p