Multi-omics Biomarker Pipeline Reveals Elevated Levels of Protein-glutamine Gamma-glutamyltransferase 4 in Seminal Plasma of Prostate Cancer Patients.

Seminal plasma, because of its proximity to prostate, is a promising fluid for biomarker discovery and noninvasive diagnostics. In this study, we investigated if seminal plasma proteins could increase diagnostic specificity of detecting primary prostate cancer and discriminate between high- and low-grade cancers. To select 147 most promising biomarker candidates, we combined proteins identified through five independent experimental or data mining approaches: tissue transcriptomics, seminal plasma proteomics, cell line secretomics, tissue specificity, and androgen regulation. A rigorous biomarker development pipeline based on selected reaction monitoring assays was designed to evaluate the most promising candidates. As a result, we qualified 76, and verified 19 proteins in seminal plasma of 67 negative biopsy and 152 prostate cancer patients. Verification revealed a prostate-specific, secreted and androgen-regulated protein-glutamine gamma-glutamyltransferase 4 (TGM4), which predicted prostate cancer on biopsy and outperformed age and serum Prostate-Specific Antigen (PSA). A machine-learning approach for data analysis provided improved multi-marker combinations for diagnosis and prognosis. In the independent verification set measured by an in-house immunoassay, TGM4 protein was upregulated 3.7-fold (p = 0.006) and revealed AUC = 0.66 for detecting prostate cancer on biopsy for patients with serum PSA ≥4 ng/ml and age ≥50. Very low levels of TGM4 (120 pg/ml) were detected in blood serum. Collectively, our study demonstrated rigorous evaluation of one of the remaining and not well-explored prostate-specific proteins within the medium-abundance proteome of seminal plasma. Performance of TGM4 warrants its further investigation within the distinct genomic subtypes and evaluation for the inclusion into emerging multi-biomarker panels.

Development and Validation of an Automated Zone Fluidics-Based Sensor for In Vitro Dissolution Studies of Captopril Using Total Error Concept.

In the present research, a zone fluidics-based automated sensor for the analysis of captopril in in vitro dissolution samples is reported. Captopril is reacted under flow conditions with Ni(II) (10 mmol L-1) in alkaline medium (0.15% v/v NH3) to form a stable derivate, which is monitored spectrophotometrically at 340 nm. The chemical and instrumental parameters were carefully investigated and optimized. The validation of the developed method was performed in the range of 5 to 120% of the expected maximum concentration using the accuracy profiles as a graphical decision-making tool. The ß-expectation tolerance intervals did not exceed the acceptance criteria of ±10%, which means that 95% of future results will be encompassed in the defined bias limits. The variation of the relative bias ranged between -2.3% and 3.5% and the RSD values for repeatability and intermediate precision were lower than 2.3% in all cases. The limit of detection (LOD), and the lower and the upper limit of quantification (LLOQ, ULOQ) were satisfactory and found to be 1%, 5% and 120% (corresponding to 0.6, 2.78 and 66.67 µg mL-1 in dissolution medium). The developed method was successfully applied for the analysis of captopril in dissolution tests of two commercially available batches.

Micelles Mediated Zone Fluidics Method for Hydrazine Determination in Environmental Samples.

An automated flow method for the determination of hydrazine based on the concept of zone-fluidics has been developed. The analyte reacts under flow conditions with p-dimethylamino benzaldehyde (25 mmol L-1) in micellar medium (100 mmol L-1 SDS) to form a stable derivative (460 nm). Micelles mediated catalysis excludes the use of highly acidic environment typical for this kind of reaction. Following careful examination of chemical and instrumental variables, the method allows the determination of hydrazine at the low micromolar level (0.3-10 µmol L-1) in water samples. Real sample analyses (drinking and boiler feed water) resulted in satisfactory results in terms of accuracy with the percent recoveries being in the range of 82-114%.

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays.

Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples revealed no statistically significant differences between patients with confirmed prostate cancer and negative biopsy. The presented multiplex targeted proteomic assays are an alternative analytical tool to study the biological and pathological roles of human KLKs.

Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility.

BACKGROUND: TEX101 is a cell membrane protein exclusively expressed by testicular germ cells and shed into seminal plasma. We previously verified human TEX101 as a biomarker for the differential diagnosis of azoospermia, and developed a first-of-its-kind TEX101 ELISA. To demonstrate the clinical utility of TEX101, in this work we aimed at evaluating ELISA performance in a large population of fertile, subfertile, and infertile men. METHODS: Mass spectrometry, size-exclusion chromatography, ultracentrifugation, and immunohistochemistry were used to characterize TEX101 protein as an analyte in seminal plasma. Using the optimized protocol for seminal plasma pretreatment, TEX101 was measured by ELISA in 805 seminal plasma samples. RESULTS: We demonstrated that TEX101 was present in seminal plasma mostly in a free soluble form and that its small fraction was associated with seminal microvesicles. TEX101 median values were estimated in healthy, fertile pre-vasectomy men (5436 ng/mL, N = 64) and in patients with unexplained infertility (4967 ng/mL, N = 277), oligospermia (450 ng/mL, N = 270), and azoospermia (0.5 ng/mL, N = 137). Fertile post-vasectomy men (N = 57) and patients with Sertoli cell-only syndrome (N = 13) and obstructive azoospermia (N = 36) had undetectable levels of TEX101 (≤0.5 ng/mL). A cut-off value of 0.9 ng/mL provided 100% sensitivity at 100% specificity for distinguishing pre- and post-vasectomy men. The combination of a concentration of TEX101 > 0.9 ng/mL and epididymis-specific protein ECM1 > 2.3 µg/mL provided 81% sensitivity at 100% specificity for differentiating between non-obstructive and obstructive azoospermia, thus eliminating the majority of diagnostic testicular biopsies. In addition, a cut-off value of ≥0.6 ng/mL provided 73% sensitivity at 64% specificity for predicting sperm or spermatid retrieval in patients with non-obstructive azoospermia. CONCLUSIONS: We demonstrated the clinical utility of TEX101 ELISA as a test to evaluate vasectomy success, to stratify azoospermia forms, and to better select patients for sperm retrieval.

A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9.

BACKGROUND: Kallikrein 9 (KLK9) is a member of the human kallikrein-related peptidases family, whose physiological role and implications in disease processes remain unclear. The active form of the enzyme is predicted to have chymotryptic activity. In the present study, we produced for the first time the active recombinant protein and monoclonal antibodies, and developed novel immunoassays for the quantification of free and bound KLK9 in biological samples. METHODS: The coding sequence of mature KLK9 isoform (mat-KLK9) was expressed in an Expi293F mammalian system and the synthesized polypeptide was purified through a two-step protocol. The purified protein was used as an immunogen for production of monoclonal antibodies in mice. Hybridomas were further expanded and antibodies were purified. Newly-produced monoclonal antibodies were screened for reaction with the KLK9 recombinant protein by a state-of-the-art immunocapture/parallel reaction monitoring mass spectrometry-based methodology. RESULTS: Anti-KLK9 antibodies were combined in pairs, resulting in the development of a highly sensitive (limit of detection: 15 pg/mL) and specific (no cross-reactivity with other KLKs) sandwich-type ELISA. Highest KLK9 protein levels were found in tonsil and sweat and lower levels in the heart, kidney and liver. Hybrid immunoassays using an anti-KLK9 antibody for antigen capture and various anti-serine protease inhibitor polyclonal antibodies, revealed the presence of an a1-antichymotrypsin-bound KLK9 isoform in biological samples. CONCLUSIONS: The ELISAs for free and bound forms of KLK9 may be highly useful for the detection of KLK9 in a broad range of biological samples, thus enabling the clarification of KLK9 function and use as a potential disease biomarker.

Determination of glutathione and cysteine in yeasts by hydrophilic interaction liquid chromatography followed by on-line postcolumn derivatization.

In the present study, we report a new method for the determination of two primary thiols, cysteine (CYS) and glutathione (GSH), by hydrophilic interaction LC. The polar analytes are separated isocratically using a mobile phase consisting of 65% acetonitrile/35% ammonium acetate (15 mmol/L, pH 2.0) and are detected at 285 nm following on-line postcolumn derivatization by the thiol-selective reagent methyl propiolate. The main figures of merit included linearity in the range of 5-200 µmol/L and an LOD 0.6 µmol/L for both compounds. The absence of matrix effect allowed the determination of CYS and GSH in various yeast samples. GSH was present in most of the samples at levels ranging between 0.9 and 3.1 mg/g, whereas CYS was determined in only one sample at a significantly lower concentration. In terms of accuracy, the percent recoveries ranged between 91.2 and 105.6% for GSH, and 91.6 and 106.9% for CYS.

Establishing preanalytical stability of vitamin A and vitamin E.

Vitamin A and Vitamin E, a group of lipid-soluble vitamins, can be degraded by photolysis and photooxidation after exposure to light. As an essential area of the preanalytical stage, inappropriate storage conditions of patient samples could lead to inaccurate results. In this study, we evaluated three of the most common preanalytical storage conditions (RT, +4°C, and -20 °C) in the workflow in the clinical laboratory setting using both clear and amber Eppendorf tubes. Analyte stability in each storage condition was tested with five patient pools. After 24 hrs at RT, a significant decrease in vitamin A and E was observed for samples stored in clear Eppendorf tubes (19.2 % and 17.9 %, for vitamin A and E, respectively), compared with samples stored in amber Eppendorf tubes (9.1 % and 7.3 %), respectively. After the specimen was stored at 4 °C for 7 days, vitamin A concentrations decreased in clear tubes by 14.7 % and in amber tubes by 12.7 %. Similarly, vitamin E concentrations decreased in both clear and amber tubes by 11.2 % and 13.4 %, respectively. Both vitamin A and vitamin E demonstrated acceptable stability in clear and amber Eppendorf tubes at -20 °C for up to 22 days. It is preferable to use amber tubes to protect vitamin A and E from light during short term storage. Storage of vitamin A and E specimens beyond 7 days should be at -20 °C.

Automated pre-column derivatization of thiolic fruit-antibrowning agents by sequential injection coupled to high-performance liquid chromatography using a monolithic stationary phase and an in-loop stopped-flow approach.

The present study reports the very first application of ethyl propiolate (EP) as an advantageous pre-column derivatization reagent for the determination of thiols by liquid chromatography (LC). Cysteine (CYS), glutathione (GSH) and N-acetylcysteine (NAC) were derivatized online under stopped-flow conditions in a sequential injection (SI) system coupled to HPLC. The formed derivatives were separated isocratically with a monolithic stationary phase (100×4.6 mm id) and UV detected at 285 nm. Critical parameters that affected the online pre-column derivatization reaction (e.g. the reaction time and the amount concentration of EP) and the separation (e.g. pH and the composition of the mobile phase) were investigated. The developed analytical scheme offers a total analysis time of less than 10 min, limits of detection in the range of 0.24-0.35 µmol/L and satisfactory linearity up to 200 µmol/L for all analytes. The proposed method was applied to the analysis of the selected thiols--that are often employed as antibrowning agents--in fresh fruit samples.

Biochemical characterization of human tissue kallikrein 15 and examination of its potential role in cancer.

OBJECTIVE: Human tissue kallikrein 15 (KLK15) is the last cloned member of the KLK-related gene family. Despite being implicated in multiple cancers, its pathophysiological role remains unknown. We aimed to biochemically characterize KLK15 and preliminarily study its role in cancer. DESIGN & METHODS: Recombinant KLK15 protein was produced, purified to homogeneity and quantified by mass spectrometry (parallel reaction monitoring analysis). We profiled the enzymatic activity of KLK15 using fluorogenic peptide substrates, and performed kinetic analysis to discover the cleavage sites. As KLK15 has mainly been associated with prostate cancer, we used a degradomic approach and subsequent KEGG pathway analysis to identify a number of putative protein substrates in the KLK15-treated prostate cancer cell line PC3. RESULTS: We discovered trypsin-like activity in KLK15, finding that it cleaves preferentially after arginine (R). The enzymatic activity of KLK15 was regulated by different factors such as pH, cations and serine protease inhibitors. Notably, we revealed that KLK15 most likely interacts with the extracellular matrix (ECM) receptor group. CONCLUSION: To our knowledge, this is the first study that experimentally verifies the trypsin-like activity of KLK15. We show here for the first time that KLK15 may be able to cleave many ECM components, similar to several members of the KLK family. Thus the protease could potentially be linked to tumorigenesis by promoting metastasis via this mechanism.

Selective fluorimetric method for the determination of histamine in seafood samples based on the concept of zone fluidics.

In the present article we report our results on the development of a selective automated method for the determination of histamine in seafood using the concept of zone fluidics. The method is based on the sequential on-line reaction of the analyte with o-phthalaldehyde in the absence of a nucleophilic reagent, followed by acidification. The careful selection of the chemical and instrumental variables enabled the determination of the analyte with adequate sensitivity at the low micromolar level and with specificity against other biogenic amines and amino acids such as histidine. The LOD was 0.05 µmol L(-1) (0.6 mg kg(-1)) and linearity was obeyed in the range of 0.5-15 µmol L(-1) (5.5-170 mg kg(-1)). The proposed method offers a satisfactory sampling rate of 15 h(-1) and adequate accuracy and precision for the analysis of seafood products after minimum sample preparation and without employing a separation technique.

Zwitterionic hydrophilic interaction chromatography coupled with post-column derivatization for the analysis of glutathione in wine samples.

In this study the development, validation and application of a new chromatographic method for the determination of glutathione (GSH) in wine samples is presented. The separation of the GSH was carried out using a sulfobetaine-based hydrophilic interaction chromatography (HILIC) analytical column whereas its detection was carried out spectrofluorimetrically (λext/λem=340/455 nm) after post-column derivatization with o-phthalaldehyde. GSH was separated efficiently from matrix endogenous compounds of wines by using a mobile phase of 15 mmol L(-1) CH3COONH4 (pH=2.5)/CH3CN, 35/65% (v/v). The parameters of the post-column reaction (pH, amount concentration of the reagent and buffer solution, flow rate, length of the reaction coil) were investigated. The linear determination range for GSH was 0.25-5.0 µmol L(-1) and the LOD was 19 nmol L(-1). No matrix effect was observed, while the accuracy was evaluated with recovery experiments and was ranged between 89% and 108%.

Automated determination of total captopril in urine by liquid chromatography with post-column derivatization coupled to on-line solid phase extraction in a sequential injection manifold.

The present study reports a new liquid chromatographic (HPLC) method for the determination of the anti-hypertension drug captopril (CAP) in human urine. After its separation from the sample matrix in a reversed phase HPLC column, CAP reacts with the thiol-selective reagent ethyl-propiolate (EP) in a post-column configuration and the formed thioacrylate derivative is detected at 285 nm. Automated 4-fold preconcentration of the analyte prior to analysis was achieved by an on-line solid phase extraction (SPE) step using a sequential injection (SI) manifold. The Oasis HLB SPE cartridges offered quantitative recoveries and effective sample cleaning by applying a simple SPE protocol. The limits of detection and quantitation were 10 µg L(-1) and 35 µg L(-1) respectively. The percent recoveries for the analysis of human urine samples ranged between 90 and 96% and 95 and 104% using aqueous and matrix matched calibration curves respectively.

Automated tagging of pharmaceutically active thiols under flow conditions using monobromobimane.

The thiol-specific derivatization reagent monobromobimane (MBB) is applied--for the first time--under flow conditions. Sequential injection analysis allows the handling of precise volumes of the reagent in the micro-liter range. The effect of the main chemical and instrumental variables was investigated using captopril (CAP), N-acetylcysteine (NAC) and penicillamine (PEN) as representative pharmaceutically active thiols. Previously reported hydrolysis of MBB due to interaction with nucleophilic components of the buffers was avoided kinetically under flow conditions. The proposed analytical scheme is suitable for the fluorimetric determination of thiols at a sampling rate of 36 h(-1).