Sampling and Analytical Strategies for Biomarker Discovery Using Mass Spectrometry

There is an often unspoken truth behind the course of scientific investigation that involves not what is necessarily academically worthy of study, but rather what is scientifically worthy in the eyes of funding agencies. The perception of worthy research is, as cost is driven in the simplest sense in economics, often driven by demand. Presently, the demand for novel diagnostic and therapeutic protein biomarkers that possess high sensitivity and specificity is placing major impact on the field of proteomics. The focal discovery technology that is being relied on is mass spectrometry (MS), whereas the challenge of biomarker discovery often lies not in the application of MS but in the underlying proteome sampling and bioinformatic processing strategies. Although biomarker discovery research has been historically technology-driven, it is clear from the meager success in generating validated biomarkers that increasing attention must be placed at the pre-analytic stage, such as sample retrieval and preparation. As diseases vary, so do the combinations of sampling and sample analyses necessary to discover novel biomarkers. In this review, we highlight different strategies used toward biomarker discovery and discuss them in terms of their reliance on technology and methodology

Proteomic Analysis of Ovarian Cancer Proximal Fluids: Validation of Elevated Peroxiredoxin 1 in Patient Peripheral Circulation

Background: Epithelial ovarian cancer (EOC) is the deadliest gynecologic malignancy in the United States. Unfortunately, a validated protein biomarker-screening test to detect early stage disease from peripheral blood has not yet been developed. The present investigation assesses the ability to identify tumor relevant proteins from ovarian cancer proximal fluids, including tissue interstitial fluid (TIF) and corresponding ascites, from patients with papillary serous EOC and translates these findings to targeted blood-based immunoassays. Methodology/Principal Findings: Paired TIF and ascites collected from four papillary serous EOC patients at the time of surgery underwent immunodepletion, resolution by 1D gel electrophoresis and in-gel digestion for analysis by liquid chromatography-tandem mass spectrometry, which resulted in an aggregate identification of 569 and 171 proteins from TIF and ascites, respectively. Of these, peroxiredoxin I (PRDX1) was selected for validation in serum by ELISA and demonstrated to be present and significantly elevated (p = 0.0188) in 20 EOC patients with a mean level of 26.0 ng/mL (±9.27 SEM) as compared to 4.19 ng/mL (±2.58 SEM) from 16 patients with normal/benign ovarian pathology. Conclusions/Significance: We have utilized a workflow for harvesting EOC-relevant proximal biofluids, including TIF and ascites, for proteomic analysis. Among the differentially abundant proteins identified from these proximal fluids, PRDX1 was demonstrated to be present in serum and shown by ELISA to be elevated by nearly 6-fold in papillary serous EOC patients relative to normal/benign patients. Our findings demonstrate the facile ability to discover potential EOC-relevant proteins in proximal fluids and confirm their presence in peripheral blood serum. In addition, our finding of elevated levels of PRDX1 in the serum of EOC patients versus normal/benign patients warrants further evaluation as a tumor specific biomarker for EOC. © 2011 Hoskins et al

Preformulation and stability in biological fluids of the retrocyclin RC-101, a potential anti-HIV topical microbicide

<p>Abstract</p> <p>Background</p> <p>RC-101, a cationic peptide retrocyclin analog, has <it>in vitro </it>activity against HIV-1. Peptide drugs are commonly prone to conformational changes, oxidation and hydrolysis when exposed to excipients in a formulation or biological fluids in the body, this can affect product efficacy. We aimed to investigate RC-101 stability under several conditions including the presence of human vaginal fluids (HVF), enabling the efficient design of a safe and effective microbicide product. Stability studies (temperature, pH, and oxidation) were performed by HPLC, Circular Dichroism, and Mass Spectrometry (LC-MS/MS). Additionally, the effect of HVF on formulated RC-101 was evaluated with fluids collected from healthy volunteers, or from subjects with bacterial vaginosis (BV). RC-101 was monitored by LC-MS/MS for up to 72 h.</p> <p>Results</p> <p>RC-101 was stable at pH 3, 4, and 7, at 25 and 37°C. High concentrations of hydrogen peroxide resulted in less than 10% RC-101 reduction over 24 h. RC-101 was detected 48 h after incubation with normal HVF; however, not following incubation with HVF from BV subjects.</p> <p>Conclusions</p> <p>Our results emphasize the importance of preformulation evaluations and highlight the impact of HVF on microbicide product stability and efficacy. RC-101 was stable in normal HVF for at least 48 h, indicating that it is a promising candidate for microbicide product development. However, RC-101 stability appears compromised in individuals with BV, requiring more advanced formulation strategies for stabilization in this environment.</p

Supramolecular organizations in the aerobic respiratory chain of Escherichia coli

The organization of respiratory chain complexes in supercomplexes has been shown in the mitochondria of several eukaryotes and in the cell membranes of some bacteria. These supercomplexes are suggested to be important for oxidative phosphorylation efficiency and to prevent the formation of reactive oxygen species. Here we describe, for the first time, the identification of supramolecular organizations in the aerobic respiratory chain of Escherichia coli, including a trimer of succinate dehydrogenase. Furthermore, two heterooligomerizations have been shown: one resulting from the association of the NADH:quinone oxidoreductases NDH-1 and NDH-2, and another composed by the cytochrome bo3 quinol:oxygen reductase, cytochrome bd quinol:oxygen reductase and formate dehydrogenase (fdo). These results are supported by blue native-electrophoresis, mass spectrometry and kinetic data of wild type and mutant E . coli strains

Liquid Tissue: Proteomic Profiling of Formalin-Fixed Tissues

Identification and quantitation of candidate biomarker proteins in large numbers of individual tissues is required to validate specific proteins, or panels of proteins, for clinical use as diagnostic, prognostic, toxicological, or therapeutic markers. Mass spectrometry (MS) provides an exciting analytical methodology for this purpose. Liquid Tissue MS protein preparation allows researchers to utilize the vast, already existing, collections offormalin-fixed paraffin-embedded (FFPE) tissues for the procurement of peptides and the analysis across a variety of MS platforms

Mitochondrial respiration - an important therapeutic target in melanoma

The importance of mitochondria as oxygen sensors as well as producers of ATP and reactive oxygen species (ROS) has recently become a focal point of cancer research. However, in the case of melanoma, little information is available to what extent cellular bioenergetics processes contribute to the progression of the disease and related to it, whether oxidative phosphorylation (OXPHOS) has a prominent role in advanced melanoma. In this study we demonstrate that compared to melanocytes, metastatic melanoma cells have elevated levels of OXPHOS. Furthermore, treating metastatic melanoma cells with the drug, Elesclomol, which induces cancer cell apoptosis through oxidative stress, we document by way of stable isotope labeling with amino acids in cell culture (SILAC) that proteins participating in OXPHOS are downregulated. We also provide evidence that melanoma cells with high levels of glycolysis are more resistant to Elesclomol. We further show that Elesclomol upregulates hypoxia inducible factor 1-α (HIF-1α), and that prolonged exposure of melanoma cells to this drug leads to selection of melanoma cells with high levels of glycolysis. Taken together, our findings suggest that molecular targeting of OXPHOS may have efficacy for advanced melanoma. © 2012 Barbi de Moura et al

Loss of the integrin-activating transmembrane protein Fam38A (Piezo1) promotes a switch to a reduced integrin-dependent mode of cell migration

Lung cancer is one of the most common fatal diseases in the developed world. The disease is rarely cured by currently available therapies, with an overall survival rate of ∼10%. Characterizing novel proteins that offer crucial insights into the processes of lung tumour invasion and metastasis may therefore provide much-needed prognostic markers, and influence therapeutic strategies. Aberrant function of the integrin family of heterodimeric cell surface receptors is a common theme in cancer--investigation into novel integrin activity regulators may offer crucial insights into the processes of tumour invasion and metastasis and may reveal insights into potential therapeutic targets. We previously described that depletion of the novel multi-transmembrane domain protein Fam38A, located at the endoplasmic reticulum (ER), inactivates endogenous beta1 integrin affinity, reducing cell adhesion. We now show that depletion of Fam38A, also now known as Piezo1, causes anchorage independence and a switch to a reduced integrin-dependent mode of cell migration/invasion, a novel phenotype for this integrin-regulating protein. Normal lung epithelial cells show increased rates of migration by 2D time-lapse microscopy and increased capacity to invade into matrigel, despite having decreased integrin affinity. We confirm greatly depleted Fam38A expression in small cell lung cancer (SCLC) lines where a form of reduced integrin-dependent migration, i.e. amoeboid migration, is a known phenotype. We propose that loss of Fam38A expression may cause increased cell migration and metastasis in lung tumours

Identifier mapping performance for integrating transcriptomics and proteomics experimental results

Background\ud Studies integrating transcriptomic data with proteomic data can illuminate the proteome more clearly than either separately. Integromic studies can deepen understanding of the dynamic complex regulatory relationship between the transcriptome and the proteome. Integrating these data dictates a reliable mapping between the identifier nomenclature resultant from the two high-throughput platforms. However, this kind of analysis is well known to be hampered by lack of standardization of identifier nomenclature among proteins, genes, and microarray probe sets. Therefore data integration may also play a role in critiquing the fallible gene identifications that both platforms emit.\ud \ud Results\ud We compared three freely available internet-based identifier mapping resources for mapping UniProt accessions (ACCs) to Affymetrix probesets identifications (IDs): DAVID, EnVision, and NetAffx. Liquid chromatography-tandem mass spectrometry analyses of 91 endometrial cancer and 7 noncancer samples generated 11,879 distinct ACCs. For each ACC, we compared the retrieval sets of probeset IDs from each mapping resource. We confirmed a high level of discrepancy among the mapping resources. On the same samples, mRNA expression was available. Therefore, to evaluate the quality of each ACC-to-probeset match, we calculated proteome-transcriptome correlations, and compared the resources presuming that better mapping of identifiers should generate a higher proportion of mapped pairs with strong inter-platform correlations. A mixture model for the correlations fitted well and supported regression analysis, providing a window into the performance of the mapping resources. The resources have added and dropped matches over two years, but their overall performance has not changed.\ud \ud Conclusions\ud The methods presented here serve to achieve concrete context-specific insight, to support well-informed decisions in choosing an ID mapping strategy for "omic" data merging

Experimental investigation on the effect of wear flat inclination on the cutting response of a blunt tool in rock cutting

A vast majority of experimental researches focuses on the cutting action of a sharp cutter, while there has been limited experimental work devoted to the study of the contact process at the wear flat-rock interface. The specific objective of this study is to determine the effect of the wear flat inclination angle ( β ) with respect to the cutter velocity vector ( vv ) on both the contact stress ( σ ) and friction coefficient ( μ ) mobilized at the wear flat-rock interface. An extensive and comprehensive set of cutting experiments was carried out on thirteen different sedimentary quarry rock samples using a state-of-the-art rock cutting equipment. A unique cutter holder was purposely designed and manufactured along with a precise experimental protocol implemented in order to change the back rake angle and therefore the inclination β by steps of 0.10∘ . The experimental observations confirm the existence of three regimes of frictional contact (identified as elastic, elasto-plastic and plastic) for all rock samples. Further, the results suggest that the scaled contact stress is predominantly controlled by a dimensionless number η=E∗tanβq with E∗ the plane strain elastic modulus and q the rock strength

CMB-S4: Forecasting Constraints on Primordial Gravitational Waves

CMB-S4---the next-generation ground-based cosmic microwave background (CMB) experiment---is set to significantly advance the sensitivity of CMB measurements and enhance our understanding of the origin and evolution of the Universe, from the highest energies at the dawn of time through the growth of structure to the present day. Among the science cases pursued with CMB-S4, the quest for detecting primordial gravitational waves is a central driver of the experimental design. This work details the development of a forecasting framework that includes a power-spectrum-based semi-analytic projection tool, targeted explicitly towards optimizing constraints on the tensor-to-scalar ratio, $r$, in the presence of Galactic foregrounds and gravitational lensing of the CMB. This framework is unique in its direct use of information from the achieved performance of current Stage 2--3 CMB experiments to robustly forecast the science reach of upcoming CMB-polarization endeavors. The methodology allows for rapid iteration over experimental configurations and offers a flexible way to optimize the design of future experiments given a desired scientific goal. To form a closed-loop process, we couple this semi-analytic tool with map-based validation studies, which allow for the injection of additional complexity and verification of our forecasts with several independent analysis methods. We document multiple rounds of forecasts for CMB-S4 using this process and the resulting establishment of the current reference design of the primordial gravitational-wave component of the Stage-4 experiment, optimized to achieve our science goals of detecting primordial gravitational waves for $r > 0.003$ at greater than $5\sigma$, or, in the absence of a detection, of reaching an upper limit of $r < 0.001$ at $95\%$ CL.Comment: 24 pages, 8 figures, 9 tables, submitted to ApJ. arXiv admin note: text overlap with arXiv:1907.0447