A multiplexed, automated immuno-matrix assisted laser desorption/ionization mass spectrometry assay for simultaneous and precise quantitation of PTEN and p110 alpha in cell lines and tumor tissues

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110 alpha is the catalytic alpha-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110 alpha protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110 alpha concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110 alpha-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110 alpha protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 mu g of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.Proteomic

The Effect of Pre-Analytical Variability on the Measurement of MRM-MS-Based Mid- to High-Abundance Plasma Protein Biomarkers and a Panel of Cytokines

Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies

Serum S100A6 Concentration Predicts Peritoneal Tumor Burden in Mice with Epithelial Ovarian Cancer and Is Associated with Advanced Stage in Patients

BACKGROUND:Ovarian cancer is the 5th leading cause of cancer related deaths in women. Five-year survival rates for early stage disease are greater than 94%, however most women are diagnosed in advanced stage with 5 year survival less than 28%. Improved means for early detection and reliable patient monitoring are needed to increase survival. METHODOLOGY AND PRINCIPAL FINDINGS:Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of S100A6 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. S100A6 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100A6 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as S100A6 was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease. CONCLUSIONS:S100A6 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. S100A6 is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100A6 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers

Mycorrhizas in South American Anthropic Environments

The agricultural expansion has leaded to increase the irrigated cropland area and the use of fertilizers, resulting in water degradation, increased energy use, and common pollution. Of particular concern is the increased interest to reduce the environmental impacts of high quantities of water dedicated to irrigation by agricultural activities We are now truly recognizing the importance of sustainable measures in agriculture such as conservation of the vegetation cover and management approach to understand surface and deep soil responses to global change. The agroecology management based on key processes from natural ecosystems can help to solve some agricultural difficulties. Increasing studies on the Arbuscular mycorrhizal fungi (AMF) has showed their importance for soil ecology and studies on their biodiversity have spread in some agro-ecosystems such as corn and soybean monocultures. Therefore, it is needed to deeply study the mycorrhizal functions under global change. In this chapter, we examine the major developments and advances on mycorrhizal fungi based on recent research from South American countries. New reports on the occurrence of mycorrhizas in Amazonian dark earth, as well as the inoculum production of arbuscular mycorrhizal fungi native of soils under native forest covers, have resulted in a more detailed understanding of the soil biology from South America. Reports from Amazonian dark earth or “Terra preta do índio” soil has stimulated the use of biochar worldwide as a soil conditioner that can add value to non-harvested agricultural products and promote plant growth. Few reports from Brazil showed that the addition of inorganic fertilizer, compost and chicken manure resulted in increases in plant cover and plant species richness. In this sense, the biochar/mycorrhizae interactions also can be prioritized for sequestration of carbon in soils to contribute to climate change mitigation

Comparison of cytokine levels between tube types and processing protocols.

<p>Concentration of 20 cytokines in pg/ml from plasma collected in K₂EDTA tubes, P100 tubes processed with different protocols and CTAD tubes. All tubes were processed immediately after collection (T0). Brackets depict groups compared using paired t-tests and for which statistical significance * p<0.05 was found.</p

Cytokine levels at time 0.

<p>Concentration of cytokines in pg/ml from plasma collected in K₂EDTA (white bars) and P100 (black bars) tubes processed immediately after collection (T0) using protocol A. The levels of 20 cytokines detected using the Bio-Plex® Assay are shown (RANTES levels not included in the figure, concentration is off scale). Paired t-test comparisons were performed for each cytokine between tube types. * p<0.05, <b>+</b> cytokines not detected in K₂EDTA samples.</p

Percent differences in peptides measured in P100 and kEDTA tubes compared to CTAD tubes.

*<p>p<0.05.</p>**<p>p<0.001.</p

Systematic Optimization of the iMALDI Workflow for the Robust and Straightforward Quantification of Signaling Proteins in Cancer Cells

Purpose Immuno-MALDI (iMALDI) combines immuno-enrichment of biomarkers with MALDI-MS for fast, precise, and specific quantitation, making it a valuable tool for developing clinical assays. iMALDI assays are optimized for the PI3-kinase signaling pathway members phosphatase and tensin homolog (PTEN) and PI3-kinase catalytic subunit alpha (p110 alpha), with regard to sensitivity, robustness, and throughput. A standardized template for developing future iMALDI assays, including automation protocols to streamline assay development and translation, is provided. Experimental Design Conditions for tryptic digestion and immuno-enrichment (beads, bead:antibody ratios, incubation times, direct vs. indirect immuno-enrichment) are rigorously tested. Different strategies for calibration and data readout are compared. Results Digestion using 1:2 protein:trypsin (wt:wt) for 1 h yielded high and consistent peptide recoveries. Direct immuno-enrichment (antibody-bead coupling prior to antigen-enrichment) yielded 30% higher peptide recovery with a 1 h shorter incubation time than indirect enrichment. Immuno-enrichment incubation overnight yielded 1.5-fold higher sensitivities than 1 h incubation. Quantitation of the endogenous target proteins is not affected by the complexity of the calibration matrix, further simplifying the workflow. Conclusions and Clinical Relevance This optimized and automated workflow will facilitate the clinical translation of high-throughput sensitive iMALDI assays for quantifying cell-signaling proteins in individual tumor samples, thereby improving patient stratification for targeted treatment.Proteomic