Serum Protein Adsorption Modulates the Toxicity of Highly Positively Charged Hydrogel Surfaces.

Hydrogels formed from peptide self-assembly are a class of materials that are being explored for their utility in tissue engineering, drug and cell delivery, two- and three-dimensional cell culture, and as adjuvants in surgical procedures. Most self-assembled peptide gels can be syringe-injected in vivo to facilitate the local delivery of payloads, including cells, directly to the targeted tissue. Herein, we report that highly positively charged peptide gels are inherently toxic to cells, which would seem to limit their utility. However, adding media containing fetal bovine serum, a common culture supplement, directly transforms these toxic gels into cytocompatible materials capable of sustaining cell viability even in the absence of added nutrients. Multistage mass spectrometry showed that at least 40 serum proteins can absorb to a gel's surface through electrostatic attraction ameliorating its toxicity. Further, cell-based studies employing model gels having only bovine serum albumin, fetuin-A, or vitronectin absorbed to the gel surface showed that single protein additives can also be effective depending on the identity of the cell line. Separate studies employing these model gels showed that the mechanism(s) responsible for mitigating apoptosis involve both the pacification of gel surface charge and adsorbed protein-mediated cell signaling events that activate both the PI3/Akt and MAPK/ERK pathways which are known to facilitate resistance to stress-induced apoptosis and overall cell survival.

Exploring Biological and Geological Age-related Changes through Variations in Intra- and Intertooth Proteomes of Ancient Dentine.

Proteomic analyses are becoming more widely used in archeology not only due to the greater preservation of proteins in ancient specimens than DNA but also because they can offer different information, particularly relating to compositional preservation and potentially a means to estimate biological and geological age. However, it remains unclear to what extent different burial environments impact these aspects of proteome decay. Teeth have to date been much less studied than bone but are ideal to explore how proteins decay with time due to the negligible turnover that occurs in dentine relative to bone. We investigated the proteome variability and deamidation levels of different sections of molar teeth from archeological bovine mandibles as well as their mandibular bone. We obtained a greater yield of proteins from the crown of the teeth but did not find differences between the different molars analyzed within each mandible. We also obtained the best variety of protein from a well-preserved mandible that was not the youngest one in terms of chronological age, showing the influence of the preservation conditions on the final proteomic outcome. Intriguingly, we also noticed an increase in abundance levels of fetuin-A in biologically younger mandibles as reported previously, but the opposite trend in tooth dentine. Interestingly, we observed higher glutamine deamidation levels in teeth from the geologically oldest mandible despite it being the biologically youngest specimen, showing that the archeological age strongly impacts on the level of deamidations observed, much more so than biological aging. This indicates that the glutamine deamidation ratio of selected peptides may act as a good predictor of the relative geochronological age of archeological specimens.

Targeted Measurements of O- and N-Glycopeptides Show That Proteins in High Density Lipoprotein Particles Are Enriched with Specific Glycosylation Compared to Plasma.

High density lipoprotein (HDL) particles are believed to be protective due to their inverse correlation with the prevalence of cardiovascular diseases. However, recent studies show that in some conditions such as heart disease and diabetes, HDL particles can become dysfunctional. Great attention has been directed toward HDL particle composition because the relative abundances of HDL constituents determine HDL's functional properties. A key factor to consider when studying the structure and composition of plasma particles is the protein glycosylation. Here, we profile the O- and N-linked glycosylation of HDL associated-proteins including the truncated form of Apo CIII and their glycan heterogeneity in a site-specific manner. Apolipoprotein CIII, fetuin A, and alpha 1 antitrypsin are glycoproteins associated with lipoproteins and are implicated in many cardiovascular and other disease conditions. A targeted method (UHPLC-QQQ) was used to measure the glycoprotein concentrations and site-specific glycovariations of the proteins in human plasma and compared with HDL particles isolated from the same plasma samples. The proteins found in the plasma are differentially glycosylated compared to those isolated in HDL. The results of this study suggest that glycosylation may play a role in protein partitioning in the blood, with possible functional implications.

Rapid sandwich ELISA-based in vitro diagnostic procedure for the highly-sensitive detection of human fetuin A.

A rapid sandwich enzyme-linked immunosorbent assay (ELISA)-based in vitro diagnostic (IVD) procedure has been developed for human fetuin A (HFA), an important disease biomarker for inflammatory diseases as well as malignancies. In this simplified and cost-effective procedure, the EDC-activated anti-HFA antibody (Ab) was admixed with 1% (v/v) 3-aminopropyltriethoxysilane (APTES) in 1:1 (v/v) and dispensed in a KOH-pretreated microtiter plate (MTP). APTES formed a stable complex with the capture antibody that was in turn covalently bonded on the KOH-treated surface in 30 min. The resulting immunoassay (IA) format detects HFA with a dynamic range of 0.1-243 ng mL(-1), and a limit of detection (LOD) and analytical sensitivity of 0.3 ng mL(-1) and 1.0 ng mL(-1), respectively. For the determination of HFA spiked in diluted human whole blood and serum, and HFA in ethylenediaminetetraacetic acid (EDTA)-plasma of patients, the obtained analytical precision is similar to that of the conventional sandwich ELISA. The anti-HFA Ab-bound MTPs, stored at 4 °C in 0.1M PBS, pH 7.4, retained its biological activity for 8 weeks, thereby demonstrating excellent storage stability. This generic sandwich ELISA procedure can be extended for rapid, simplified and cost-effective detection of other disease biomarkers.