Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes

The influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling targets uncovered by comparing the phosphoproteomes generated by two commonly used methods—strong cation exchange chromatography-based phosphoproteomics (SCXPhos) and single-run high-throughput phosphoproteomics (HighPhos). Phosphoproteomes of embryonic stem cells exposed to ionizing radiation (IR) profiled by both methods achieved equivalent coverage (around 20,000 phosphosites), whereas a combined dataset significantly increased the depth (>30,000 phosphosites). While both methods reproducibly quantified a subset of shared IR-responsive phosphosites that represent DNA damage and cell-cycle-related signaling events, most IR-responsive phosphoproteins (>82%) and phosphosites (>96%) were method-specific. Both methods uncovered unique insights into phospho-signaling mediated by single (SCXPhos) versus double/multi-site (HighPhos) phosphorylation events; particularly, each method identified a distinct set of previously unreported IR-responsive kinome/phosphatome (95% disparate) directly impacting the uncovered biology.

Ion-Exclusion/Cation-Exchange Chromatography Using Dual-Ion-Exchange Groups for Simultaneous Determination of Inorganic Ionic Nutrients in Fertilizer Solution Samples for the Management of Hydroponic Culture

In this study, ion-exclusion/cation-exchange chromatography (IEC/CEC) using dual-ion-exchange groups (carboxy and sulfo groups) for the simultaneous determination of anions (SO42−, Cl−, NO3−, and HPO42−) and cations (Na+, NH4+, K+, Mg2+, and Ca2+) was developed. By using the combination of dual-ion-exchange groups, simultaneous separation of inorganic ions with HPO42− was achieved that was impossible by the conventional IEC/CEC based on the single-ion-exchange group (carboxy group). This method was applied to the monitoring of inorganic ionic nutrients in fertilizer solution samples in hydroponic culture. As a result, a higher peak resolution of inorganic anions and cations with phosphate ion using IEC/CEC with dual-ion-exchange groups was achieved in the absence of matrix effects. In addition, the developed method helps to understand the behavior of ionic nutrients in fertilizer solution during hydroponic cultivation and is potentially useful for the individual fertilization of ionic nutrients.

Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification

Background The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar. Results The pure enzyme without the inhibitor barstar was exported to the extracellular space through the periplasm and then purified from the cell-free supernatant. Cation exchange chromatography was employed as a primary purification step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units mL− 1), in principle its application to plasmid DNA purification could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production. Conclusions The current work focusses on the downstream purification strategies for a recombinant RNase and sets a framework for higher scale production if specific productivity is increased by optimal hosts and/or re-engineered plasmids. Also important is to curtail the massive enzyme loss during purification by cation exchange chromatography. Application of even a relatively small amount of recombinant RNase would contribute to greatly reducing the initial RNA levels in alkaline lysates thereby augmenting further downstream plasmid purification steps.