Research Tools & Reagents

Liquid chromatography/mass spectrometry (LC/MS) is increasingly being used for metabolic analysis due in part to its widespread availability and compatibility with biological samples. This invention provides ionizable, isotopic labeling reagents and labeling reaction products, answering the current need for relative quantification using mass spectrometry.

Absolute quantification of an analyte relies upon the addition of an internal standard differing only in its isotopic form. However, it is impractical to add an isotopic standard for every compound when performing more comprehensive metabolic profiling. Relative quantification between samples is more amenable to analyzing broad classes of compounds, and it often provides very useful biological information.

Heavy and light isotopic forms of methylacetimidate have been synthesized and used as labeling reagents for quantification of amine-containing molecules, such as biological samples. Heavy and light isotopic forms of formaldehyde and cholamine have also been synthesized and used independently as labeling reagents for quantification of amine-containing and carboxylic acid-containing molecules, such as these found in biological samples. Advantageously, the labeled end-products are positively charged under normal acidic conditions involving conventional liquid chromatography mass spectrometry (LC/MS) applications.

This invention improves the precision of relative quantification by minimizing or negating errors associated with run-to-run irreproducibility. Such errors can arise from variations in mass spectrometric detection sensitivity, such as those caused by ionization suppression in electrospray, or from retention time differences between runs. The isotopic pair of labeled compounds co-elute within a single run. Therefore, they have identical retention times and are electrosprayed from identical solution conditions.

Dr. van der Donk from the University of Illinois has characterized expression vectors and a family of proteases that are useful in removing affinity tags after protein expression. Affinity tag removal after protein expression is sometimes necessary for the protein of interest to be functional and active. However during this tag removal, some amino acids not native to the protein of interest can be left behind. Dr. van der Donk's invention eliminates the possibility of extraneous amino acids being left on the final protein product, which is an improvement over existing enzymatic tag removal techniques. This invention has identified a class of lanthipeptides, class II LanPs that show substrate specificity and can serve as efficient sequence-specific traceless proteases, expanding and advancing the current toolbox of proteases.

The general goal of Dr. van der Donk's research is to understand enzymatic transformations that are of environmental or pharmaceutical importance. The van der Donk lab utilizes both synthetic organic chemistry and molecular biology to reach this goal. Specifically, the lab uses genome mining strategies to discover new natural products, use microbiology and genomic tools to determine their mode of action, use chemical biology techniques to study their biosynthesis, and use synthetic chemistry to improve their activities and therapeutic properties.

Researchers from the University of Illinois have developed a new synthetic tissue microenvironment that may give cancer researchers the next-best look at tumor growth and behavior. The 3D patterned vasculature tumor microenvironment is created by changing the flow rates of fluid channels and the structures are made of alginate that was mixed with breast cancer cells in the outer layer and macrophages on the inner layer. The 3D vascularization, a network of capillaries that carry drugs and other materials are created can go from straight, to snakelike, to any shape, that offers modelling for metastasis, because the vessel architecture can be tuned on the fly. 

The technology accurately predicted the clinically measured EC50 of three drugs: Gefitinib, zoledronic acid, and RAC inhibitor. This quick process to create synthetic tumor microenvironments lends to tremendous potential such as model systems for high-throughput screening for drug efficacy, as well as flowable and vascularized lab-on-a-fiber platforms. Finally, biopsied cancer cells lends to personalized cancer treatments with this device that could realistically and quickly recreate microenvironments found across biology.

Blood transfusions provide important medical benefits primarily centered on the replacement of lost blood in trauma and surgical patients.

However, the availability of blood transfusions in both hospital and field settings is limited by the safety and availability of the donated blood supply. Therefore, blood substitutes have long been sought as one way to alleviate these safety and availability concerns.

To date, current blood substitutes have met with modest clinical success against a backdrop of varied concerns regarding cardiac function, renal toxicity, and molecular stability. Recombinant oxygen-binding heme proteins incorporating circularly permuted globins address these issues through the promotion of cross-linking and the formation of hemoglobin multimers.

Recombinant DNA sequences coding for the hemoglobin protein have been modified to introduce new peptide linkers and termini. The resulting genetic cross-linking and covalent binding promote stability within individual hemoglobin molecules and the formation of high-weight hemoglobin multimers.

Applications

• As a blood substitute in the treatment of traumatic injury in both hospital and field settings

Benefits

• Increased safety and availability of blood substitutes for transfusion
• Potential for increased stability and reduced toxicity in a recombinant protein due to the modifications that promote subunit cross-linking and the formation of hemoglobin multimers