PTMScan Proteomics Lecture

Charles L. Farnsworth, PhD | 12/15/15 | 10:15AM | Olson Pavilion 8-260

PTMScan Proteomics Lecture

The ability to identify and quantify changes in protein modification state levels in cell lines or tissue samples is critical for gaining insight into the biology and chemistry of biological systems. PTMScan® Proteomics Technologies, developed at Cell Signaling Technology, provide comprehensive analytical profiling of post-translational modifications (PTMs), including Ser, Thr, and Tyr phosphorylation, as well as acetylation, ubiquitination, and methylation. Discovery-mode studies, carried out with PTMScan® Discovery Technology, identify PTMs throughout the proteome in a variety of biological model systems and disease states. Alternatively, targeted-mode studies, performed using PTMScan® Direct Technology, allow for targeted screening of known PTMs on protein targets within a defined group of signaling pathways in response to a drug treatment or in the context of a specific disease state. Both technologies will be outlined with an emphasis on their direct application to drug discovery research.

New Article Published with Richard Silverman

Mechanism of Inactivation of Neuronal Nitric Oxide Synthase by (S)-2-Amino-5-(2-(methylthio)acetimidamido)pentanoic Acid.

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and the second messenger nitric oxide. Three mechanistic pathways are proposed for the inactivation of neuronal NOS (nNOS) by (S)-2-amino-5-(2-(methylthio)acetimidamido)pentanoic acid (1): sulfide oxidation, oxidative dethiolation, and oxidative demethylation. Four possible intermediates were synthesized. All compounds were assayed with nNOS, their IC50, KI, and kinact values were obtained, and their crystal structures were determined. The identification and characterization of the products formed during inactivation provide evidence for the details of the inactivation mechanism. On the basis of these studies, the most probable mechanism for the inactivation of nNOS involves oxidative demethylation with the resulting thiol coordinating to the cofactor heme iron. Although nNOS is a heme-containing enzyme, this is the first example of a NOS that catalyzes an S-demethylation reaction; the novel mechanism of inactivation described here could be applied to the design of inactivators of other heme-dependent enzymes.

Northwestern Team Develops System for Simplifying Sample Cleanup in Top-Down Proteomics

The lab of Northwestern University researcher Neil Kelleher has developed an on-line system for clean up of top-down proteomics samples following gel eluted liquid fraction entrapment electrophoresis (GELFrEE) fractionation. The system, detailed in a paper published this month in the Journal of Proteome Research, uses asymmetrical flow field-flow fractionation (AF4) to address what is a key challenge in top-down proteomic workflows, and could make top-down approaches more widely shareable as well as improve their applicability to clinical work, Philip Compton, director of instrumentation at NorthWestern's Proteomics Center of Excellence and an author on the paper, told GenomeWeb.

Wisconsin Human Proteomics Symposium

Post-Translational Modifications in Human Disease

Wisconsin Human Proteomics Symposium

The Human Proteomics Program at the University of Wisconsin-Madison and the BioPharmaceutical Technology Center Institute are pleased to offer this excellent symposium. The theme of this year's exciting program is Post-Translational Modifications in Human Disease. Speakers will present a range of topics in cutting-edge proteomics technology developments for qualitative and quantitative analysis of post-translational modifications. Applications to human diseases, for example, cancer and cardiovascular, infectious and neurodegenerative diseases, will be discussed. Meeting attendees are encouraged to present their recent work in scientific poster sessions. This year, a poster contest is included and three winners will receive their awards and provide brief presentations during a special afternoon session.

Top-Down Proteomics for Biomarker Research

There’s been considerable excitement of late about the clinical potential of next-generation DNA sequencing. After all, many diseases, such as cancer, sickle cell anemia and Huntington’s disease, are encoded in mutations or variations in genomic DNA. Yet these diseases physically manifest themselves in the presence and absence of proteins and small molecules. Thus, focused efforts are on cataloging and characterizing these molecules to identify molecular indicators (read: biomarkers) of disease, prognosis, therapeutic response and progression. At the protein level, such studies are conducted using mass spectrometry, and they can be done in either of two ways. The simplest and most widely used approach is “bottom-up proteomics.” Here, protein extracts—representing, say, cancerous and normal tissues—are broken down into peptides with a proteinase, chromatographically separated and then analyzed in a mass spectrometer. The goal is to identify peptides (and thus, the proteins they represent) whose abundance or post-translational modification (PTM) changes as a result of disease or treatment.