ISPPP 2009 Short Course Description and Schedule

Sunday, October 25, 2009 - Note that all courses will be held simultaneously starting at 1:00 PM and continuing to approximately 4:30 PM

1. The role of Mass Spectrometry in Glycomics and Glycoproteomics

Professor Ron Orlando, Complex Carbohydrate Research Center, University of Georgia (USA)

Glycosylation is one of the two most common post-translational modifications found on proteins. Glycan structures and sites of glycosylation have been shown to change with the state/condition of the cell in which the proteins are produced. For example, it has been known for over 40 years that cancerous cells attach different glycans than those of corresponding “normal” cells from the same tissue/organ. Since many glycoproteins are excreted, altered glycosylation has the potential to be used as a biomarker for cancer. Numerous other disease states, ranging from arthritis to alcoholism, are also characterized by altered glycoprotein glycans, as is normal cell growth, differentiation, and development. Identifying glycan structures and how these structures change as cells differentiate or as tumor cells progress, for example, is the focus of an emerging field called glycomics. This workshop will focus on the role of mass spectrometry in the emerging field of glycomics and glycoproteomics. An overview will be presented on the biosynthetic pathway that leads to protein glycosylation and how this, in turn, leads to diverse structures of glycoprotein glycans. Other topics that will be discussed include: the analytical challenges of characterizing glycoproteins and their glycans; the methods used to determine glycan structure, sites of glycosylation, and identify glycans present at individual glycosylation sites. Approaches used for comparative glycomic studies will also be covered. Many of the techniques discussed are applicable to both whole cell glycoprotein extracts (i.e., glycomics) as well as the characterization of purified glycoproteins. Although the emphasis of this workshop will be on N-linked glycosylation, the methodology discussed can be extrapolated to other types of glycosylation.

2. Preparative-scale Separation of Biomolecules
Professor Alois Jungbauer, University of Natural Resources and Applied Life Sciences, Vienna, Austria

Chromatographic methods play a pivotal role in biotechnology and biopharmaceutical technology, particularly for high molecular mass compounds such as proteins and plasmids. The high level of purity can be only achieved by chromatographic methods. Beside bulk contaminants traces of bioactive compounds such as endotoxins, DNA and other adventitious agents must be efficiently removed from the process solution. In the workshop special emphasis will be put on the description of the characteristics of chromatography media used in bioseparation and how they differ from analytical media and media used for separation of small molecules. Process optimization, scale up and important design criteria will be discussed. The influence of mobile phase composition on resolution , and guidelines for the optimization of selectivity will be presented. An overview on novel stationary phases for protein and polynucelotide separation and examples for novel bioseparation processes using these phases will be given. The difference and applicability of monoliths, beads with a porous shell and polymer grafted beads will be elaborated. In the second part of the workshop the progress on biorecognition for affinity chromatography will be discussed.

3. Affinity Chromatography

Professor David S. Hage, Dept. of Chemistry, University of Nebraska (USA).

Affinity chromatography is a type of liquid chromatography that relies on the use of a biologically-related ligand as a stationary phase for the selective retention of sample components. This method has been popular for decades as a means for the rapid and selective isolation of proteins and other biological agents. This technique has also seen increased use in recent years as a tool for the selective analysis of chemicals, sample preparation in multidimensional analytical techniques, and the study of biological interactions.

In this course we will discuss the basic principles of affinity chromatography and key factors to consider in the development of an affinity-based separation. A survey of popular types of affinity chromatography and their applications will also be presented, including bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, lectin affinity chromatography, dye ligand/biomimetic affinity chromatography, and the use of affinity chromatography in chiral separations. Several examples illustrating the use of affinity chromatography in preparative or analytical work and multidimensional methods will also be described during this discussion.

4. 2D Liquid chromatography: Basic operating principles, advantages, key applications, instrumentation, methods development, and implementation.

Dr. Mark R. Schure, Dow Advanced Materials, Springhouse, Pennsylvania (USA)

Multidimensional Liquid Chromatography is a technique that keeps growing and developing. The two-dimensional comprehensive version of this technique, which we will call 2DLC, is a very interesting technique where sample zones are fractionated over two different types of retention mechanisms, from two independent columns. For many bioseparations, one needs to have at least two different retention mechanisms to spread the separation over an area (two-dimensional), rather than along a one-dimensional separation axis, as is the normal way with HPLC. Often people talk about how 2DLC can increase peak capacity so that more peaks can be resolved. This is often true but not always.

In this course we will review the basic operation of 2DLC and discuss the key biochemistry applications where one needs to use this type of technology. The present state of instrumentation will be surveyed in separate groups:

1) Commercially available as complete turn-key systems
2) How to add 2DLC to an existing HPLC with an add-on kit

We will not discuss how to build a system from the ground up. The various methods development processes will be taught. A survey of the present state-of-the art work in 2DLC will be given with emphasis on the application. Students will be expected to be familiar with HPLC.

The ISPPP Symposium reserves the right, without notice, to modify or amend the roster of Sunday short courses and/or presenters. Any changes will be updated on the web site.