[Program | Abstracts: I, II, III, IV | Exhibitors ]

SYMPOSIUM IV: Bioinformatics and Mass Spectrometry in Proteomics

Organizers: Nikolaj Blom, Danish Technical University and Allan Stensballe, University of Southern Denmark

Abstracts

The Synergy between Bioinformatics and Proteomics Research

Pierre-Alain Binz¹, Markus Müller¹, Christine Hoogland¹, Daniel Walther¹, Steven Gay¹, Robin Gras¹, Willy V. Bienvenut², Carla Pasquarello², Salvo Paesano², Garry Corthals², Jean-Charles Sanchez², Amos Bairoch^1,3, Denis F. Hochstrasser^2,3, Ron D. Appel^1,3

¹ Swiss Institute of Bioinformatics, Geneva, Switzerland. ² Geneva Proteome Centre, Central Clinical Chemistry Laboratory, Geneva University Hospital, Geneva, Switzerland. ³ Geneva University, Geneva, Switzerland.

Studying the abundance, structure or function of proteins in biological samples has always been forcing significant advances in technology. In contrast to DNA and RNA, proteins highlight very heterogeneous physico-chemical properties and display higher degrees of biological complexity. In addition, and as a consequence, current proteomics technologies do not allow reaching the throughput of genomics. Protein chemists and now “proteomicists” are continuously requiring instrumental devices and laboratory protocols that aim to be more sensitive, more specific, less expensive, more automated, of higher quality or easier to handle. As a consequence, large amounts of very heterogeneous data can be produced in a single lab today. All this information has to be treated with appropriate bioinformatics tools in order to extract the most relevant biological interpretation. Therefore, during last few years, we have observed developments of complementary workflows that focus on various methods for sample preparation, protein or peptide separation and digestion, mass spectrometric analysis, and bioinformatics interpretation. We will discuss some aspects of dynamic interaction between technology and bioinformatics development and the biological relevance of the interpretation of the results generated.

Bioinformatics of PTMs - post-translational modifications

Nikolaj Blom, CBS, DTU, Denmark

Prediction of protein PTMs is becoming a serious research tool, not only for studying modifications of a protein, but also for larger-scale systems biology studies, e.g. large-scale protein function prediction. Many PTMs occur at specific, yet variable motifs, in the target proteins. In contrast to simple consensus patterns, machine learning techniques, such as artificial neural networks, are often well suited to integrate the subtleties of sequence variations. We present our latest results in predicting kinase-specific phosphorylation sites, N-glycosylation sites and propeptide cleavage sites.

Prediction of Human Protein Function from Post-translational Modifications and Localization Features.

Lars Juhl Jensen, CBS, DTU, Denmark.

Of the 30,000 to 40,000 genes believed to be present in the human genome not more than half can be assigned a functional role based on homology to known proteins. Traditionally, protein function has been viewed as something directly related to the conformation of the poly-peptide chain. However, as the 3D structure currently is quite hard to calculate from the sequence, a computational strategy for the elucidation of orphan protein function may benefit also from the prediction of functional attributes (such as post- translational modifications (PTMs)) which are more directly related to the linear sequence of amino acids. As a protein will have to operate using the same cellular machinery for modification and sorting as all the other proteins do, on can expect some conservation of essential types of PTMs among proteins with similar function.

For any function prediction method, the ability to correctly assign the relationship depends strongly on the function classification scheme used. We predict a scheme of 12 cellular functions which is closely related to the 14 class classification originally proposed by Riley for the E.coli genome. We also use the same approach for prediction of enzyme classes as well as several Gene Ontology classes. Furthermore, the method trained on human proteins has proven to be able to generalize to most if not all eukaryotes.

Mass Spectrometric characterization of post-translational modifications in proteome analysis

Martin R. Larsen, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense Denmark.

Modification of specific amino acids in proteins is a well known phenomenon in all organisms. Such modifications can have a wide range of effects on the proteins in the cell, e.g., change of the function, solubility, stability, localization or interaction with other proteins/components etc. Lately, an increased number of diseases have been associated with abnormalities in the level of modifications of different proteins, e.g. diabetes mellitus. In order to understand such diseases, and find possible ways of treatment, a complete characterization of the modified proteins of interest is essential. Currently, liquid chromatography mass spectrometry (LC-MS) is not able to identify global changes in the level of protein modifications but only in the expression level. Thus, 2 D gel electrophoresis is still the method of choice for this purpose, as most protein modifications change the molecular weight and/or pI of the protein, resulting in separation of the modified forms.

In this presentation, examples of the characterization of phosphorylated and glycosylated proteins directly from the polyacrylamide gel matrix will be given. After in-gel enzymatic digestion of the relevant protein, the resulting peptides are characterized by mass spectrometry using a variety of optimized sample preparation methods prior to mass analysis, to enrich for modified peptides. The sample preparation methods take advantages of the use of GELoader tip micro-columns packed with a variety of chromatographic material for purification of different compounds. In addition, LC-MS operating in different scanning modes will be shown for elucidation of different modifications. The full characterization includes protein identification, site-specific assignment of phosphorylation and glycosylation site(s) and elucidation of the glycan structure.

Integration of Biology, Mass Spectrometry and Bioinformatics in a biotech company

Thomas Kofoed, Head of Analytical Chemistry, ACE BioSciences, Odense, Denmark

How to integrate the data from biology and mass spectrometry laboratories into a well functioning and easy to use bioinformatics toolbox. This will be discussed and exemplified with results from a study of the bacterium Campylobacter Jejuni.

Modificomix: Combined Phosphoprotein & Glycoprotein analysis by mass spectrometry

Allan Stensballe; University of Southern Denmark, Odense, Denmark

The cellular proteome is highly complex and dynamic due to sequence variations and post-translational modifications of most proteins. Mass spectrometry is the method of choice for identification and investigation of posttranslationally modified proteins because it provides the sensitivity, selectivity and specificity required to characterize covalently modified peptides. One of the most challenging tasks in the field of proteomics today is the global analysis of protein phosphorylation in a cell or cell culture at a given time (phosphoproteomics). Here a mass spectrometry based strategy for primarily phosphorylation-specific but also glycosylation-specific protein analysis will be presented. This strategy encompasses differential peptide mass mapping, affinity purification of modified peptides as well as nanoflow LC-MS/MS employing online precursor ion discovery which enable specific detection of post-translationally modified peptides. Automated data analysis of nanoflow LC-MS/MS data enable protein identification as well as site-specific knowledge of post-translationally modified residues.