µ°°×ÖÊ×éѧ:Ñо¿·½·¨ÓëʵÑé·½°¸

µ°°×ÖÊ×éѧ:Ñо¿·½·¨ÓëʵÑé·½°¸ ½ÚÑ¡

PART I INTRODUCTION Chapter 1 Introduction to Proteomics Friedrich Lottspeich Summary In this chapter£¬ the evolvement of proteomics from classical protein chemistry is depicted. The challenges of complexity and dynamics led to several new approaches and to the firm belief that a valuable proteomics technique has to be quantitative. Protein-based vs. peptide-based techniques£¬ gel-based vs. non-gel-based proteomics£¬ targeted vs. general proteomics£¬ isotopic labeling vs. label-free techniques£¬ and the importance of informatics are summarized and compared. A short outlook into the near future is given at the end of the chapter. Key words: History £¬ Quantitative proteomics £¬ Targeted proteomics £¬ Isotopic labeling £¬ Protein-based proteomics £¬ Peptide-based proteomics 1. The History and the Challenge In the end of the last century£¬ a change of paradigm from the pure function driven biosciences to systematic and holistic approaches has taken place. Following the successful genomics projects£¬ classical protein chemistry has evolved into a high throughput and systematic science£¬ called proteomics. Starting in 1995£¬ the first attempts to deliver a ¡°protein complement of the genome¡± used the established high-resolving separation techniques like two-dimensional (2D) gel electrophoresis and almost exclusively identified the proteins by the increasingly powerful mass spectrometry. Soon£¬ fundamental and technical challenges were recognized. Unlike the genome£¬ the proteome is dynamic£¬ responding to any change in genetic and environmental parameters. Furthermore£¬ the proteome appears to be orders of magnitude more complex than a genome owing to splicing and editing processes at the RNA level and owing to all the posttranslational events on the protein level£¬ like limited processing£¬ post-translational modifications£¬ and degradation. The situation is even more difficult£¬ since many important proteins are only present in a few copies/cells and have to be identified and quantified in the presence of a large excess of many other proteins. The dynamic range of the abundant and the minor proteins often exceeds the capabilities of all analytical methods. So far£¬ only few solutions are available to handle the complexity and dynamic range. One is to reduce the complexity of the proteome and to separate the low abundant proteins from the more abundant ones. This£¬ for example£¬ can be achieved by multidimensional separation steps. But£¬ unpredictable losses of proteins and a large number of resulting fractions make this approach time-consuming and thus also very costly. Alternatively£¬ the proteome to be investigated can be simplified by starting with a specific biological compartment or by reducing the complexity using a suitable sample preparation (e.g. enzyme ligand chips£¬ functionalized surface chips£¬ class-specific antibodies). Successful examples are the analysis of functional complexes or most interaction proteomics approaches. In another approach£¬ a selective detection is performed£¬ which visualizes only a certain number of proteins that exhibit specific common properties. This can be achieved by antibodies£¬ selective staining protocols£¬ protein ligands£¬ or selective mass spectrometry techniques like MRM (multiple reaction monitoring) or SRM (single reaction monitoring) ( 1 ) . The most straightforward application of this approach is ¡°targeted proteomics£¬¡± which monitors a small set of well-known proteins/peptides. However£¬ in the later years of the past century£¬ the main focus of proteomics projects was to decipher the constituents of a proteome. It was realized only slowly that for solving biological problems and realizing the potential of holistic approaches£¬ the changes and the dynamics of changes on the protein level have to be monitored quantitatively. 2. Gel-Based Proteomics Since 1975 by their introduction in by O¡¯Farrel ( 2 ) and Klose ( 3 ) £¬ 2D gels have fascinated many scientists owing to their separation power. The combination of a concentrating technique£¬ i.e. isoelectric focusing£¬ with a separation according to molecular mass£¬ i.e. SDS gel electrophoresis£¬ provides a space for resolving more than 10£¬000 different compounds. Consequently£¬ 2D gels were the method of choice when dealing with very complex protein mixtures like proteomes. Unfortunately£¬ gel-based proteomics had inherent limitations in reproducibility and dynamic range. Standard operating procedures had to be carefully followed to get almost reproducible results even within one lab. Results produced from identical samples in different labs were hardly comparable on a quantitative level. A significant improvement was the introduction of the DIGE technique (GE Healthcare)£¬ a multiplexed fluorescent Cy-Dye staining of different proteome states£¬ which eliminated to a large extent the technical irreproducibility (4) . With the cysteine-modifying ¡°DIGE saturation labeling£¬¡± impressive proteome visualizatio