Proteomic mass spectrometry

The sustained attractiveness of photolabeling is apparent from its prominence in studies of y-secretase, an intramembrane protease that contributes to forming amyloid-p peptides and is a major target in Alzheimer s disease [60-62]. y-Secretase is a complex of at least four different polypeptides, and is difficult to engage with high-resolution structural methods. However, in a case of this kind that involves a known target, immunodetection of proteins can often specify the target of y-secretase inhibitor photoaffinity probes such as 19, and proteomic mass spectrometry is not needed. 

Godovac-Zimmermann, J. and Brown, L.R. (2001) Perspectives for mass spectrometry and functional proteomics. Mass Spectrometry Reviews 20, 1-57. 

Goshe MB, Smith RD Stable isotope-coded proteomic mass spectrometry. Curr. Opin. Biotechnol. (2003) 14 101-109. 

Proteomic Mass Spectrometry, The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, United Kingdom. 

Key words Small molecules, Drugs, Target identification. Metabolites, Natural products, Proteomics, Mass spectrometry, Immunoblotting... 

James, P, Proteome Research Mass Spectrometry (Principles and Practice), Springer-Verlag, Heidelberg,... 

The Tools of Proteomics A variety of methods and techniques including two-dimensional gel electrophoresis (2DE), capillary liquid chromatography, stable isotope labeling, and mass spectrometry has been developed for qualitative and quantitative protein... 

Proteomics. Figure 2 A strategy for mass spectrometry (MS)-based identification of proteins. 

Tao WA, Aebersold R (2003) Advances in quantitative proteomics via stable isotope tagging and mass spectrometry. Curr Opin Biotechnol 14 110-118... 

A new chapter on the primary structure of proteins, which provides coverage of both classic and newly emerging proteomic and genomic methods for identifying proteins. A new section on the appHcation of mass spectrometry to the analysis of protein structure has been added, including comments on the identification of covalent modifications. 

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

Jensen P.K., Pasa-Tolic L., Anderson G. A., Horner J. A., Lipton M.S., Bruce J.E., and Smith R.D., Probing proteomes using capillary isolectric focusing-elec-trospray ionization Fourier transform ion cyclotron resonance mass spectrometry, Anal. Chem. 71, 2076, 1999. 

Current proteomics studies rely almost exclusively on 2D gel electrophoresis to resolve proteins before MALDI-TOF or ESI-MS/MS approaches. A drawback of the 2D gel approach is that it is relatively slow and work intensive. In addition, the in-gel proteolytic digestion of spots followed by mass spectrometry is a one-at-a-time method that is not well suited for high throughput studies. Therefore, considerable effort is being directed towards alternate methods for higher throughput protein characterization. 

Post-translational modification of proteins plays a critical role in cellular function. For, example protein phosphorylation events control the majority of the signal transduction pathways in eukaryotic cells. Therefore, an important goal of proteomics is the identification of post-translational modifications. Proteins can undergo a wide range of post-translational modifications such as phosphorylation, glycosylation, sulphonation, palmitoylation and ADP-ribosylation. These modifications can play an essential role in the function of the protein and mass spectrometry has been used to characterize such modifications. 

Another means of moving beyond pure protein preparations to high-throughput characterization of proteomes is to enrich for phosphopeptides from complex mixtures by metal affinity chromatography (Andersson and Porath, 1986). Using this method, protein mixtures are proteolyzed to create peptides and phosphorylated peptides are enriched by metal affinity chromatography and subsequently identified by mass spectrometry. This method is limited, however, because in many cases phosphopeptides absorb poorly or nonphosphorylated peptides absorb nonspecifically to the metal affinity resins (Ahn and Resing, 2001). 

Gauss, C., Kalkum, M., Lowe, M., Lehrach, H., and Klose, J. (1999). Analysis of the mouse proteome. (I) Brain proteins Separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation. Electrophoresis 20, 575-600. 

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