Proteome coverage proteomics

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. 

Gaucher, S.P., Taylor, S.W., Fahy, E., Zhang, B., Wamock, D.E., Ghosh, S.S., Gibson, B.W. (2004). Expanded coverage of the human heart mitochondrial proteome using multidimensional liquid chromatography coupled with tandem mass spectrometry. J. Proteome Res. 3, 495-505. 

Olivova, P., Gilar, M., Dorschel, C. A., Gebler, J.C. (2005). Improved peptide identification and protein coverage for proteomic samples using alternative 2D-HPLC MS/MS approaches. ASMS, 2005, San Antonio, TX Poster TP29. 

Whitelegge, J.P, Zhang, H., Aguilera, R., Taylor, R.M., Cramer, W.A. (2002). Full subunit coverage liquid chromatography electrospray ionization mass spectrometry (LCMS+) of an oligomeric membrane protein cytochrome b(6)f complex from spinach and the cyanobacterium Mastigocladus laminosus. Mol. Cell. Proteomics. 1, 816-827. 

Figure 20.11 Coverage of protein ErbB2 by shotgun proteomic discovery of sample ...

Figure 20.11 Coverage of protein ErbB2 by shotgun proteomic discovery of sample fixed for various times, including fresh. The color gradient represents the increasing abundance of the peptides. All were identified at an FDR <1%. Reproduced with permission from Reference 20.

Antibody coverage of the human proteome is estimated to be about 5 to 10% of all human proteins and isoforms (Valle and Jendoubi, 2003). A major bottleneck in the use of protein expression arrays is the lack of such a comprehensive set of these capture agents (Hanash, 2003). Since an equivalent of the polymerase chain reaction (PCR) process for mass amplification of low abxmdant proteins does not exist, the remaining library of proteome capture ligands will need to be generated by other means such as recombinant protein expression systems (Cahill, 2001). 

Coverage The use of large format gels, pre-firactionation and multiple use of narrow range isoelectric focusing gels enable more comprehensive proteomic studies... 

A minority of proteins make up a large proportion of the total protein abundance in most samples, and thus, the coverage of a proteome is highly... 

A growing number of researchers are focusing on the use of top-down proteomics, a relatively new approach compared to bottom-up, in which structure of proteins is studied through measurement of their intact mass followed by direct ion dissociation in the gas phase. The main advantages over the bottom-up approach are that higher sequence coverage is obtained, it permits... 

In P. aeruginosa, despite considerably lower levels of LasR when compared to the E. coli system, the same protocol resulted in detection of a band by in-gel fluorescence, which could be excised and the identification of LasR achieved by proteomic MS methods with 32% total sequence coverage. The methodology was also extended to allow analysis by flow cytometry. In this way, the authors were able to demonstrate that cell density had an effect on LasR labelling. As expected, fluorescence increased with cell density up to the point at which quorum was achieved (around OD600 = 6), beyond which fluorescence began to weaken. 

Fischer, F., Wolters, D., Rogner, M. and Poetsch, A. (2006) Toward the complete membrane proteome high coverage of integral membrane proteins through transmembrane peptide detection. Mol. Cell. Proteomics 5, 444-453. 

Miguet, L., Pacaud, K., Felden, C., Hugel, B., Martinez, M.C., Freyssinet, J.M., Herbrecht, R., Potier, N., van Dorsselaer, A. and Mauvieux, L. (2005) Proteomic analysis of malignant lymphocyte membrane microparticles using double ionization coverage optimization. Proteomics 6, 153-171. 

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