Proteomics Western blots

Figure 12.2. Identification of immunogenic proteins by 2DE/MALDI TOF-MS and Western blots probed with antisera from immunized animals or humans. (Reprinted from Khan, A. S. et al., 2006. Proteomics and Bioinformatics Strategies to Design Countermeasures Against Infectious Threat Agents. J. Chem. Inf. Model. 46 111-115. With permission.) (See color insert.)...

Conrad, . C. Malakowsky, C. A. Talent, J. Rong, D. Lakdawala, S. Gracy, R. W. Chemiluminescent standards for quantitative comparison of two-dimensional electrophoresis western blots. Proteomics (Germany) 2001,1(3), 365-369. 

Figure 18.2 Redox proteomics and oxidatively modified proteins in the brain. Redox proteomics has the potential of detecting disease markers and identifying potential targets for drug therapy in neurodegenerative disorders. Redox proteomics involves the separation of brain proteins followed by detection, usually immunochemically, of oxidatively modified proteins, either from a two-dimensional western blot or from column eluents. Subsequent mass spectrometric analysis of tryptic digests and database searching leads to protein identification.

McDonough, J. L., Neverova, I., and Van Eyk, J. E. (2002) Proteomic analysis of human biopsy samples by single two-dimensional electrophoresis Coomassie, silver, mass spectrometry, and Western blotting. Proteomics 2, 978-987... 

Amott D, Kishiyama A, Luis EA, Ludlum SG, Marsters JC Jr, Stults JT. Selective detection of membrane proteins without antibodies a mass spectro-metric version of the Western blot. Mol Cell Proteomics 2002 1 148-156. 

The SI LAC approach has also been used to investigate metastatic prostate cancer development at the protein level (Everley et al., 2004). The fact that proteins showed altered concentration ratios by quantitative MS was confirmed by western blotting. In addition, proteomic approaches for quantitation of protein phosphorylation via SILAC combined with MS analysis have been described (Gruhler et al., 2005 Ibarrola et al., 2003, 2004). A recent study reports on identification as well as relative quantitation of in vivo mefhylation sites of proteins in HeLa cells by stable isotope labeling wifh C Hj-methionine (Ong et al., 2004). 

Table 12.1 RCC biomarkers identified by classical proteomics. IHC Immunohistochemistry, NB northern blot, V/6 western blot.

Onono, F.K., et al. (2010). A tagging-via-substrate approach to detect the farnesylated proteome using Two-dimensional electrophoresis coupled with western blotting. Mol Cell Proteom 9 742-751. 

Gerbil cortex 2-DE MALDI-TOF LC-MS/MS Western blot Redox proteome (Boyd-Kimball et al. 2005)... 

Protein measurement [66, 67] Western blots with appropriate antibodies Proteomics Identification often uncertain, needs integration with other techniques Proteomic techniques need latest analytical tools... 

Although mass spectrometry has made significant strides in sensitivity and proteome coverage, a recent report by Weissman and co-workers indicates that significant challenges remain [72]. With the use of a complete fusion, tandem-affinity-purification (TAP)-tag library for the genome of Saccharomyces cere-visiae, protein expression levels were quantitatively determined with Western blots by chemiluminescent detection. These results were compared to MS-based (MudPIT) results [54,73], and the MS data were strongly biased toward the detection of abundant proteins. For the 75% of the proteome that is represented by proteins present at fewer than 5000 copies per cell, only 8% were... 

---