Mapping, electrophoresis

Pietrogrande, M.C., Marchetti, N., Dondi, F., Righetti, P.G. (2002). Spot overlapping in two-dimensional polyacrylamide gel electrophoresis separations a statistical study of complex protein maps. Electrophoresis 23, 283. 

A novel subfractionation approach for mitochondrial proteins a three-dimensional mitochondrial proteome map. Electrophoresis 22, 950-959. 

Marengo E, Robotti E, Gianotti V, Righetti PG, Cecconi D, Domenici E. A new integrated statistical approach to the diagnostic use of two-dimensional maps. Electrophoresis 2003 24(l-2) 225-36. 

Hanson, B. J., B. Schulenberg, W. F. Patton, and R. A. Capaldi. 2001, A novel subfractionation approach for mitochondrial proteuis a three-dimensional mitochondrial proteome map. Electrophoresis 22 950-959. 

R. R. Ogorzalek Loo, C. Mitchell, et al.. Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels implications for proteome mapping. Electrophoresis 18, 382-390 (1997). 

M. Stromqvist, Peptide mapping using combinations of size-exclusion chromatography, reversed-phase chromatography and capillary electrophoresis , 7. Chromatogr. 667 304-310(1994). 

Microtubule-associated proteins bind to microtubules in vivo and subserve a number of functions including the promotion of microtubule assembly and bundling, chemomechanical force generation, and the attachment of microtubules to transport vesicles and organelles (Olmsted, 1986). Tubulin purified from brain tissue by repeated polymerization-depolymerization contains up to 20% MAPs. The latter can be dissociated from tubulin by ion-exchange chromatography. The MAPs from brain can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 

The extraordinary complexity of human genes and their products has encouraged the development of extremely high-resolution analytical methods.75 Capillary electrophoresis is competitive with slab gel methods, with resolution up to the order of about 1,000 base pairs for sequencing, sizing, and detection of mutation. Reversed phase HPLC is useful for restriction digest mapping and MALDI-MS up to about 1000 base pairs. 

Palmieri, R., Peptide mapping by capillary electrophoresis, Appl. Brief DS-774, Beckman Instruments, Fullerton, CA, 1990. 

Suzuki, S., Kakehi, K., and Honda, S., Two-dimensional mapping of N-gly-cosidically linked asialo-oligosaccharides from glycoproteins as reductively pyridylaminated derivatives using dual separation modes of high-performance capillary electrophoresis, Anal. Biochem., 205, 227, 1992. 

Bonneil, E., Mercier, M. and Waldron, K.C., Reproducibility of a solid-phase trypsin microreactor for peptide mapping by capillary electrophoresis, Anal. Chim. Acta, 404, 29, 2000. 

Protein expression mapping by 2D gel electrophoresis and mass spectrometry... 

The use of 2D gel electrophoresis and mass spectrometry to identify proteins was discussed in Chapter 2. Protein expression mapping involves the use of these methodologies to compare expression patterns in different cell types or in the same cell type that has been exposed to different... 

The utility of protein expression mapping using 2D gel electrophoresis and mass spectrometry has been demonstrated for several experimental systems. One application has been to assess the differences in protein expression between normal and cancerous cells. For example, expression mapping has been used to identify protein markers for bladder cancer (Ostergaard et al., 1999). This was accomplished by identifying proteins released into the urine of patients with and without bladder cancer using 2D electrophoresis and mass spectrometry. 

Figure 3.1. Protein expression mapping using 2-D electrophoresis and mass spectrometry. The purpose is to compare protein expression patterns between cell types or in the same cell type under different growth conditions. Proteins are extracted from the different cell types and separated by 2D gel electrophoresis. Image analysis programs are used to compare the spot intensities between gels and identify proteins that are differentially expressed. The protein of interest is excised from the gel and its identity is determined by mass spectrometry. The power of the method increases greatly if the identity of a large number of proteins on the gel is known and present in a database because information can then be obtained without further mass spectrometry.

The difficulty with protein arrays is that proteins do not behave as uniformly as nucleic acid. Protein function is dependent on a precise, and fragile, three-dimensional structure that may be difficult to maintain in an array format. In addition, the strength and stability of interactions between proteins are not nearly as standardized as nucleic acid hybridization. Each protein-protein interaction is unique and could assume a wide range of affinities. Currently, protein expression mapping is performed almost exclusively by two-dimensional electrophoresis and mass spectrometry. The development of protein arrays, however, could provide another powerful... 

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