Applications to Protein Analysis

Figeys, D., and Aebersold, R. (1999). Microfabricated modules for sample handling, sample concentration and flow mixing application to protein analysis by tandem mass spectrometry. /. Biomech. Eng. 121, 7—12. 

Rohlicek V, Deyl Z (1989) Simple apparatus for capillary zone electrophoresis and its application to protein analysis. J Chromatogr 494 87-99. 

Clore, G. M., Gronenbom, A. M. Theory of the time dependent transferred nuclear Overhauser effect applications to stmctural analysis of ligand-protein complexes in solution. J. Magn. Reson. 1983, 53, 423 2. 

Tejero-Diez, P., Rodriguez-Sanchez, P., Diez-Guerra, F.J. (1999). Microscale purification of proteins exhibiting anomalous electrophoretic migration application to the analysis of GAP-43 phosphorylation. Anal. Biochem. 274, 278-282. 

Electrophoresis is for separating ions, since only ions will migrate under the influence of an electric field, negative ions to the positive electrode and positive ions to the negative electrode. Scientists have found electrophoresis especially useful in biochemistry experiments in which charged amino acid molecules and other biomolecules need to be separated. Thus, application to protein and nucleic acid analysis has been popular (see Chapter 16). 

Many of the advantages that MALDI offers for peptide analysis are equally applicable to proteins. Protein analysis is similar to peptide analysis, in which ionization usually occurs through the addition of one, two, or three protons. However, since proteins are significantly bigger than peptides, ion detection is typically less efficient. Therefore, while peptides are measured at the femtomole or even attomole level with MALDI, proteins are usually measured at the high femtomole to low picomole level. 

Further improvement of microchemical methods for proteinaceous media was based on immunological techniques. The high specificity of the antigen-antibody reaction enables the discrimination of the same protein coming from different species, or the detection of multiple antigens in the same sample. Application to the analysis of artwork has been reported in two types of immunological techniques immunofluorescence microscopy (IFM), and enzyme-linked immunosorbent assays (ELISA) [31]. 

The wavelengths of IR absorption bands are characteristic of specific types of chemical bonds. In the past infrared had little application in protein analysis due to instrumentation and interpretation limitations. The development of Fourier transform infrared spectroscopy (FUR) makes it possible to characterize proteins using IR techniques (Surewicz et al. 1993). Several IR absorption regions are important for protein analysis. The amide I groups in proteins have a vibration absorption frequency of 1630-1670 cm. Secondary structures of proteins such as alpha(a)-helix and beta(P)-sheet have amide absorptions of 1645-1660 cm-1 and 1665-1680 cm, respectively. Random coil has absorptions in the range of 1660-1670 cm These characterization criteria come from studies of model polypeptides with known secondary structures. Thus, FTIR is useful in conformational analysis of peptides and proteins (Arrondo et al. 1993). 

Fenn JB, Mann M, Meng CK Electrospray ionization for mass spectrometry of large biomolecules. Science (1989) 246 64-71. Patrick JS, Lagu AL Review applications of capillary electrophoresis to the analysis of biotechnology-derived therapeutic proteins. Electrophoresis (2001) 22 4179-4196. Sowell J, Salon J, Strekowski L, et al Covalent and noncovalent labeling schemes for near-infrared dyes in capillary electrophoresis protein applications. Methods Mol. Biol. (2004) 276 39-75. Moini M Capillary electrophoresis mass spectrometry and its application to the analysis ofbiological mixtures. Anal. Bio-anal. Chem. (2002) 373 466 180. Nemunaitis J, Holmlund JT, Kraynak M, et al. Phase I evaluation of ISIS 3521, an antisense oligodeoxynucleotide to protein kinase C-a, in patients with advanced cancer./. Clin. Oncol. (1999) 17 3586-3595. De Frutos M, Cifuentes A, Diez-Masa JC Differences in capillary electrophoresis profiles of urinary and recombinant erythropoietin. Electrophoresis (2003) 24 678-680. 

C3. Consden, R., and Stam er, W. M., lonophoresis of sugars on paper and some applications to the analysis of protein polysaccharide complexes. Nature 169, 783 (1952). 

Wojciechowski M, Lesyng B (2004) Generalized Bom Model, Analysis, Refinement and Applications to Proteins, J. Phys. Chem. B 108 18368-18376... 

One of the most powerful separation techniques for purity and impurity analysis for the bioanalyst is capillary electrophoresis (CE). However, it is a relatively new analytical tool and its methodology is evolving at a rapid pace, so there is limited reference to its application to protein impurity analysis in the literature. Nonetheless, this is only a temporary respite. In the future, CE will become a standard and routine analytical technique for the analysis of protein impurities in recombinant pharmaceuticals. 

Sequence similarity database searching and protein sequence analysis constitute one of the most important computational approaches to understanding protein structure and function. Although most computational methods used for nucleic acid sequence analysis are also applicable to protein sequence studies, how to capture the enriched features of amino acid alphabets (Chapter 6) poses a special challenge for protein analysis. 

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