Protein characterizing

The secondary stmcture elements are then identified, and finally, the three-dimensional protein stmcture is obtained from the measured interproton distances and torsion angle parameters. This procedure requites a minimum of two days of nmr instmment time per sample, because two pulse delays are requited in the 3-D experiment. In addition, approximately 20 hours of computing time, using a supercomputer, is necessary for the calculations. Nevertheless, protein stmcture can be assigned using 3-D nmr and a resolution of 0.2 nanometers is achievable. The largest protein characterized by nmr at this writing contained 43 amino acid units (51). However, attempts ate underway to characterize the stmcture of interleukin 2 [85898-30-2] which has over 150 amino acid units. 

Polyacrylamide gel electrophoresis is one of the most commonly used electrophoretic methods. AnalyMcal uses of this technique center around protein characterization, for example, purity, size, or molecular weight, and composition of a protein. Polyacrylamide gels can be used in both reduced and nonreduced systems as weU as in combination with discontinuous and ief systems (39). 

Mann, M., and Wilm, M., 1995. Electro.spray ma.ss. spectrometry for protein characterization. Trends in Biochemical Sciences 20 219-224. A review of die ba.sic application of ma.ss. spectrometric methods to the analysis of protein. sequence and. structure. 

This review will focus on the large family of EF-hand proteins characterized by a common structural motif, the EF-hand (Fig 1). 

Light scattering (nephelometry) was used as a detection system for gly-cosaminoglycans from urine, eluted from a DEAE Sephadex (Pharmacia Biotechnology Uppsala, Sweden) A-25 column.68 This technique has been more recently applied to protein characterization.69 Interferometry was used for analysis of dextran eluted from a size exclusion column.70 One of the problems of electrochemical detection is that it is relatively insensitive to polymers. Because many of the materials discussed below (DNA, proteins, and polysaccharides) are polymeric, a brief mention of some alternative... 

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. 

Number of Proteins and Residues in Databases of Intrinsically Disordered Protein Characterized by Various Methods... 

Ducret, A., Van Oostveen, I., Eng, J.K., Yates, J.R., 3rd, Aebersold, R. (1998). High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry. Protein Sci. 7, 706-719. 

In addition to the determination of molar mass distributions and various molar mass averages there are many experiments, requiring sometimes sophisticated data evaluation, that can be carried out with an analytical ultracentrifuge. Examples are the analysis of association, the analysis of heterogeneity, the observation of chemical reactions, and protein characterization, to mention only a few. A detailed discussion is beyond the scope of this article, but there is excellent literature available [77-79,81,87-89]... 

Pennington, S.R., Wilkins, M.R., Hochstrasser, D.F. and Dunn, M.J. (1999) Proteome analysis from protein characterization to biological function. Trends in Cell Biology, 1, 168-173. 

Newsholme SJ et al. Two-dimensional electrophoresis of liver proteins characterization of a drug-induced hepatomegaly in rats. Electrophoresis 2000 21 2122-2128. 

Usui, K., Ojima, T., Takahashi, M., Nokihara, K. and Mihara, H. (2004a). Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns. Biopolymers 76, 129-139. 

This multidimensional protein identification technology (MudPIT) specifically incorporates a strong cationic exchange (SCX) column in tandem with an RP column to achieve maximal resolution and exquisite sensitivity. MudPIT is effective for studying complex proteomes such as mammalian cellular samples. It has been applied to large-scale protein characterization with identification of up to 1484 proteins from yeast in a single experiment.12... 

V. Applications of Protein Characterization with Mass Spectrometry ... 

The identification of the fold is, however, only a minor part of protein characterization. Function is a loosely defined term, but must be viewed within a particular context, e.g., protein function can only take place with an interaction partner or within cellular cascades and networks. Fold predictions and homology searches can only give partial answers to such higher order functions. Thus, independent functional features have to be collected and put into context. Such features include not only molecular properties, but also cellular roles, expression patterns, dysfunctions, pathway context, and subcellular localization. The latter can be predicted by exploiting a variety of methods and localization sites. Kenta Nakai reviews many such sites and their implementation... 

Californium plasma desorption ( Cf-PD) dates back to 1973 [4-6,22,154-156] and was the first method to yield quasimolecular ions of bovine insulin. [157] Practically, Cf-PD served for protein characterization, a field of application which is now almost fully transferred to MALDI or ESI (Chaps. 10,11). [158]... 

ESI is not only a versatile tool for any aspects of peptide and protein characterization including their complete sequencing, it also offers numerous other fields of application [5,17] some of which are highlighted below. 

Valaskovic M, KeUeher NK, McLafferty FW. 1996. Attomole protein characterization by capillary electrophoresis—mass spectrometry. Science 273 1199-1202. 

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