Proteins characterization

Proteins are the unique products of gene expression, either produced on a continuing basis (constitutive proteins) reflecting the ongoing needs or synthesized in response to specific environmental stimuli (inducible proteins). Hence, proteins can serve as fingerprints of the nucleic acid segment (gene) that encoded them and, by extrapolation, the sequence of nucleic acids. 

Once extracted from the cell, protein separation is performed using one- or two-dimensional polyacrylamide gel electrophoresis (PAGE). Here, the protein extracts are placed onto a semisolid support (gel) and separated based on their relative migration within a lane induced by an electrical current. Once separated, the various bands representing different proteins can be stained for visualization. The presence or absence of different proteins can then be used to identify similarities and differences between isolates. These methods have been used in the identification of lactic acid bacteria isolated from wines (Couto and Hogg, 1994 Patarata et al., 1994). 

In addition to gel electrophoresis, proteins can also be characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Developed by Karas etal. (1987), this system uses a laser to irradiate intact cells, which ionizes surface cell proteins that are then characterized. The patterns of proteins yield a profile unique to the microorganism and are therefore used for identification purposes. MALDI-TOF-MS offers the advantage of rapid results with minimal sample preparation and reagents. However, the cost of equipment makes these methods impractical for wineries. 

Electrophoresis separation can be monodimensional (ID) or bidimensional (2D). In ID electrophoresis, proteins are separated in one dimension on the basis of only one property, such as charge (isoelectric point) or size. 2D electrophoresis combines the ID electrophoresis 

Lately, the discontinuous Laemmli system buffer (containing 0.1% w/v SDS) is most commonly used for SDS-PAGE. The method has been widely used for the characterization of the protein profiles of processed whey, meat, cereal, legume, and seed proteins (Roy et al., 2007 Miyamoto et al, 2009 Boye et al, 2010b Sikes et al., 2010 Warchalewski and Gralik, 2010) as well as the analysis of membrane proteins (Braun et al, 2009). Further information on SDS-PAGE electrophoresis can be found elsewhere (Amersham, 1999a). 

Immunoelectrophoresis is a specific method characterized by the presence of polyclonal monospecific antibodies against a specific protein in the agarose gel. The running buffer used in this method should have a pH corresponding to the isoelectric point of the antibody. The fixation of the latter in the gel leads to the formation of a precipitate comprised of the antigen and the specific antibody, which may be visualized by using a Coomassie blue stain or some other suitable technique (Westermeier and Scheibe, 2009). Immunoelectrophoresis has been used for the qualitative and quantitative analysis of individual proteins in complex mixture (Hansen and Larsen, 2008). 

Heat treatment is one of the most commonly used techniques in food processing, and is frequently responsible for the transition of proteins from the native folded state to the denatured unfolded state. Thermal denaturation of proteins involves a structural change which affects the nutritional quality of foods (Plum, 2009). 

Differential scanning calorimetry (DSC) is a suitable method for the characterization of thermal and thermodynamic stabilities of the protein. DSC provides several tools for the study of the thermal properties of proteins under controlled heating and cooling rates and can be used to determine the apparent specific heat of proteins (O Brien and Haq, 2(X)4). 

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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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