Protein fragmentation methods

Abraham, D. J., Leo, A. J. Extension of the fragment method to calculate amino acid zwitterion and side chain partition coefficients. Proteins 1987, 2,130-152. 

Another method that has been applied by our group to the study of enzymatic reactions is the Effective Fragment Potential (EFP) method [19]. The EFP method (developed at Mark Gordon s group at Iowa State University) allows the explicit inclusion of environment effects in quantum chemical calculations. The solvent, which may consist of discrete solvent molecules, protein fragments or other material, is treated explicitly using a model potential that incorporates electrostatics, polarization, and exchange repulsion effects. The solute, which can include some... 

With advances in multidimensional NMR technology and the development of methods for uniform labeling of proteins with 15N and 13C, direct NMR studies on unfolded states of full-length proteins, rather than protein fragments, became possible. These studies were aided by the advent of high-field NMR spectrometers, which provide the necessary dispersion and sensitivity. High sensitivity is a critical factor in the study of unfolded and partly folded proteins, since NMR experiments must often be performed at very low concentrations to prevent aggregation. 

Soltes, L., Sebille, B. (1997). Reversible binding interactions between the tryptophan enantiomers and albumins of different animal species as determined by novel high performance liquid chromatographic methods an attempt to localize the d- and L-tryptophan binding sites on the human serum albumin polypeptide chain by using protein fragments. Chirality 9, 373-379. 

In a more modified approach, differential display proteomics can also be done with no separation of proteins. This is called the protein chip approach. In this method, a variety of bait proteins such as antibodies, peptides, or protein fragments may be immobilized in an array format on specially treated surfaces. The surface is then probed with the samples of interest. Proteins that bind to the relevant target can then be analyzed by direct MALDI readout of the bound material (Nelson, 1997 Davies et ah, 1999). Lor example, well-characterized antibodies can be used as bait. Protein samples from two different cell states are then labeled by different fluorophores, mixed together, and used as probe. In such a case, the fluorescent color acts as an indicator for any change in the abundance of the protein that remains bound to the chip (Lueking et ah, 1999). A number of technical problems would still need to be overcome before applying this technique for large-scale analysis of proteins. 

Helix propensities have been measured by different groups for all 20 proteinogenic amino acids using statistical surveys of protein helices/8,91 host-guest analysis/101 small model peptides and protein fragments/11-141 site-directed mutagenesis/151 and calculation studies/16171 The results from each method are different, although some correlations can be made. 

Many groups have used electrophoresis to enhance a primary chromatographic separation. These techniques can be considered to be two-dimensional, but they are not comprehensive, usually due to the loss of resolution in the interface between the two methods. For instance, capillary electrophoresis was used in 1989 by Grossman and co-workers to analyze fractions from an HPLC separation of peptide fragments. In this study, CE was employed for the separation of protein fragments that were not resolved by HPLC. These two techniques proved to be truly orthogonal, since there was no correlation between the retention time in HPLC and the elution order in CE. The analysis time for CE was found to be four times faster than for HPLC (12), which demonstrated that CE is a good candidate for the second dimension in a two-dimensional separation system, as will be discussed in more detail later. 

Reversible (noncovalent) binding events can be detected with nondenaturing ESI-MS.151-541 A considerable advantage of the method is that the stoichiometry of the binding event is measured directly. As a consequence, it is possible to observe directly if two fragments bind simultaneously. There is also direct information on compound solubility and integrity, in addition to the state of the target protein. The method suffers, however, from a few major... 

Routinely, common chemical and enzymatic techniques are used to obtain protein fragments. Unfortunately, when enzymatic digestion techniques and nanograms quantities of proteins are used, the method become ineffective due to dilution and reduced enzymatic activity. An alternative approach to overcome this problem is the use of proteolytic enzymes immobilized to a solid support and a small-bore reactor column. Using trypsin immobilized to agarose, tryptic digests of less than 100 ng of protein can be reproducible obtained (49). 

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