Peptides, derivatives separation

Polypeptide Synthesis and Analysis. Sihca or controUed-pore glass supports treated with (chloromethyl)phenylethyltrimethoxysilane [68128-25-6] or its derivatives are replacing chloromethylated styrene—divinylbenzene (Merrifield resin) as supports in polypeptide synthesis. The sdylated support reacts with the triethyl ammonium salt of a protected amino acid. Once the initial amino acid residue has been coupled to the support, a variety of peptide synthesis methods can be used (34). At the completion of synthesis, the anchored peptide is separated from the support with hydrogen bromide in acetic acid (see Protein engineering Proteins). 

Besides sensitive methods for the analysis of proteins, bioinformatics is one of the key components of proteome research. This includes software to monitor and quantify the separation of complex samples, e.g., to analyze 2DE images. Web-based database search engines are available to compare experimentally measured peptide masses or sequence ions of protein digests with theoretical values of peptides derived from protein sequences. Websites for database searching with mass spectrometric data may be found at http //www.expasy.ch/tools, http //prospector.ucsf. edu/ and http //www.matrixscience.com. 

Sweedler reported a two-dimensional separation, where fluorescein thiocarbamyl derivatives of peptides were separated by capillary zone electrophoresis in the first dimension (Liu and Sweedler, 1996). The outlet of the capillary was wiped across the top of an SDS-PAGE gel, where peptides were then separated based on their size. 

Following cleavage with hydrogen fluoride, the various classes of peptides were separated in a one-step purification procedure on a tertiary or quaternary amine column. After removal of the Sulfmoc group with 5% TEA, homogeneous Leu-Ala-Gly-Val, for example, was obtained. The Sulfmoc procedure was also very effective for purification of synthetic thymosin oq (28 residues). 87 This was the first use of an Fmoc derivative for selective and reversible orthogonal peptide purification. 

The electronic property of the amino acid on the C-terminus also has an effect on antioxidant activity (Li et al., 2011), that is, the larger the electronic property, the higher is the activity. The C-terminus is a polar position that is thus affected by its electrostatic potential, to some extent therefore, the amino acids Trp, Glu, Leu, lie, Met, Val, Tyr, etc. are suitable at the C-terminus. Some researchers have speculated that the identity of the amino acid on the C-terminus would play an important role in its activity. Suetsuna (2000) separated and identified a radical scavenging peptide, Tyr-Phe-Tyr-Pro-Glu-Leu, from casein hydrolysate, and it was confirmed that the Glu-Leu on the C-terminus mainly contributed to its antioxidant activity. Kim et al. (2009) speculated that the hydrophobic property of the amino acid on the C-terminus, for example, Val and Leu, had a distinct effect on the activity, as determined from the analysis of antioxidative peptides derived from venison hydrolysate. 

Separation of diastereoisomeric peptides by HPLC is more common. Since each diastereo-isomer has different physicochemical and biological properties, this is of great interest. Separations of diastereoiosomeric di- and tripeptides have usually been performed on reversed-phase columns. Cahill et al. (119) separated diastereoisomeric amino acids and derivatized dipeptides using esters of the /V-hydroxysuccinamide of f-butyl carbonyl-L-amino acid on Cl8 and C8 columns. Linder et al. (120) separated amino acid and peptide derivatives on an RP-C8 column, adding a metal chelate. Mixtures of DL and LD-dipeptides can be separated by RP-HPLC into two peaks, one containing LL- and DD-isomers, the other containing LD and DL-isomers. Sep-... 

FIGURE 1 Example of a gel-free-oriented proteomics nano-LC/MS-MS workflow in which bacterial culture proteins digested to tryptic peptides are separated via LC and peptides subsequently analyzed by mass spectrometry. In the process, the spectrometer rapidly cycles every few seconds and examines a size window in which peptide-derived MSI ions are analyzed to define MS/MS (MS2) spectra. The MS/MS (MS2) spectrum generated for each peptide then enters a bioinformatic pipeline for sequence identification, statistical validation, and quantification. 

Heegaard et al. [8] seem to be the first who used this approach to separate free synthetic peptide derived from the heparin-binding region of human serum amyloid P component from its complex with anionic carbohydrates. Quantification of the peptide in a series of affinity mixtures was accomplished by a comparison of the relevant peak areas with the calibration curve. Using UV detection, the authors obtained a dissociation constant of about 10 s M, which is in the range of the sensitivity such detection can provide. 

In the first study appearing in the literature on wine peptides, Acedo et al. (1994) applied RP-HPLC to separate peptides from wine after the formation of peptide derivatives with o-phthaldialdehyde. Also an RP-HPLC procedure using a Nova-Pak Cig column under gradient conditions was applied to analyze peptides... 

The study of the proteome of the recombinant adenovirus type 5 vectors demonstrated an important apphcation of separation techniques in combination with MS methods in the drug discovery process. With completely sequenced adenovirus genome available, this approach provides a chemically well-dehned method of characterization of structural proteins of recombinant adenoviral vectors. The information of protein MWs, tryptic peptide mass mapping, and sequence tags of tryptic peptides derived from HPLC/MS resulted in the identification of 17 adenoviral proteins/polypeptides in the purified virion. The rapid and accurate identification of viral proteins from recombinant adenoviruses in this study is significant since it provides direct evidence of the maturation stage of adenoviruses, which is closely related to viral infectivity and efficacy in gene therapy. 

Urea affects the gel as well as the state of aggregation of solutes. Stepa-now et al. (1961) have shown that formation of peptide-peptide complexes may be avoided in phenolate or alkaline urea solutions. Two peptides derived from tobacco mosaic virus protein could be separated with Sephadex only in the presence of 8 M urea. Urea need only be included in the sample solution, not in the eluting solvent (0.01 M sodium hydroxide). Upon filtration on a Sephadex G-50 column, urea and the two peptides moved as well-separated zones. The authors did not comment on the choice of G-.50. It was probably found to be superior to G-25 since urea seems to close the pores and meshes of a Sephadex gel. The reduction in effective pore and mesh size is perhaps caused by urea being bound to the carbohydrate network since the swelling is in fact increased in strong urea solutions. 

Table II summarizes those amino acids that contain more than an un-reactive aliphatic chain, namely a reactive site which may be a functional group in the traditional sense such as sulfhydryl, thiomethyl, hydroxyl, carboxyl, carbamide, amino, or guanido, or may be an activated aromatic ring or heterocycle such as the phenolic part of tyrosine, the pyrrole unit in troptophan, and the imidazole part in histidine. Phenylalanine would only be considered in this connection as the reactive di- or tetrahydro derivative ring. iV-Peptides derived from proline and hydroxyproline are in a separate class because they are tertiary amides carrying no proton at the nitrogen atom. It may be possible to utilize this special feature for a preferential cleavage under proper conditions.

To a cooled (—10 °C) soln of the protected amino acid or peptide derivative (2 mmol) in CH2CI2 (50 mL) was added dropwise IM BBr3 in CH2CI2 (lOmL, lOmmol) with stirring. After 1 h at —10°C and 2h at 25 °C, the reaction was terminated by careful dropwise addition of H2O (50 mL). The layers were separated, the organic phase was washed with H2O (3 x 25 mL), and the combined aqueous layers were concentrated. The residue was taken up in H2O and purified by chromatography. 

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