Peptides HPLC separation

However, the use of a HPLC separation step enabled a remarkable acceleration of the deconvolution process. Instead of preparing all of the sublibraries, the c(Arg-Lys-O-Pro-O-P-Ala) library was fractionated on a semipreparative HPLC column and three fractions as shown in Fig. 3-2 were collected and subjected to amino acid analysis. According to the analysis, the least hydrophobic fraction, which eluted first, did not contain peptides that included valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine residues and also did not exhibit any separation ability for the tested racemic amino acid derivatives (Table 3-1). 

In addition to the development of the powerful chiral additive, this study also demonstrated that the often tedious deconvolution process can be accelerated using HPLC separation. As a result, only 15 libraries had to be synthesized instead of 64 libraries that would be required for the full-scale deconvolution. A somewhat similar approach also involving HPLC fractionations has recently been demonstrated by Griffey for the deconvolution of libraries screened for biological activity [76]. Although demonstrated only for CE, the cyclic hexapeptides might also be useful selectors for the preparation of chiral stationary phases for HPLC. However, this would require the development of non-trivial additional chemistry to appropriately link the peptide to a porous solid support. 

The proteolytic digestion of j6-lactoglobulin was carried out with trypsin which, as indicated in Table 5.4 above, is expected to cleave the polypeptide backbone at the carboxy-terminus side of lysine (K) and arginine (R). On this basis, and from the known sequence of the protein, nineteen peptide fragments would be expected, as shown in Table 5.7. Only 13 components were detected after HPLC separation and, of these, ten were chosen for further study, as shown in Table 5.8. 

Matrix-associated laser desorption ionization with a time-of-flight mass analyser (MALDl-ToF) was used to examine the crude tryptic peptide mixture from a number of the proteins, without HPLC separation, to provide a mass map, i.e. a survey of the molecular weights of the peptides generated by the digestion process. 

The peptides were separated by reverse-phase HPLC. N-terminus (N-ter) was obtained with the entire PEy. ... 

Stoll, D.R., Carr, P.W. (2005). Fast, comprehensive two-dimensional HPLC separation of tryptic peptides based on high temperature HPLC. J. Am. Chem. Soc. 127, 5034-5035. 

A typical HPLC separation using a 15-cm column of 15,000 theoretical plates produces peak capacity (Giddings, 1991) of about 80-100 under isocratic conditions and up to 150 under gradient conditions in 1 h(Eq. 7.3, n peak capacity, A number of theoretical plates of a column, and fR and t retention time of the last and the first peak of the chromatogram, respectively). An increase in the number of separated peaks per unit time can be achieved by increased separation speed made possible by monolithic silica columns (Deng et al., 2002 Volmer et al., 2002). This has also been shown for peptides and proteins (Minakuchi et al., 1998 Leinweber et al., 2003). 

Proteomics ultimately hinges upon protein identification to reveal the meaning behind the masses, spots, or peaks detected by other means. Because fraction collection is a natural component of HPLC separations, intact proteins can be readily collected either for direct analysis or for proteolytic digestion and identification using peptide mass fingerprinting (PMF) in conjunction with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 

Determination of iodo amino acids by HPLC with inductively coupled plasma (ICP)-MS detection had LOD 35-130 pg of I, which is about one order of magnitude lower than with UVD usually applied for these compounds175. Amino acids and peptides containing sulfur, such as cysteine, cystine, methionine and glutathione, can be determined after HPLC separation by pulsed electrochemical detection, using gold electrodes176. 

Ilisz, I., Berkecz, R., and Peter, A., HPLC separation of amino acid enantiomers and small peptides on macrocyclic antibiotic-based chiral stationary phases a review, J. Sep. ScL, 29, 1305, 2006. 

Three approaches can be employed to separate peptide stereoisomers and amino acid enantiomers separations on chiral columns, separations on achiral stationary phases with mobile phases containing chiral selectors, and precolumn derivatization with chiral agents [111]. Cyclodextrins are most often used for the preparation of chiral columns and as chiral selectors in mobile phases. Macrocyclic antibiotics have also been used as chiral selectors [126]. Very recently, Ilsz et al. [127] reviewed HPLC separation of small peptides and amino acids on macrocyclic antibiotic-based chiral stationary phases. 

Figure 15 Representative RP-HPLC Separation of the Tryptic/Chymotryptic Peptides of Bovine Thyrotropin p-Subunit bTSHfS Labeled In Situ with the Fluorescent Reagent 5-[(Iodoacetamidoethyl)amino]naphthalene-l-sulfonic acid (5-l-AEDANS) MI a b...

In the case of peptide separation by HPLC, separation modes are combined in series. This approach is called tandem LC. For instance, ion exchange associated with RP is used for peptide separation. Multidimensional protein identification technique (MudPIT) involving use of microcapillary columns (SCX cationic column and RP column) linked in series and eluted into MS is preferred for separation of complex peptide mixtures (Figure 5.4). 

Although the silica-based columns are the most widely used in RP-HPLC separations of peptides, the use of polymeric carriers (polystyrene divinylbenzene) and composite materials (silica particles with a polymeric coating), which are more chemically stable in that they do not break down at pH values higher than 8 as silica does, is gaining currency (54,55). The mobile phase usually consists of a mixture of water and an organic solvent, generally acetonitrile, methanol, or... 

The greatest advance in the analysis of peptides has been the coupling with spectrometric techniques, both for identification and for characterization. Their high accuracy, sensitivity, and, in some instances, tolerance to solvents make mass spectrometers ideal detectors for analyses of HPLC-separated peptides. 

One of the problems attaching to the HPLC separation of peptides is the analysis of stereoisomers (enantiomers and diastereoisomers), that is, of peptides that differ only in the configuration of their amino acid residues. 

O-Phthaldialdehyde (OPA) is an amine detection reagent that reacts in the presence of 2-mercaptoethanol to generate a fluorescent product (for preparation, see Section 4.1, 2-mercaptoethanol) (Fig. 91). The resultant fluorophore has an excitation wavelength of 360 nm and an emission point at 455 nm. OPA can be used as a sensitive detection reagent for the HPLC separation of amino acids, peptides, and proteins (Fried et al., 1985). It is also possible to measure the amine content in proteins and other molecules using a test tube or microplate format assay with OPA. Detection limits are typically in the microgram per milliliter range for proteins. 

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. 

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