Peptides column selection

Other reviews of multidimensional separations have been published. These include a book on polymer characterization by hyphenated and multidimensional techniques (Provder et al., 1995), a review on polymer analysis by 2DLC (van der Horst and Schoenmakers, 2003), and two reviews on two-dimensional techniques in peptide and protein separations (Issaq et al., 2005 Stroink et al., 2005). Reviews on multidimensional separations in biomedical and pharmaceutical analysis (Dixon et al. 2006) and multidimensional column selectivity (Jandera, 2006) were recently published. Suggested nomenclature and conventions for comprehensive multidimensional chromatography were published in 2003 (Schoenmakers et al., 2003), and a book chapter in the Advances in Chromatography series on MDLC was published in 2006 (Shalliker and Gray 2006). 

In common with other application areas of chromatographic separation, a considerable amount of effort has been expended recently on the development of different elution conditions and types of stationary phases for peptide separations in attempts to maximize column selectivities without adversely affecting column efficiences. Peptide retention will invariably be mediated by the participation of electrostatic, hydrogen bonding, and hydrophobic interactions in the distribution phenomenon. The nature of the predominant distribution mechanism will be dependent on the physical and chemical characteristics of the stationary phase as well as the nature of the molecular forces which hold the solute molecules within the mobile and stationary zones. The retention of the solute in all HPLC modes can be described by the equation... 

For the affinity column selection library, a solution-phase peptide library (a mixture) is loaded onto a column with an immobilized target protein. This method has been applied successfully to retrieve an antibody-specific binding peptide from a mixture of peptides. It also was reported to identify peptide motifs for SH2 domains and kinase domains of protein tyrosine kinases. 

Figure 5 Inhibition of [ H]-cefadroxil uptake into Caco-2 cells by stereoisomers of alanyl peptides and selected P-lactam antibiotics. In addition, influx inhibition by the dipeptides glycyl-sarcosine (Gly-Sar) and glycyl-glutamine (Gly-Gln) and the P-lactams, amoxicillin and benzylpenicillin, is presented. Uptake of 1 mM cefadroxil into Caco-2 cells was measured at apical buffer pH 6.5 for 30 min at 37°C in the absence (control) or presence of lOmM of the inhibitors. The aminopeptidase inhibitor, amasatin, was present at a concentration of 100 pM in each experiment. Values are expressed as the mean SD of 3-6 monolayers. Hatched columns ( / ) no significant inhibition open columns 50% inhibition back hatched columns ( ) 60% inhibition. (Adapted from Ref. 16.)... 

The properties of the peptide or protein of interest that are most important in column selection are as follows pi, molecular weight, hydrophobicity, reactivity. 

This result is important to fully understanding the biochemical and ultra-structural origin of peaks and the physiological basis for variation. It not only helps in designing the analytical strategy (e.g., in selection of cleanup columns) but, more important, in making a decision on whether the marker should be used for strain or species identification or for biodetection. For example, there are a number of low-molecular weight peptides (1500-8000 kDa) present in... 

FIGURE 12.5 Human serum tryptic digest analysis. Fractionation in the first LC dimension was performed using a C18 column at pH 10. Fractions were analyzed using NanoEase 0.3 x 150 mm Atlantis d18 column. Approximately 66 lg (400 pmole of semm albumin peptides) was injected on column. Arrow points to a selected albumin peptide illustrating a local column mass overloading. Ten-5mm wide fractions were collected in 1st LC dimension. 

Since amino acids and nucleotides are all polar and hydrophilic, they will be eluted quickly by the column. The mobile phase (see below) is also selected on the basis of polarity, with a medium- to high-polarity solvent required. The opposite of reverse phase chromatography is normal phase, where the column packing is medium to high polarity and the mobile phase is nonpolar. This technology is generally not applied to the analysis of polar molecules such as amino acids or nucleotides. Some peptides are more hydrophobic, making this method potentially more useful for peptides than for amino acids or nucleotides. 

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