Dear Jahnu,

The following Review article entitled " Analysis of Endogenous D-Amino Acid-Containing Peptides in Metazoa" by Sweedler and coworkers published in Bioanal Rev. 2009 Dec; 1(1): 7–24 depicts all methods available for verification of D and L stereo-isomers in a peptide sequence:

Non-Chiral Separations

Many D-amino acid-containing peptides (DAACPs) were discovered because they possess different properties from the all-L isomers. Intrinsic differences in these peptide diastereomers—pKa values, polarities, hydrodynamic radii—have been exploited in many DAACP separations using high performance liquid chromatography (HPLC), with several widely adopted for capillary electrophoresis (CE).

High Performance Liquid Chromatography

HPLC is the most commonly used separation technique for peptides. Therefore, it should not be surprising that it is the most prevalent method in DAACP investigations, partly owing to its ability to discriminate between diastereomers using non-chiral (such as C18) columns and chiral stationary phase-based columns, its relative low cost, and high throughput. Except for rare cases where diastereomers exhibit similar retention behavior using reversed-phase (RP)-HPLC , most known DAACPs have been separated using achiral columns in RP-HPLC. Ion-exchange chromatography has also been successful in this application but is not as often used.

Capillary Electrophoresis

CE separations are based on charge and molecular shape, and are categorized into several modes. Many peptide diastereomers, especially small ones, can be resolved in CE using simple phosphate buffer systems without adding chiral selectors. Enkephalin-related peptides, including two pairs of diastereomers, have been separated using CE, with sodium dihydrogen phosphate as the buffer system. A decrease in pH shortens separation time and improves separation efficiency, and in this case, a pH slightly higher than 3 was found to be optimal.

Non-Chiral Separations

Many DAACPs were discovered because they possess different properties from the all-L isomers. Intrinsic differences in these peptide diastereomers—pKa values, polarities, hydrodynamic radii—have been exploited in many DAACP separations using high performance liquid chromatography (HPLC), with several widely adopted for capillary electrophoresis (CE).

Chiral Separations

Although peptide diastereomers can be discriminated from each other based on differences in a variety of physicochemical properties such as pKa, shape, and hydrophobocity, the use of chiral selectors often makes separations easier. Diastereomeric analytes form transient complexes based on the affinity of the analyte to the chiral selector, and thus result in different retention behaviors or electrophoretic mobilities. 

 High Performance Liquid Chromatography

While enantiomeric separations require a chiral selector, as described above, diastereomers can be resolved using RP or ion-exchange columns without a chiral additive or stationary phase. The addition of a chiral selector such as a CSP often improves the resolution between peptides and usually does not require peptide derivatization, and therefore has become an effective means of DAACP/LAACP separation. Because the goals of characterizing endogenous DAACPs are often to understand peptide function, and the goals of synthetic DAACP/LAACP separations are to understand and optimize the separation approaches, most comparisons have involved synthetic peptide mixtures. In what follows, several of the more common CSP categories are briefly described in relation to DAACP separations

Capillary Electrophoresis

CE is a small-volume separation approach with high separation efficiency, well suited to separating mass-limited samples such as endogenous peptides. Chiral separations use mobile phase additives added to the background electrolyte. Commonly used CMPAs include cyclodextrins, chiral ion pair complexes, chiral ligand-exchange complexes, and others. Because the selector is added to the electrolyte, one can easily optimize the additive composition and concentration, unlike a system that requires a new stationary phase.

Micellar Electrokinetic Chromatography

By adding surfactants into the background electrolyte, CE can be operated in a different mode with a chromatographic principle, known as MEKC. This mode is not as often used for DAACP/LAACP applications. The examples mentioned here mainly include the use of derivatized peptides. Wan et al. reported the separation of nine peptides with γ-cyclodextrin as the chiral selector. These peptides were derivatized with 9-fluorenylmethyl chloroformate and subjected to MEKC separations. Two other peptides, Leu-Ala and Leu-Leu, were separated with β-cyclodextrin after derivatization. With the use of surfactants, separation based on both chiral selectivity and hydrophobicity occurred, resulting in a separation power that chiral selectors alone could not have achieved. In their later work, this same group evaluated the concentration of teicoplanin and acetonitrile on the resolution and retention time of di- and tri-peptide separations.

Capillary Electrochromatography

Interestingly, the roles of LC and CE in chiral separations are distinct and yet somewhat complementary to each other. CEC can be considered a hybrid of these approaches, as it contains features of both LC and CE and so should be mentioned. In one report, the chiral peptide separation was achieved using a capillary packed with teicoplanin aglycone as a CSP. The diastereomeric dipeptides and tripeptides investigated were baseline-resolved. For these peptides, RP separations worked well, with the organic modifier having an important impact on the separation.

Mass Spectrometry

While MS can be interfaced with LC and CE separations, the ability to separate DAACPS from LAACPs is based on the differences in elution time offered by the separation. Can MS be used to characterize DAACPs directly? After all, MS is perhaps the detector of choice for peptide characterization as it allows structural characterization of the peptide, including most PTMs and sequences. How does one use MS to determine the chirality of an amino acid residue? Several studies have shown that differences in the chemical properties of two diastereomers can be detected by monitoring the stability of complexes formed with chiral selectors, and that tandem-MS provides different fragmentation results based on the chirality of the amino acid residues.

Nuclear Magnetic Resonance

NMR can determine the three dimensional structure of a peptide and so can be used to resolve the chirality of peptide residues. Typically used to study molecular interactions between analytes such as peptides and chiral selectors, it does not appear that NMR has been used for DAACP peptide discovery as yet, partially because of its intrinsic low sensitivity compared to the separations- and MS-based approaches. It also has been applied to studying peptide-receptor interactions and will become an indispensable tool to examine the differences in interactions between an endogenous DAACP and an LAACP with their cognate receptors. As one example of the use of NMR, achatin-I (GdFAD), isolated from A. fulica, was found to have different chromatographic behavior from its all-L isomer. An 1H-NMR spectral study demonstrated the points of interaction as the α-methine proton of the phenylalanine with the α-methane proton and the methyl protons of alanine in achatin-I appearing upfield from the L-isomer. In another interesting study from the peptide toxin field, an NMR experiment demonstrated the necessity of having a D-Phe13 in the conotoxin peptide, conomarphin, for forming a tight loop in the middle of the peptide. The toxin counterpart with L-Phe13 did not form this loop, perhaps indicating the reason why a D-Phe is required to obtain the specific functions of this peptide.

Peptide Digestion

Acid Hydrolyzation

In multiple instances of DAACP discovery, acid hydrolysis followed by derivatization and separation is common because the separation of amino acid enantiomers is a relatively mature technique. The limited number of possible amino acids makes the analysis of peptide hydrolysates more convenient. As a classic example, to confirm that [D-Leu2]deltorphin was a DAACP, the peptide was first hydrolyzed, amino acids derivatized with a chiral reagent, 1-fluoro-2,4-dinitro-5-L-alanine and separated by RP-HPLC, and configuration of each residue determined.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2871709/

For references and more on this topic, please use the following link:

Rafik

Dear Jahnu,

The following Review article entitled " Analysis of Endogenous D-Amino Acid-Containing Peptides in Metazoa" by Sweedler and coworkers published in Bioanal Rev. 2009 Dec; 1(1): 7–24 depicts all methods available for verification of D and L stereo-isomers in a peptide sequence:

Non-Chiral Separations

Many D-amino acid-containing peptides (DAACPs) were discovered because they possess different properties from the all-L isomers. Intrinsic differences in these peptide diastereomers—pKa values, polarities, hydrodynamic radii—have been exploited in many DAACP separations using high performance liquid chromatography (HPLC), with several widely adopted for capillary electrophoresis (CE).

High Performance Liquid Chromatography

HPLC is the most commonly used separation technique for peptides. Therefore, it should not be surprising that it is the most prevalent method in DAACP investigations, partly owing to its ability to discriminate between diastereomers using non-chiral (such as C18) columns and chiral stationary phase-based columns, its relative low cost, and high throughput. Except for rare cases where diastereomers exhibit similar retention behavior using reversed-phase (RP)-HPLC , most known DAACPs have been separated using achiral columns in RP-HPLC. Ion-exchange chromatography has also been successful in this application but is not as often used.

Capillary Electrophoresis

CE separations are based on charge and molecular shape, and are categorized into several modes. Many peptide diastereomers, especially small ones, can be resolved in CE using simple phosphate buffer systems without adding chiral selectors. Enkephalin-related peptides, including two pairs of diastereomers, have been separated using CE, with sodium dihydrogen phosphate as the buffer system. A decrease in pH shortens separation time and improves separation efficiency, and in this case, a pH slightly higher than 3 was found to be optimal.

Non-Chiral Separations

Many DAACPs were discovered because they possess different properties from the all-L isomers. Intrinsic differences in these peptide diastereomers—pKa values, polarities, hydrodynamic radii—have been exploited in many DAACP separations using high performance liquid chromatography (HPLC), with several widely adopted for capillary electrophoresis (CE).

Chiral Separations

Although peptide diastereomers can be discriminated from each other based on differences in a variety of physicochemical properties such as pKa, shape, and hydrophobocity, the use of chiral selectors often makes separations easier. Diastereomeric analytes form transient complexes based on the affinity of the analyte to the chiral selector, and thus result in different retention behaviors or electrophoretic mobilities. 

 High Performance Liquid Chromatography

While enantiomeric separations require a chiral selector, as described above, diastereomers can be resolved using RP or ion-exchange columns without a chiral additive or stationary phase. The addition of a chiral selector such as a CSP often improves the resolution between peptides and usually does not require peptide derivatization, and therefore has become an effective means of DAACP/LAACP separation. Because the goals of characterizing endogenous DAACPs are often to understand peptide function, and the goals of synthetic DAACP/LAACP separations are to understand and optimize the separation approaches, most comparisons have involved synthetic peptide mixtures. In what follows, several of the more common CSP categories are briefly described in relation to DAACP separations

Capillary Electrophoresis

CE is a small-volume separation approach with high separation efficiency, well suited to separating mass-limited samples such as endogenous peptides. Chiral separations use mobile phase additives added to the background electrolyte. Commonly used CMPAs include cyclodextrins, chiral ion pair complexes, chiral ligand-exchange complexes, and others. Because the selector is added to the electrolyte, one can easily optimize the additive composition and concentration, unlike a system that requires a new stationary phase.

Micellar Electrokinetic Chromatography

By adding surfactants into the background electrolyte, CE can be operated in a different mode with a chromatographic principle, known as MEKC. This mode is not as often used for DAACP/LAACP applications. The examples mentioned here mainly include the use of derivatized peptides. Wan et al. reported the separation of nine peptides with γ-cyclodextrin as the chiral selector. These peptides were derivatized with 9-fluorenylmethyl chloroformate and subjected to MEKC separations. Two other peptides, Leu-Ala and Leu-Leu, were separated with β-cyclodextrin after derivatization. With the use of surfactants, separation based on both chiral selectivity and hydrophobicity occurred, resulting in a separation power that chiral selectors alone could not have achieved. In their later work, this same group evaluated the concentration of teicoplanin and acetonitrile on the resolution and retention time of di- and tri-peptide separations.

Capillary Electrochromatography

Interestingly, the roles of LC and CE in chiral separations are distinct and yet somewhat complementary to each other. CEC can be considered a hybrid of these approaches, as it contains features of both LC and CE and so should be mentioned. In one report, the chiral peptide separation was achieved using a capillary packed with teicoplanin aglycone as a CSP. The diastereomeric dipeptides and tripeptides investigated were baseline-resolved. For these peptides, RP separations worked well, with the organic modifier having an important impact on the separation.

Mass Spectrometry

While MS can be interfaced with LC and CE separations, the ability to separate DAACPS from LAACPs is based on the differences in elution time offered by the separation. Can MS be used to characterize DAACPs directly? After all, MS is perhaps the detector of choice for peptide characterization as it allows structural characterization of the peptide, including most PTMs and sequences. How does one use MS to determine the chirality of an amino acid residue? Several studies have shown that differences in the chemical properties of two diastereomers can be detected by monitoring the stability of complexes formed with chiral selectors, and that tandem-MS provides different fragmentation results based on the chirality of the amino acid residues.

Nuclear Magnetic Resonance

NMR can determine the three dimensional structure of a peptide and so can be used to resolve the chirality of peptide residues. Typically used to study molecular interactions between analytes such as peptides and chiral selectors, it does not appear that NMR has been used for DAACP peptide discovery as yet, partially because of its intrinsic low sensitivity compared to the separations- and MS-based approaches. It also has been applied to studying peptide-receptor interactions and will become an indispensable tool to examine the differences in interactions between an endogenous DAACP and an LAACP with their cognate receptors. As one example of the use of NMR, achatin-I (GdFAD), isolated from A. fulica, was found to have different chromatographic behavior from its all-L isomer. An 1H-NMR spectral study demonstrated the points of interaction as the α-methine proton of the phenylalanine with the α-methane proton and the methyl protons of alanine in achatin-I appearing upfield from the L-isomer. In another interesting study from the peptide toxin field, an NMR experiment demonstrated the necessity of having a D-Phe13 in the conotoxin peptide, conomarphin, for forming a tight loop in the middle of the peptide. The toxin counterpart with L-Phe13 did not form this loop, perhaps indicating the reason why a D-Phe is required to obtain the specific functions of this peptide.

Peptide Digestion

Acid Hydrolyzation

In multiple instances of DAACP discovery, acid hydrolysis followed by derivatization and separation is common because the separation of amino acid enantiomers is a relatively mature technique. The limited number of possible amino acids makes the analysis of peptide hydrolysates more convenient. As a classic example, to confirm that [D-Leu2]deltorphin was a DAACP, the peptide was first hydrolyzed, amino acids derivatized with a chiral reagent, 1-fluoro-2,4-dinitro-5-L-alanine and separated by RP-HPLC, and configuration of each residue determined.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2871709/

For references and more on this topic, please use the following link:

Rafik

MALDI TOF-TOF will be a good method. The parental peaks will demonstrate the incorporation of several D- amino acids in the whole sequences. The fragmentation pattern will indicate the specific locations of individual D- amino acid and provide verification of the sequence.

Thank you Rafik Karaman.

Thank you Xin Qi.

will polarimetric estimation give a better idea about the sequence?

Dear Jahnu,

Polarimetry rotation will give you information about the optical rotation of the whole peptide (if it is + or -) but it dos not give any indication what amino acid is L and what is R. Thus, no indication about the sequence will be achieved using this method.

Hoping this will clarify the point,

Rafik