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Chromatographic Techniques
Classical techniques like PAGE, sodium dodecyl sulfate PAGE (SDS-PAGE) and
2D-PAGE have been used previously, although they are less accurate in identifying
biomolecules compared to CE or HPLC coupled to mass spectrometry (Bernal et al.
2011). Based on the peptide physiochemical properties, different chromatographic
purification techniques can be used. For example, to retain hydrophilic peptides,
hydrophilic interaction chromatography (HILIC) is a better option. Size exclusion
chromatography (SEC) is often used for routine and validation analyses because of
its reproducibility and speed but coupling this technique with MS is a challenge
(Capriotti et al. 2016). Separation of large number of peptides in a sample is performed by HPLC because of its efficiency, versatility and automation capability
(Hernandez-Ledesma et al. 2013). To analyze milk-derived bioactive peptides,
reversed-phase HPLC (RP-HPLC) is commonly employed to separate small peptides (Recio and Lopez-Fandino 2010); this technique is more effective because
most bioactive peptides are small in size (Roberts et al. 1999). In RP-HPLC, retention time depends on the amino acid composition of small peptides while for larger
peptides, retention time is influenced by molecular weight and conformational
effects. Because RP-HPLC profiles of milk protein hydrolysates cover thousands of
peptides of different chemical and genetic variants, statistical tools help researchers
obtain only relevant information from such large data (Coker et al. 2005). HPLC
technique is often combined with conventional UV and fluorescence detectors.
Sometimes, it’s necessary to use more than one HPLC step i.e. a combination of
techniques to achieve separation (Sandra et al. 2009). An example of a study where
RP-HPLC was used to separate and quantify genetic variants of casein and whey
protein was done on water buffalo milk. They reported separation of all major protein fractions with improved resolution, and in a shorter time (Bonfatti et al. 2013).
Juan et al. (2009) also separated and quantified six major bovine proteins in milk
using RP-HPLC.
Mass Spectrometry (MS)
In recent past, MS and proteomics technologies has been considered as gold standard for peptide analysis. This method allows peptide identification in a mixture,
therefore, bypassing the cumbersome Edman’s degradation based method and does
not either require a single pure peptide (Rizzello et al. 2016). The last decade has
seen a notable development of the MS technique, which is widely being accepted as
a tool in identifying bioactive peptides (Picariello et al. 2012). MS has been helpful
in determining molecular mass, protein sequences, protein conformations and
detection of new genetic variants (del Mar Contreras et al. 2008). It has also been
combined with traditional identification methods like Edman degradation
S. Maqsood et al.
Chromatographic Techniques
Classical techniques like PAGE, sodium dodecyl sulfate PAGE (SDS-PAGE) and
2D-PAGE have been used previously, although they are less accurate in identifying
biomolecules compared to CE or HPLC coupled to mass spectrometry (Bernal et al.
2011). Based on the peptide physiochemical properties, different chromatographic
purification techniques can be used. For example, to retain hydrophilic peptides,
hydrophilic interaction chromatography (HILIC) is a better option. Size exclusion
chromatography (SEC) is often used for routine and validation analyses because of
its reproducibility and speed but coupling this technique with MS is a challenge
(Capriotti et al. 2016). Separation of large number of peptides in a sample is performed by HPLC because of its efficiency, versatility and automation capability
(Hernandez-Ledesma et al. 2013). To analyze milk-derived bioactive peptides,
reversed-phase HPLC (RP-HPLC) is commonly employed to separate small peptides (Recio and Lopez-Fandino 2010); this technique is more effective because
most bioactive peptides are small in size (Roberts et al. 1999). In RP-HPLC, retention time depends on the amino acid composition of small peptides while for larger
peptides, retention time is influenced by molecular weight and conformational
effects. Because RP-HPLC profiles of milk protein hydrolysates cover thousands of
peptides of different chemical and genetic variants, statistical tools help researchers
obtain only relevant information from such large data (Coker et al. 2005). HPLC
technique is often combined with conventional UV and fluorescence detectors.
Sometimes, it’s necessary to use more than one HPLC step i.e. a combination of
techniques to achieve separation (Sandra et al. 2009). An example of a study where
RP-HPLC was used to separate and quantify genetic variants of casein and whey
protein was done on water buffalo milk. They reported separation of all major protein fractions with improved resolution, and in a shorter time (Bonfatti et al. 2013).
Juan et al. (2009) also separated and quantified six major bovine proteins in milk
using RP-HPLC.
Mass Spectrometry (MS)
In recent past, MS and proteomics technologies has been considered as gold standard for peptide analysis. This method allows peptide identification in a mixture,
therefore, bypassing the cumbersome Edman’s degradation based method and does
not either require a single pure peptide (Rizzello et al. 2016). The last decade has
seen a notable development of the MS technique, which is widely being accepted as
a tool in identifying bioactive peptides (Picariello et al. 2012). MS has been helpful
in determining molecular mass, protein sequences, protein conformations and
detection of new genetic variants (del Mar Contreras et al. 2008). It has also been
combined with traditional identification methods like Edman degradation
S. Maqsood et al.