Two-dimensional high-performance thin-layer chromatography for the characterization of milk peptide properties and a prediction of the retention behavior – a proof-of-principle study

High-Performance Thin-Layer Chromatography-Immunostaining as a Technique for the Characterization of Whey Protein Enrichment in Edam Cheese

“…In addition, spiking of a cheese sample (six weeks ripening, 30% HH milk) with the peptide VYVEELKPTPEGDLEILLQK directly on the HPTLC plate did not result in a separation of the bands (data not shown). When calculating the probable position of the two peptides on the HPTLC plate on the basis of their amino acid composition, or more precisely on the basis of their percentage of non-polar amino acids, an R f value of 0.33 could be obtained for the complete peptide VYVEELKPTPEGDLEILLQK and an R f value of 0.36 for the semi-tryptic peptide VYVEELKPTP [ 33 ]. Both calculated R f values are higher than the measured R f value of 0.25.…”

“…Mass spectrometric identification of the amino acid sequences of selected peptide bands was performed according to a recently described protocol [ 33 ]. Briefly, the fluorescamine-derivatized peptide bands of tryptically digested β-LG ( n = 1) were scraped out from the plate with a scalpel, and the peptides were re-solvated with a solution of 60% ( v / v ) acetonitrile and 0.1% ( v / v ) formic acid in water.…”

“…Especially, novel cheese products, in which whey proteins are enriched, have not yet been analyzed in this way. HPTLC, in combination with different detection methods such as MS, ninhydrin or fluorescamine staining (FS), immunostaining (IS), or aptastaining, has been already successfully applied exemplarily for the analysis of intact and tryptically hydrolyzed milk proteins [ 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. It is a cost-effective method as many samples can be analyzed simultaneously with only small solvent consumption.…”

“…With HPTLC, there is also no loss of analytes (especially posttranslational modifications) during the chromatographic separation, as happening during LC in the pre-column or at the beginning of the separation column [ 33 , 40 ]. With regard to bioactivity, it is not possible to determine the allergenicity or the presence of epitopes of the analytes in high-performance liquid chromatography (HPLC), but using HPTLC-immunostaining (HPTLC-IS) [ 35 ].…”

Multivariate optimization of microwave-assisted enzyme digestion of α-casein for generation of bioactive peptides

“…Regarding peptides determination, the most commonly technique is chromatography [20], e.g., gel chromatography [21], ion-exchange chromatography (IEC) [20], and reverse-phase high-performance liquid chromatography (HPLC) [15]. Another versatile technique is high-per-formance thin layer chromatography (HPTLC) used as an alternative or complementarily tool to HPLC for separating hydrophilic and small peptides based on parameters such as size, charge, and polarity [22][23][24]. Due to the lack of important chromophore groups, peptides detection requires pre-or post-chromatography derivati-zation the direct coupling with mass spectrometry however neither process can detect bioactivity, of the peptides generated [25,26].…”