Immunological analysis of food proteins using high-performance thin-layer chromatography-immunostaining.

The chromatographic analysis of intact proteins is still challenging, especially when biological functions as antigenicity of proteins or peptides are in the focus. Traditional immunoassays provide information about the entirety of antigenic proteins/peptides, e.g., in ELISA assays. On the other hand, when focusing on the investigation of (cross) reactivity of antibodies, Western blot following gel-electrophoresis represents the method of choice. However, gel-electrophoresis is limited by the molecular weight and therefore, not suitable for peptides ≤3kDa or proteins ≥250kDa. Furthermore, for gaining detailed information about the protein sequence (e.g., via mass spectrometric analysis), a so called in-gel digest needs to be performed following electrophoretic separation and is therefore elaborate and accompanied by a significant loss of structural, and even more severe, conformational information. Here, protein analysis using HPTLC seems to be a promising alternative due to the high level of variability regarding the chromatographic system (multiple mobile and stationary phases, even mixed) and manifold detection as well as hyphenation possibilities. This study exemplarily focused on the immunological investigation of proteins in milk following thin-layer chromatographic separation. The detection of these antigens is mandatory, as they might trigger allergenic reactions in sensitized people. Besides the proof of its applicability on different stationary phase materials, the newly developed immunoassay can be used as an approach for semi-quantitative estimation of antigenic proteins. In addition to the analysis of intact food allergens, also analyzing peptides thereof is worth considering which can be realized using HPTLC-immunostaining as well.

HPTLC-aptastaining - Innovative protein detection system for high-performance thin-layer chromatography.

Protein analysis using high-performance thin-layer chromatography (HPTLC) is not commonly used but can complement traditional electrophoretic and mass spectrometric approaches in a unique way. Due to various detection protocols and possibilities for hyphenation, HPTLC protein analysis is a promising alternative for e.g., investigating posttranslational modifications. This study exemplarily focused on the investigation of lysozyme, an enzyme which is occurring in eggs and technologically added to foods and beverages such as wine. The detection of lysozyme is mandatory, as it might trigger allergenic reactions in sensitive individuals. To underline the advantages of HPTLC in protein analysis, the development of innovative, highly specific staining protocols leads to improved sensitivity for protein detection on HPTLC plates in comparison to universal protein derivatization reagents. This study aimed at developing a detection methodology for HPTLC separated proteins using aptamers. Due to their affinity and specificity towards a wide range of targets, an aptamer based staining procedure on HPTLC (HPTLC-aptastaining) will enable manifold analytical possibilities. Besides the proof of its applicability for the very first time, (i) aptamer-based staining of proteins is applicable on different stationary phase materials and (ii) furthermore, it can be used as an approach for a semi-quantitative estimation of protein concentrations.

High-performance thin-layer chromatography as a fast screening tool for phosphorylated peptides.

This study aimed at developing a rapid chromatographic assay to monitor phosphorylation sites in peptides. For the analysis of nociceptive signal transduction pathways, the detection of phosphorylated proteins/peptides plays a fundamental role. To get further insights in the phosphorylation mechanism of protein kinase C-ε (PKC-ε) and protein kinase A (PKA), potential targets were divided into subsections resulting in peptides that contain only one possible phospho-binding site. The use of high-performance thin-layer chromatography (HPTLC) offers the possibility of a high throughput of samples and the advantage of a quick sample clean-up. A combined strategy of an effect-directed overlay procedure on the TLC plate using specific antibodies (immunostaining, HPTLC-IS) as well as a parallel, direct mass spectrometric methodology by HPTLC-MALDI-TOF-MS was developed. With regard to HPTLC-IS, validation of the data exhibited a lower limit of detection than the traditionally used protein derivatization reagent fluorescamine. Besides the identification of the phosphorylated peptides, a semi-quantitative estimation can be performed with HPTLC-IS.

Development of optimized mobile phases for protein separation by high performance thin layer chromatography.

In recent years, protein chemistry tends inexorably toward the analysis of more complex proteins, proteoforms, and posttranslational protein modifications. Although mass spectrometry developed quite fast correspondingly, sample preparation and separation of these analytes is still a major issue and quite challenging. For many years, electrophoresis seemed to be the method of choice; nonetheless its variance is limited to parameters such as size and charge. When taking a look at traditional (thin-layer) chromatography, further parameters such as polarity and different mobile and stationary phases can be utilized. Further, possibilities of detection are manifold compared to electrophoresis. Similarly, two-dimensional separation can be also performed with thin-layer chromatography (TLC). As the revival of TLC developed enormously in the last decade, it seems to be also an alternative to use high performance thin-layer chromatography (HPTLC) for the separation of proteins. The aim of this study was to establish an HPTLC separation system that allows a separation of protein mixtures over a broad polarity range, or if necessary allowing to modify the separation with only few steps to improve the separation for a specific scope. Several layers and solvent systems have been evaluated to reach a fully utilized and optimized separation system.