J Chromatogr A 1653, 462442 (2021). Samples were peptides obtained through tryptic hydrolysis of the 5 most abundant milk proteins: α-lactalbumin (α-LA), β-lactoglobulin (β-LG), α-, β- and κ-casein (CA). As standards, synthetic whey and pea (Pisum sativum, Fabaceae) peptides (selected based on the in silico tryptic digest of α-LA, β-LG, legumin A, and vicilin with one or zero miscleavages) were only used in the last assay for prediction of the RF values of peptides with known amino-acid (AA) sequences. Two-dimensional HPTLC on silica gel (pre-washed with methanol and activated 10 min at 100°), first with basic mobile phase sec-butanol – pyridine – ammonia – water 39:34:10:26, and (after 12h drying) in the orthogonal direction with acidic mobile phase sec-butanol – pyridine – acetic acid – water 11:8:2:5. Derivatization for peptides and proteins by immersion into fluorescamine (0.05 % in acetone); visualization under UV 254 nm and 365 nm. Computer-assisted determination of the x- and y-coordinates of the derivatized zones. Repeatability (n=8) of the 2D-HPTLC was statistically tested with the Kolmogorov-Smirnov test for normal distribution and with Dixon’s Q test for outliers. Relative standard deviation (RSD) for the RF values was 12.9 % for the first dimension (y-coordinates) and 16.5 % for the second dimension (x-coordinates). According to their higher intensity and sharpness, 15 – 20 detected zones from each protein hydrolyzate were selected, manually scraped from the derivatized layer, dissolved in formic acid solution (0.1 % in acetonitrile – water 3:2), mixed with an equal volume of matrix (dihydroxybenzoic acid 2 % in acetonitrile – water 3:7), crystallized on air on a ground steel target, before being desorbed by the laser beam of the MALDI-TOF-MS/MS (matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry). Direct hyphenation of HPTLC to MS was not performed, to avoid zone diffusion during plate coating with the matrix and to circumvent the stronger binding of polar peptides on the layer.  The MS spectra were acquired in positive reflector mode in m/z range 340 – 4000 (10 – 2500 for fragments), using an external peptide as calibration standard. Identification of 51 from the 85 selected peptides according to AA sequences was performed, using software programs allowing m/z calculation of protein fragments and estimation of cleavage sites. Correlation of the retention behaviour of the peptides with their properties (molecular weight MW, isoelectric point IEP, charges, polarity) was tested with Student’s two-sided t-test after calculation of Pearson’s correlation coefficients. The correlation was significant with IEP, percentages of anionic AA and of non-polar AA; but not with the following properties: MW, percentages of cationic AA and of uncharged polar AA. Finally, based on the correlation results, regression formulas were found to calculate the x- and y-coordinates of any known peptide from the percentage of non-polar AA (or vice-versa). The prediction power of these formulas was verified by repeating the complete 2D-HPTLC-MS experiment with the standard peptides of whey and of peas, and measuring the absolute and relative deviations between the actual x- and y-coordinates and the predicted values. The absolute deviations were higher in the lower RF zones. The average, relative RF value deviations (range 22.1 – 25.7 %) were not different between whey and pea peptides.

Biochem. Biophys. Res. Commun. 394, 733-736 (2010). TLC of liposomes containing membrane protein integrase (MPIase), on silica gel with ethanol - chloroform - water 7:3:4. Detection by spraying with a solution of anisaldehyde - concentrated sulphuric acid - acetic acid 3:4:1. The hRf value of MPIase was 35.

Acta Chimica, 128, 751-773 (1991). TLC of HBsAG segments (HBsAG = hepatitis B-virus) on silica with solvent systems, which were made up by mixing ethyl acetate with a stock solution of pyridine – acetic acid – water 20:6:11 in various proportions. Visualization by spraying with tolidine – KI after chlorination.

J. Chromatogr. 698, 145-162 (1995). A review with 99 references on the methods for the electrophoretic recovery of proteins from polyacrylamide gel, including principles, advantages and disadvantages, and updated information on related techniques.

J. Chromatogr. B 737 (1/2), 71-77 (2000). Description of a fast and effective three-step purification procedure for a fragment of FKBP52, consisting of affinity chromatography, anion-exchange and gel-permeation chromatography. Verification of the purity of the products by SDS-PAGE.

CBS 110, 10-11 (2013). New HPTLC silica gel plates specially suited for coupling with MS and TLC plates for coupling with NMR. These MS-grade HPTLC plates are low in impurities and show less background noise, the layer thickness is 100 µm. HPTLC of human insulin and its secondary components on MS-grade silica gel with 2-butanol - pyridine - ammonia 25 % - water 39:34:10:26, detection under UV 366 nm after treatment with fluorescamine and by elution with the TLC-MS Interface and Q-TOF MS analysis in ESI positive mode, the eluent was acetonitrile - water 19:1. Separation and identification of insulin and desamido insulin was achieved, even though there is only 1 Da mass difference.

Anal. Biochem. 206, 168-171 (1992). Description of a rapid immunochromatographic method for the analysis of protein antigents, based on „sandwich“ assay format using monoclonal antibodies of two distinct specificities, one covalently immobilized to a defined detection zone on a porous membrane while other serve as a label. The sample is mixed with the coating, and the mixture is then passed along a porous membrane by capillary action past the antibodies in the detection zone, giving a blue color. Detection limit, below the nanomolar range for the antigen, human chlorionic gonadotropin.

J. Chromatogr. 698, 89-105 (1995). A review with 54 references on the basic principles of affinity electrophoresis and quantitative affinity electrophoresis with the latest applications, especially the thermodynamic analysis of biospecific interactions by affinity electrophoresis and the development of two-dimensional affinity heterogeneity, lectin affinity electrophoresis for characterizing glycoprotein and newly developed capillary gel affinity electrophoresis.