Determination of the protein content of complex samples by aromatic amino acid analysis, liquid chromatography-UV absorbance, and colorimetry

Abstract

Fast and accurate determination of the protein content of a sample is an important and non-trivial task of many biochemical, biomedical, food chemical, pharmaceutical, and environmental research activities. Different methods of total protein determination are used for a wide range of proteins with highly variable properties in complex matrices. These methods usually work reasonably well for proteins under controlled conditions, but the results for non-standard and complex samples are often questionable. Here, we compare new and well-established methods, including traditional amino acid analysis (AAA), aromatic amino acid analysis (AAAA) based on the amino acids phenylalanine and tyrosine, reversed-phase liquid chromatography of intact proteins with UV absorbance measurements at 220 and 280 nm (LC-220, LC-280), and colorimetric assays like Coomassie Blue G-250 dye-binding assay (Bradford) and bicinchoninic acid (BCA) assay. We investigated different samples, including proteins with challenging properties, chemical modifications, mixtures, and complex matrices like air particulate matter and pollen extracts. All methods yielded accurate and precise results for the protein and matrix used for calibration. AAA, AAAA with fluorescence detection, and the LC-220 method yielded robust results even under more challenging conditions (variable analytes and matrices). These methods turned out to be well-suited for reliable determination of the protein content in a wide range of samples, such as air particulate matter and pollen.

Phytochemical Profiling, Isolation, and Pharmacological Applications of Bioactive Compounds from Insects of the Family Blattidae Together with Related Drug Development

“…This method can also be used in ultraviolet spectrophotometry, and the maximum absorption wavelength of the obtained blue-violet substance is 570 nm [92]. Among the 20 natural amino acids, only tyrosine, tryptophan, and phenylalanine have maximum absorbances in the ultraviolet region, and their wavelengths are 275 nm, 280 nm, and 257 nm, respectively [93]. In this way, these three amino acids can be firstly identified by UV absorption spectroscopy, and the remaining amino acids can be identified by UV after color development.…”

Efficient Purification of Cowpea Chlorotic Mottle Virus by a Novel Peptide Aptamer

“…Purity assessment of biomolecules is often performed using reversed-phase liquid chromatography with UV detection, e.g., at 220 nm [55]. At this wavelength, peptidic amide bonds have a strong absorbance.…”

High-Purity Corundum as Support for Affinity Extractions from Complex Samples

“…Aromatic amino acid analysis (AAAA) was performed as previously described. [31,32] A dispersion of 1 mL of protein-functionalized corundum containing 1 g corundum particles was transferred into hydrolysis glass tubes, and 6 mL of 37% hydrochloric acid containing 17 mg/mL of DL-methionine were added. The air in the glass tubes was displaced by an argon stream applied for 60 seconds.…”