UVB pretreatment of ß-lactoglobulin affects the temperature-induced formation of functional amyloid-like aggregates and promotes oxidative degradation.

Unfolding in combination with or without acid hydrolysis is crucial for the formation of functional amyloid (fibrillar) or amyloid-like (worm-like) ß-lactoglobulin (BLG) aggregates, which can be induced through temperature treatment for several hours at pH 2-4. A preceding conformational destabilization of BLG might affect its aggregation. We investigated ultraviolet (UV) B radiation as conformational perturbing treatment to facilitate temperature-induced protein aggregation. 2-h UVB pretreated BLG (UV-BLG) exhibited an accelerated worm-like aggregation at pH 3.5, while at pH 2 the formation of fibrils was decelerated. The UV-induced conformational destabilization lowered the thermal stability and thus facilitates unfolding during thermal treatment. Thereby, the formation of covalent and non-covalent intermolecular interactions was favored, which promoted assembly of intact proteins resulting in worm-like aggregates. The oxidative degradation of UV-BLG was suggested to alter fibrillation-prone protein regions and thereby impede peptide assembly.

Modification and oxidative degradation of ß-lactoglobulin by UVB irradiation.

Ultraviolet (UV) B irradiation induces protein modification, especially the conformational rearrangement of proteins, and is therefore promising as a non-thermal and non-chemical functionalization technique. Nevertheless, UVB irradiation introduces radicals and oxidizes side chains resulting in the loss of food quality. Thus, assessing the UVB irradiation-based functionalization of ß-lactoglobulin (BLG) versus its oxidative degradation is of interest. UVB irradiation of up to 8 h was successfully applied to loosen the rigid folding of BLG and increase its flexibility. Thereby, the cysteine at position 121 and hydrophobic regions became surface-exposed as indicated by the increase in accessible thiol groups and increased surface hydrophobicity. Furthermore, we demonstrated the cleavage of the "outer" disulfide bond C66-C160 by LC-MS/MS after tryptic digestion of BLG. The 2-h-irradiated BLG showed adequate conformational rearrangement for protein functionalization while being minimally oxidized.

A novel screening method for free non-standard amino acids in human plasma samples using AccQ·Tag reagents and LC-MS/MS.

There are at least 500 naturally occurring amino acids, of which only 20 standard proteinogenic amino acids are used universally across all organisms in the synthesis of peptides and proteins. Non-standard amino acids can be incorporated into proteins or are intermediates and products of metabolic pathways. While the analysis of standard amino acids is well-defined, the analysis of non-standard amino acids can be challenging due to the wide range of physicochemical properties, and the lack of both reference standards and information in curated databases to aid compound identification. It has been shown that the use of an AccQ·Tag™ derivatization kit along with LC-MS/MS is an attractive option for the analysis of free standard amino acids in complex samples because it is fast, sensitive, reproducible, and selective. It has been demonstrated that the most abundant quantitative transition for MS/MS analysis of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids corresponds to the fragmentation of the molecule at the 6-aminoquinoline carbonyl group producing a common m/z 171 fragment ion and occurs at similar mass spectrometry collision energy and cone voltages. In this study, the unique properties of AQC derivatized amino acids producing high intensity common fragment ions, along with chromatographic separation of amino acids under generic chromatography conditions, were used to develop a novel screening method for the detection of trace levels of non-standard amino acids in complex matrices. Structural elucidation was carried out by comparing the MS/MS fragment ion mass spectra generated with in silico predicted fragmentation spectra to enable a putative identification, which was confirmed using an appropriate analytical standard. This workflow was applied to screen human plasma samples for bioactive thiol-group modified cysteine amino acids and S-allylmercaptocysteine (SAMC), S-allylcysteine sulfoxide (SACS or alliin) and S-propenylcysteine (S1PC) are reported for the first time to be present in human plasma samples after the administration of garlic supplements.