Towards characterization of cell culture conditions for reliable proteomic analysis: in vitro studies on A549, differentiated THP-1, and NR8383 cell lines.

Proteomic investigations result in high dimensional datasets, but integration or comparison of different studies is hampered by high variances due to different experimental setups. In addition, cell culture conditions can have a huge impact on the outcome. This study systematically investigates the impact of experimental parameters on the proteomic profiles of commonly used cell lines-A549, differentiated THP-1 macrophage-like cells, and NR8383-for toxicity studies. The work focuses on analyzing the influence at the proteome level of cell culture setup involving different vessels, cell passage numbers, and post-differentiation harvesting time, aiming to improve the reliability of proteomic analyses for hazard assessment. Mass-spectrometry-based proteomics was utilized for accurate protein quantification by means of a label-free approach. Our results showed that significant proteome variations occur when cells are cultivated under different setups. Further analysis of these variations revealed their association to specific cellular pathways related to protein misfolding, oxidative stress, and proteasome activity. Conversely, the influence of cell passage numbers on the proteome is minor, suggesting a reliable range for conducting reproducible biological replicates. Notable, substantial proteome alterations occur over-time post-differentiation of dTHP-1 cells, particularly impacting pathways crucial for macrophage function. This finding is key for the interpretation of experimental results. These results highlight the need for standardized culture conditions in proteomic-based evaluations of treatment effects to ensure reliable results, a prerequisite for achieving regulatory acceptance of proteomics data.

Influence of Hydroxycinnamic Acids on the Maillard Reaction of Arabinose and Galactose beyond Carbonyl-Trapping.

Hydroxycinnamic acids, known for their health benefits and widespread presence in plant-based food, undergo complex transformations during high-temperature processing. Recent studies revealed a high browning potential of hydroxycinnamic acids and reactive Maillard reaction intermediates, but the role of phenolic compounds in the early stage of these reactions is not unambiguously understood. Therefore, we investigated the influence of caffeic acid and ferulic acid on the nonenzymatic browning of arabinose, galactose, and/or alanine, focusing on the implications on the formation of relevant early-stage Maillard intermediates and phenol-deriving products. Contrary to previous assumptions, hydroxycinnamic acids were found to promote nonenzymatic browning instead of solely trapping reactive intermediates. This was reflected by an intense browning, which was attributed to the formation of heterogeneous phenol-containing Maillard products. Although, caffeic acid is more reactive than ferulic acid, the formation of reactive furan derivatives and of heterogeneous phenol-containing colorants was promoted in the presence of both hydroxycinnamic acids.

Advancing Nanomaterial Toxicology Screening Through Efficient and Cost-Effective Quantitative Proteomics.

Proteomic investigations yield high-dimensional datasets, yet their application to large-scale toxicological assessments is hindered by reproducibility challenges due to fluctuating measurement conditions. To address these limitations, this study introduces an advanced tandem mass tag (TMT) labeling protocol. Although labeling approaches shorten data acquisition time by multiplexing samples compared to traditional label-free quantification (LFQ) methods in general, the associated costs may surge significantly with large sample sets, for example, in toxicological screenings. However, the introduced advanced protocol offers an efficient, cost-effective alternative, reducing TMT reagent usage (by a factor of ten) and requiring minimal biological material (1 µg), while demonstrating increased reproducibility compared to LFQ. To demonstrate its effectiveness, the advanced protocol is employed to assess the toxicity of nine benchmark nanomaterials (NMs) on A549 lung epithelial cells. While LFQ measurements identify 3300 proteins, they proved inadequate to reveal NM toxicity. Conversely, despite detecting 2600 proteins, the TMT protocol demonstrates superior sensitivity by uncovering alterations induced by NM treatment. In contrast to previous studies, the introduced advanced protocol allows simultaneous and straightforward assessment of multiple test substances, enabling prioritization, ranking, and grouping for hazard evaluation. Additionally, it fosters the development of New Approach Methodologies (NAMs), contributing to innovative methodologies in toxicological research.

How processing affects marker peptide quantification - A comprehensive estimation on bovine material relevant for food and feed control.

Processing food and feed challenges official control e.g. by modifying proteins, which leads to significant underestimation in targeted, MS-based protein quantification. Whereas numerous studies identified processing-induced changes on proteins in various combinations of matrices and processing conditions, studying their impact semi-quantitatively on specific protein sequences might unveil approaches to improve protein quantification accuracy. Thus, 335 post-translational modifications (e.g. oxidation, deamidation, carboxymethylation, Amadori, acrolein adduction) were identified by bottom-up proteomic analysis of 37 bovine materials relevant in food and feed (meat, bone, blood, milk) with varying processing degrees (raw, spray-dried, pressure-sterilized). To mimic protein recovery in a targeted analysis, peak areas of marker and reference peptides were compared to those of their modified versions, which revealed peptide-specific recoveries and variances across all samples. Detailed analysis suggests that incorporating two modified versions additionally to the unmodified marker may significantly improve quantification accuracy in targeted MS-based food and feed control in processed matrices.

Browning reactions of hydroxycinnamic acids and heterocyclic Maillard reaction intermediates - Formation of phenol-containing colorants.

Non-enzymatic conversion of phenolic compounds plays an important role during thermal processing of plant-based food such as coffee, cocoa, and peanuts. However, the more prominent Maillard reaction is mainly studied at a mechanistic level for carbohydrates and amino compounds to clarify reactions that contribute to ('classic') melanoidin formation, but the role of phenolic compounds in such reactions is rarely discussed yet. To understand their contribution to non-enzymatic browning, reactions between ubiquitous phenolic acids, such as caffeic acid and ferulic acid, and prominent heterocyclic Maillard intermediates, namely furfural, hydroxymethylfurfural, and pyrrole-2-carbaldehyde were investigated. Following incubation under roasting conditions (220 °C, 0-30 min), heterogenous products were characterized by high-resolution mass spectrometry, and, after isolation, by nuclear magnetic resonance spectroscopy. By this, color precursors were identified, and it was shown that in addition to aromatic electrophilic substitution, nucleophilic and condensation reactions are key mechanisms contributing to the formation of phenol-containing melanoidins.

Colorants and Antioxidants Deriving from Methylglyoxal and Heterocyclic Maillard Reaction Intermediates.

The Maillard reaction is well known for producing antioxidant compounds alongside colored substances. Low-molecular-weight antioxidant intermediates such as maltol (MAL) or norfuraneol (NF) are well described, but it is still unclear which of these Maillard intermediates are the precursors of antioxidant and colored melanoidins-the so-called late stage Maillard reaction products. This study aimed to provide novel insights into the correlation between browning potential and antioxidant properties of reaction products formed during the heat treatment of prominent Maillard reaction intermediates. It was achieved by the incubation of binary reaction systems composed of methylglyoxal (MGO) or NF in combination with furfural (FF), MAL, and pyrrole-2-carbaldehyde (PA) at pH 5 and 130 °C for up to 120 min. Overall, it could be shown that the formation of colored products in the binary NF reaction systems was more efficient compared to those of MGO. This was reflected in an increased browning intensity of up to 400% and a lower conversion rate of NF compared to MGO. The colorants formed by NF and FF or PA (~0.34 kDa and 10-100 kDa) were also found to exhibit higher molecular weights compared to the analogue products formed in the MGO incubations (<0.34 kDa and 10-100 kDa). The incorporation of NF into these heterogenous products with FF and PA resulted in the preservation of the initial antioxidant properties of NF (p < 0.05), whereas no antioxidant products were formed after the incubation of MGO.