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.

A comparative proteomics analysis of four contact allergens in THP-1 cells shows distinct alterations in key metabolic pathways.

Allergic contact dermatitis (ACD) is the predominant form of immunotoxicity in humans. The sensitizing potential of chemicals can be assessed in vitro. However, a better mechanistic understanding could improve the current OECD-validated test battery. The aim of this study was to get insights into toxicity mechanisms of four contact allergens, p-benzoquinone (BQ), 2,4-dinitrochlorobenzene (DNCB), p-nitrobenzyl bromide (NBB) and NiSO4, by analyzing differential proteome alterations in THP-1 cells using two common proteomics workflows, stable isotope labeling by amino acids in cell culture (SILAC) and label-free quantification (LFQ). Here, SILAC was found to deliver more robust results. Overall, the four allergens induced similar responses in THP-1 cells, which underwent profound metabolic reprogramming, including a striking upregulation of the TCA cycle accompanied by pronounced induction of the Nrf2 oxidative stress response pathway. The magnitude of induction varied between the allergens with DNCB and NBB being most potent. A considerable overlap between transcriptome-based signatures of the GARD assay and the proteins identified in our study was found. When comparing the results of this study to a previous proteomics study in human primary monocyte-derived dendritic cells, we found a rather low share in regulated proteins. However, on pathway level, the overlap was high, indicating that affected pathways rather than single proteins are more eligible to investigate proteomic changes induced by contact allergens. Overall, this study confirms the potential of proteomics to obtain a profound mechanistic understanding, which may help improving existing in vitro assays for skin sensitization.

Novel indirect co-culture of immortalised hepatocytes with monocyte derived macrophages is characterised by pro-inflammatory cytokine networks.

The liver is composed of different cell populations. Interactions of different cell populations can be investigated by a newly established indirect co-culture system consisting of immortalised primary human hepatocytes and human monocyte derived macrophages (MDMs). Using the time-dependent cytokine secretion of the co-cultures and single cultures, correlation networks (including the cytokines G-CSF, CCL3, MCP-1, CCL20, FGF, TGF-ß1, GM-CSF, IL-8 IL-6, IL-1ß, and IL-18) were generated and the correlations were validated by application of IL-8 and TNF-α-neutralising antibodies. The data reveal that IL-8 is crucial for the interaction between hepatocytes and macrophages in vitro. In addition, transcriptome analyses showed that a change in the ratio between macrophages and hepatocytes may trigger pro-inflammatory signalling pathways of the acute phase response and the complement system (release of, e.g., certain cyto- and chemokines). Using diclofenac and LPS showed that the release of cytokines is increasing with higher ratios of MDMs. Altogether, we could demonstrate that the current co-culture system is better suited to mirror the in vivo situation when compared to previously established co-culture systems composed of HepG2 and differentiated THP-1 cells. Further, our data reveal that the cytokine IL-8 is crucial for the interaction between hepatocytes and macrophages in vitro.

The Contact Allergen NiSO4 Triggers a Distinct Molecular Response in Primary Human Dendritic Cells Compared to Bacterial LPS.

Dendritic cells (DC) play a central role in the pathogenesis of allergic contact dermatitis (ACD), the most prevalent form of immunotoxicity in humans. However, knowledge on allergy-induced DC maturation is still limited and proteomic studies, allowing to unravel molecular effects of allergens, remain scarce. Therefore, we conducted a global proteomic analysis of human monocyte-derived dendritic cells (MoDC) treated with NiSO4, the most prominent cause of ACD and compared proteomic alterations induced by NiSO4 to the bacterial trigger lipopolysaccharide (LPS). Both substances possess a similar toll-like receptor (TLR) 4 binding capacity, allowing to identify allergy-specific effects compared to bacterial activation. MoDCs treated for 24 h with 2.5 µg/ml LPS displayed a robust immunological response, characterized by upregulation of DC activation markers, secretion of pro-inflammatory cytokines and stimulation of T cell proliferation. Similar immunological reactions were observed after treatment with 400 µM NiSO4 but less pronounced. Both substances triggered TLR4 and triggering receptor expressed on myeloid cells (TREM) 1 signaling. However, NiSO4 also activated hypoxic and apoptotic pathways, which might have overshadowed initial signaling. Moreover, our proteomic data support the importance of nuclear factor erythroid 2-related factor 2 (Nrf2) as a key player in sensitization since many Nrf2 targets genes were strongly upregulated on protein and gene level selectively after treatment with NiSO4. Strikingly, NiSO4 stimulation induced cellular cholesterol depletion which was counteracted by the induction of genes and proteins relevant for cholesterol biosynthesis. Our proteomic study allowed for the first time to better characterize some of the fundamental differences between NiSO4 and LPS-triggered activation of MoDCs, providing an essential contribution to the molecular understanding of contact allergy.

Application of proteomics in the elucidation of chemical-mediated allergic contact dermatitis.

Allergic contact dermatitis (ACD) is a widespread hypersensitivity reaction of the skin. The cellular mechanisms underlying its development are complex and involve close interaction of different cell types of the immune system. It is this very complexity which has long prevented straightforward replacement of the corresponding regulatory in vivo tests. Recent efforts have already resulted in the development of several in vitro testing alternatives that address key steps of ACD. Yet identification of suitable biomarkers is still a subject of intense research. Search strategies for the latter encompass transcriptomics, proteomics as well as metabolomics approaches. The scope of this review shall be the application and use of proteomics in the context of ACD. This includes highlighting relevant aspects of the molecular and cellular mechanisms underlying ACD, the exploitation of these mechanisms for testing and biomarkers (e.g., in the context of the OECD's adverse outcome pathway initiative) as well as an outlook on emerging proteome targets, for example during the allergen-induced activation of dendritic cells (DCs).

Simple, Rapid, and Selective Isolation of 2S Albumins from Allergenic Seeds and Nuts.

The 2S albumins belong to the group of seed storage proteins present in different seeds and nuts. Due to their pronounced allergenic potential, which is often associated with severe allergic reactions, this protein family is of special interest in the field of allergen research. Here we present a simple, rapid, and selective method for the purification of 2S albumins directly from allergenic seeds and nuts. We systematically optimized the parameters "buffer system", "extraction temperature", "buffer molarity", and "pH " and were able to achieve 2S albumin purities of about 99% without further purification and demonstrate transferability of this method to nine different allergenic food matrices. Compared to conventional isolation routines, significant reduction of hands-on time and required laboratory equipment is achieved, but nonetheless higher protein yields are obtained. The presented method allows for the rapid purification of different 2S albumins including the corresponding isoforms from natural material.