Search

Moving from macroscale preparative systems in proteomics to micro- and nanotechnologies offers researchers the ability to deeply profile smaller numbers of cells that are more likely to be encountered in clinical settings. Herein a recently developed microscale proteomic method, microdroplet processing in one pot for trace samples (microPOTS), was employed to identify proteomic changes in ∼200 Barrett's esophageal cells following physiologic and radiation stress exposure. From this small population of cells, microPOTS confidently identified >1500 protein groups, and achieved a high reproducibility with a Pearson's correlation coefficient value of R > 0.9 and over 50% protein overlap from replicates. A Barrett's cell line model treated with either lithocholic acid (LCA) or X-ray had 21 (e.g., ASNS, RALY, FAM120A, UBE2M, IDH1, ESD) and 32 (e.g., GLUL, CALU, SH3BGRL3, S100A9, FKBP3, AGR2) overexpressed proteins, respectively, compared to the untreated set. These results demonstrate the ability of microPOTS to routinely identify and quantify differentially expressed proteins from limited numbers of cells.

A critical step in the bottom-up characterization of proteomes is the conversion of proteins to peptides, by means of endoprotease digestion. Nowadays this method typically uses overnight digestion and as such represents a considerable bottleneck for high-throughput analysis. This report describes protein digestion using an immobilized-enzyme reactor (IMER), which enables accelerated digestion times that are completed within seconds to minutes. For rapid digestion to occur, a cyclic-olefin-copolymer microfluidic reactor was constructed containing trypsin immobilized on a polymer monolithic material through a 2-vinyl-4,4-dimethylazlactone linker. The IMER was applied for the rapid offline digestion of both singular protein standards and a complex protein mixture prior to liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The effects of protein concentration and residence time in the IMER were assessed for protein standards of varying molecular weight between 11 and 240 kDa. Compared to traditional in-solution digestion, IMER-facilitated protein digestion at room temperature for 5 min yielded similar results in terms of sequence coverage and number of identified peptides. Good repeatability was demonstrated with a relative standard deviation of 6% for protein sequence coverage. The potential of the IMER was also demonstrated for a complex protein mixture in the analysis of dried blood spots. Compared to a traditional workflow a similar number of proteins could be identified, while reducing the total analysis time from 22.5 h to 4h and importantly omitting the sample-pre-treatment steps (denaturation, reduction, and alkylation). The identified proteins from two workflows showed similar distributions in terms of molecular weight and hydrophobic character. (C) 2017 The Authors. Published by Elsevier B.V.

Background/purpose Urocanic acid (UCA) absorbs ultraviolet (UV)B radiation in the epidermis which may interfere with phototherapy. Therefore, the influence of individual levels of UCA on immune reactivity and vitamin D synthesis induced by narrowband UVB radiation was assessed. Methods 28 subjects with irritant contact dermatitis of the hands were irradiated with suberythemal doses of narrowband UVB radiation on their unaffected lower forearms on three consecutive days. Stratum corneum tape strips and epidermal interstitial fluid (ISF) as well as blood samples were analyzed. Results Narrowband UVB irradiation led to the conversion of trans-UCA into its cis-isomer in the epidermis. The observed increase of 25-hydroxyvitamin D serum concentrations was inversely correlated with the baseline levels of trans-UCA. Furthermore, UVB irradiation induced significant changes in the levels of CXCL10/IP-10, CCL2/MCP-1, CCL4/MIP-1β, and the IL-1RA/IL-1α ratio. The levels of IL-1α and CXCL9/MIG showed a trend toward increase. The changes in the levels of inflammatory and immunomodulatory mediators did not depend on baseline levels of trans-UCA. Conclusion The results suggest that epidermal levels of trans-UCA affect vitamin D synthesis, but not cutaneous immune reactivity upon repeated exposure to suberythemal doses of narrowband UVB radiation. However, this requires further exploration. This article is protected by copyright. All rights reserved.

The free fatty acids (FFAs) are one of the major components of the lipids in the stratum corneum (SC), the uppermost layer of the skin. Relative composition of FFAs has been proposed as a biomarker of the skin barrier status in patients with atopic dermatitis (AD). Here, we developed an LC-ESI-MS/MS method for simultaneous quantification of a range of FFAs with long and very long chain length in the SC collected by adhesive tape (D-Squame). The method, based on derivatization with 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide, allowed highly sensitive detection and quantification of FFAs using multiple reaction monitoring. For the quantification, we applied a surrogate analyte approach and internal standardization using isotope labeled derivatives of FFAs. Adhesive tapes showed the presence of several FFAs, which are also present in the SC, a problem encountered in previous studies. Therefore, the levels of FFAs in the SC were corrected using C12:0, which was present on the adhesive tape, but not detected in the SC. The method was applied to SC samples from patients with atopic dermatitis and healthy subjects. Quantification using multiple reaction monitoring allowed sufficient sensitivity to analyze FFAs of chain lengths C16-C28 in the SC collected on only one tape strip.

SummaryBackground Fruit-derived organic compounds and detergents are relevant exposure factors for occupational contact dermatitis in the food industry. Although individuals with atopic dermatitis (AD) are at risk for development of occupational contact dermatitis, there have been no controlled studies on the effects of repeated exposure to multiple irritants, relevant for the food industry, in atopic skin. Objectives The aim of the study was to investigate the outcomes of repeated exposure to a fruit-derived organic acid and a detergent in AD compared to healthy volunteers. Methods The volunteers were exposed to 2.0% acetic acid (AcA) and/or 0.5% sodium lauryl sulfate (SLS) in controlled tandem repeated irritation test. The outcomes were assessed by measurements of erythema, transepidermal water loss (TEWL) and natural moisturizing factor (NMF) levels. Results In the AD volunteers, repeated AcA exposure led to barrier disruption and significant TEWL increase; no significant differences after the same exposure in the healthy controls were found. Repeated exposure to SLS and the irritant tandems enhanced the reactions and resulted in a significantly higher increase in TEWL in the AD compared to the control group. Cumulative irritant exposure reduced the NMF levels in both groups. Conclusions Differences in the severity of irritant-induced barrier impairment in atopic individuals contribute to the risk for occupational contact dermatitis in result of multiple exposures to food-derived irritants and detergents.

Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram-positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique interprotein cross-linked peptides with

Dermal exposure to alkaline agents may lead to skin barrier damage and irritant contact dermatitis. The objective of this study was to investigate the effects of cumulative exposure to 0.5% sodium lauryl sulphate (SLS) and 0.15% NaOH on the barrier function and natural moisturising factor (NMF) levels in atopic dermatitis and healthy volunteers with known filaggrin genotype. The skin response was monitored by measurement of erythema and transepidermal water loss. The stratum corneum NMF levels were determined by high-performance liquid chromatography. Repeated exposure to 0.5% SLS and/or 0.15% NaOH in atopic dermatitis resulted in more severe impairment of the skin barrier function. Cumulative exposure to the irritants reduced significantly NMF in both the atopic and healthy controls group. The pronounced decrease of NMF after repeated single and sequential irritant exposure may be a pathogenetically relevant factor for development of chronic irritant contact dermatitis in both healthy and atopic individuals.

The levels of natural moisturizing factors in the skin can be used as a biomarker of hydration, for studying the effect of skin irritants, or as a biomarker of loss-of-function mutations in the filaggrin gene which are the main risk factor for atopic dermatitis. In this study the chromatographic performance and recovery of natural moisturizing factors and proteins from the skin were investigated using different extraction solvents and adhesive tapes. The uppermost layer of the skin stratum corneum, collected by using commercially available D-squame and corneofix adhesive tapes, was extracted by ammonia or potassium hydroxide. Protein levels used to correct for a variable stratum corneum amount on a tape were assessed by measuring optical density of a tape or indirectly by measuring proteins by a spectrophotometric assay. The measured natural moisturizing factors, pyrrolidone-5-carboxylic acid, histidine, tyrosine, trans--urocanic acid, and cis-urocanic acid were determined by ion pair reverse phase HPLC. Sample preparation and chromatographic performance were favorable when ammonia was used as an extraction solvent. Extraction of the natural moisturizing factors with ammonia avoids a time consuming neutralization step as required with extraction procedures using strong base or acid. The only drawback of the ammonia method is incomplete extraction of proteins from the tapes ; however this can be avoided by measuring the optical density of stratum corneum-loaded tapes. The sensitivity of the method was sufficiently high to quantify the analytes even in homozygous filaggrin gene carriers. Reduced natural moisturizing factors levels found in the individuals with filaggrin gene mutation or after exposure to a skin irritant sodium lauryl sulfate were consistent with the previously reported studies.