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The promoting effect of FLG loss-of-function mutations on the development of atopic dermatitis (AD) signifies the role of filaggrin in the formation of a protective skin barrier; FLG mutations are also linked to asthma, food allergy and allergic rhinitis despite the absence of the protein in the affected tissues (lungs, intestines, and the majority of the nasal mucosa). AD patients suffer from chronic inflammation and recurrent skin infections; inflammation often precedes the appearance of spatially distant allergic manifestations. Here we show that exosome-enriched small extracellular vesicles (sEVs) secreted by filaggrin-knockdown keratinocytes are extensively remodelled as a consequence of the abnormal keratinocyte differentiation process. This alteration modulates the sEV capacity to promote type 1 and type 2 CD1a-dependent T cell responses by direct effects on self-lipid neoantigen generation; both modulating the amount of permissive (stimulatory) and non-permissive (inhibitory) CD1a ligands released from the sEV membranes by phospholipase A2. We found that this aberrant sEV lipid composition reflects a generalised cellular lipid bias with downregulation of multiple enzymes of lipid metabolic pathways, observed both in filaggrin knockdown keratinocytes in vitro, and in the skin of AD patients. Provision of modulatory ligands by sEVs secreted on a filaggrin insufficiency background, impeding both homeostatic autoreactive and protective antimicrobial CD1a-mediated type 1 and enhancing type 2 T cell responses provides basis for reduced tissue integrity and pathogen clearance and perpetuates inflammation in AD skin as well as in distant tissues to which sEVs are transferred by systemic circulation.

Top-down proteomics is a powerful tool for characterizing genetic variations and post-translational modifications at intact protein level. However, one significant technical gap of top-down proteomics is the inability to analyze a low amount of biological samples, which limits its access to isolated rare cells, fine needle aspiration biopsies, and tissue substructures. Herein, we developed an ultrasensitive top-down platform by incorporating a microfluidic sample preparation system, termed nanoPOTS (nanodroplet processing in one pot for trace samples), into a top-down proteomic workflow. A unique combination of a nonionic detergent dodecyl-β-d-maltopyranoside (DDM) with urea as protein extraction buffer significantly improved both protein extraction efficiency and sample recovery. We hypothesize that the DDM detergent improves protein recovery by efficiently reducing nonspecific adsorption of intact proteins on container surfaces, while urea serves as a strong denaturant to disrupt noncovalent complexes and release intact proteins for downstream analysis. The nanoPOTS-based top-down platform reproducibly and quantitatively identified ∼170 to ∼620 proteoforms from ∼70 to ∼770 HeLa cells containing ∼10 to ∼115 ng of total protein. A variety of post-translational modifications including acetylation, myristoylation, and iron binding were identified using only less than 800 cells. We anticipate the nanoPOTS top-down proteomics platform will be broadly applicable in biomedical research, particularly where clinical specimens are not available in amounts amenable to standard workflows.

Solitary aculeate wasps are abundant and diverse hymenopteran insects that disable prey using venom. The venom may possess neuromodulation, immunomodulatory, metabolic-modulatory and antimicrobial functions. Venom analysis of transcriptomes and proteomes has been previously performed in social and parasitoid wasp species. We develop methodologies including mass spectrometry-based shotgun proteomics to analyse the protein constituents from venom sacs of the solitary aculeate wasp Cerceris rybyensis. The venom sac constituents of C. rybyensis are discussed with respect to other wasp species.

Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh-frozen and formalin-fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue-sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom-up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys-C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue-specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm2 obtained with laser capture microdissection to much larger amounts that weight several milligrams.

Sample preparation is a key step in proteomics, however there is no consensus in the community about the standard method for preparation of proteins from clinical samples like tissues or biofluids. In this chapter, we will discuss some important steps in sample preparation used for bottom-up proteome profiling with mass spectrometry (MS). Specifically, tissues, which are an important source of biological information, are of interest because of their availability. Tissues are most often stored as fresh frozen (FF) or formalin-fixed paraffin-embedded (FFPE). While FF tissues are more readily available, paraffin embedding has historically been routinely used for tissue preservation. However, formaldehyde induced crosslinks during FFPE tissue preservation present a challenge to the protocols used for protein retrieval. Moreover, in our view, an important aspect to consider is also the amount of material available at the start of a protocol since this is directly related to the choice of protocol in order to minimize sample loss and maximize detection of peptides by MS. This “MS sensitivity” is of special importance when working with patient samples that are unique and often available in limited amounts making optimization of methods to analyze the proteins therein important given that their molecular information can be used in a patients’ diagnosis and treatment.

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.

Graphical abstract, The TAP1-TAP2 complex transports antigenic peptide substrates into the endoplasmic reticulum (ER). In ER, the peptides are further processed and loaded on the major histocompatibility class (MHC) I molecules by the peptide loading complex (PLC). The TAP transporters are linked with the PLC; a target for cancers and viral immune evasion. But the mechanisms whereby the cancer-derived mutations in TAP1-TAP2 or viral factors targeting the PLC, interfere peptide transport are only emerging. This study describes that transit of peptides through TAP can take place via two different channels (4 or 8 helices) depending on peptide length and sequence. Molecular dynamics and binding affinity predictions of peptide-transporters demonstrated that smaller peptides (8–10 mers; e.g. AAGIGILTV, SIINFEKL) can transport quickly through the transport tunnel compared to longer peptides (15-mer; e.g. ENPVVHFFKNIVTPR). In line with a regulated and selective peptide transport by TAPs, the immunopeptidome upon IFN-γ treatment in melanoma cells induced the shorter length (9-mer) peptide presentation over MHC-I that exhibit a relatively weak binding affinity with TAP. A conserved distance between N and C terminus residues of the studied peptides in the transport tunnel were reported. Furthermore, by adversely interacting with the TAP transport passage or affecting TAPNBD domains tilt movement, the viral proteins and cancer-derived mutations in TAP1-TAP2 may induce allosteric effects in TAP that block conformation of the tunnel (closed towards ER lumen). Interestingly, some cancer-associated mutations (e.g. TAP1R372Q and TAP2R373H) can specifically interfere with selective transport channels (i.e. for longer-peptides). These results provide a model for how viruses and cancer-associated mutations targeting TAP interfaces can affect MHC-I antigen presentation, and how the IFN-γ pathway alters MHC-I antigen presentation via the kinetics of peptide transport.

Developments in quantitative proteomics and data-independent acquisition (DIA) methodology is enabling quantification of proteins in biological samples. Currently, there are a few reports on DIA mass spectrometry (MS) approaches for proteome analysis of formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, to facilitate detection and quantification of immune- and glioblastoma (GBM)-relevant proteins from FFPE patient materials, we established a simple and precise DIA-MS workflow. We first evaluated different lysis buffers for their efficiency in protein extractions from FFPE GBM tissues. Our results showed that more than 1700 proteins were detected and over 1400 proteins were quantified from GBM FFPE tissue microdissections. GBM-relevant proteins (e.g., GFAP, FN1, VIM, and MBP) were quantified with high precision (median coefficient of variation

Proteomics of human tissues and isolated cellular subpopulations create new opportunities for therapy and monitoring of a patients’ treatment in the clinic. Important considerations in such analysis include recovery of adequate amounts of protein for analysis and reproducibility in sample collection. In this study we compared several protocols for proteomic sample preparation: i) filter-aided sample preparation (FASP), ii) in-solution digestion (ISD) and iii) a pressure-assisted digestion (PCT) method. PCT method is known for already a decade [1], however it is not widely used in proteomic research. We assessed protocols for proteome profiling of isolated immune cell subsets and formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that the ISD method has very good efficiency of protein and peptide identification from the whole proteome, while the FASP method is particularly effective in identification of membrane proteins. Pressure-assisted digestion methods generally provide lower numbers of protein/peptide identifications, but have gained in popularity due to their shorter digestion time making them considerably faster than for ISD or FASP. Furthermore, PCT does not result in substantial sample loss when applied to samples of 50 000 cells. Analysis of FFPE tissues shows comparable results. ISD method similarly yields the highest number of identifications. Furthermore, proteins isolated from FFPE samples show a significant reduction of cleavages at lysine sites due to chemical modifications with formaldehyde-such as methylation (+14 Da) being among the most common. The data we present will be helpful for making decisions about the robust preparation of clinical samples for biomarker discovery and studies on pathomechanisms of various diseases.

Human tissues are an important link between organ-specific spatial molecular information, patient pathology, and patient treatment options. However, patient tissues are uniquely obtained by time and location, and limited in their availability and size. Currently, little knowledge exists about appropriate and simplified protocols for routine MS-based analysis of the various types and sizes of tissues. Following standard procedures used in pathology, we selected small fresh frozen uterine tissue samples to investigate how the tissue preparation protocol affected the subsequent proteomics analysis. First, we observed that protein extraction with 0.1% SDS followed by extraction with a 30% ACN/urea resulted in a decrease in the number of identified proteins, when compared to extraction with 30% ACN/urea only. The decrease in the number of proteins was approximately 55% and 20%, for 10 and 16 μm thick tissue, respectively. Interestingly, the relative abundance of the proteins shared between the two methods was higher when SDS/ACN/urea was used, compared to the 30% ACN/urea extraction, indicating the role of SDS to be beneficial for protein solubility. Second, the influence of tissue thickness was investigated by comparing the results obtained for 10, 16, and 20 μm thick (1 mm2) tissue using urea/30% ACN. We observed an increase in the number of identified proteins and corresponding quantity with an increase in the tissue thickness. Finally, by analyzing very small amounts of tissues (∼0.2 mm2) of 10, 16, and 20 μm thickness, we observed that the increase in tissue thickness resulted in a higher number of protein identifications and corresponding quantitative values.

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.

Ceramides are a lipid subclass of the sphingolipids that show large structural diversity. Structural characterization of the ceramides (CERs) can lead to better understanding of their role and function in the biological system. Here we investigated representatives of NP (CER III, CER IIIB) and AP ceramide classes (CER VI) that contain phytosphingosine (P) backbone. Ceramides were characterized in positive ionization mode by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Fragmentation in positive ionization mode of the CER III and CER VI resulted in abundant ions assigned to phytosphingosine moiety at m/z 282, 300 and 318. HDX-MS of fragments showed increase in m/z of corresponding ions confirming the exchange of deuterium. In negative ionisation spectra multiple fragment ions were assigned to fatty acyl (RCOO–) moiety. Presence of RCOO– allowed unambiguous identification of CER III and CER IIIB which were distinguished by the presence of double bond on fatty acyl chain. This work is licensed under a Creative Commons Attribution 4.0 International License.

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.

Neoantigen-based immunotherapies promise to improve patient outcomes over the current standard of care. However, detecting these cancer-specific antigens is one of the significant challenges in the field of mass spectrometry. Even though the first sequencing of the immunopeptides was done decades ago, today there is still a diversity of the protocols used for neoantigen isolation from the cell surface. This heterogeneity makes it difficult to compare results between the laboratories and the studies. Isolation of the neoantigens from the cell surface is usually done by mild acid elution (MAE) or immunoprecipitation (IP) protocol. However, limited amounts of the neoantigens present on the cell surface impose a challenge and require instrumentation with enough sensitivity and accuracy for their detection. Detecting these neopeptides from small amounts of available patient tissue limits the scope of most of the studies to cell cultures. Here, we summarize protocols for the extraction and identification of the major histocompatibility complex (MHC) class I and II peptides. We aimed to evaluate existing methods in terms of the appropriateness of the isolation procedure, as well as instrumental parameters used for neoantigen detection. We also focus on the amount of the material used in the protocols as the critical factor to consider when analyzing neoantigens. Beyond experimental aspects, there are numerous readily available proteomics suits/tools applicable for neoantigen discovery; however, experimental validation is still necessary for neoantigen characterization.

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.

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 bacteriumBacillus subtilisas 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 inter-protein cross-linked peptides with less than a 1% false discovery rate by mass spectrometry and genome-wide data base searching. Nearly 60% of the inter-protein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and β′ subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth.

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.