No skin off your back: the sampling and extraction of sebum for metabolomics.

INTRODUCTION: Sebum-based metabolomics (a subset of "sebomics") is a developing field that involves the sampling, identification, and quantification of metabolites found in human sebum. Sebum is a lipid-rich oily substance secreted by the sebaceous glands onto the skin surface for skin homeostasis, lubrication, thermoregulation, and environmental protection. Interest in sebomics has grown over the last decade due to its potential for rapid analysis following non-invasive sampling for a range of clinical and environmental applications. OBJECTIVES: To provide an overview of various sebum sampling techniques with their associated challenges. To evaluate applications of sebum for clinical research, drug monitoring, and human biomonitoring. To provide a commentary of the opportunities of using sebum as a diagnostic biofluid in the future. METHODS: Bibliometric analyses of selected keywords regarding skin surface analysis using the Scopus search engine from 1960 to 2022 was performed on 12th January 2023. The published literature was compartmentalised based on what the work contributed to in the following areas: the understanding about sebum, its composition, the analytical technologies used, or the purpose of use of sebum. The findings were summarised in this review. RESULTS: Historically, about 15 methods of sampling have been used for sebum collection. The sample preparation approaches vary depending on the analytes of interest and are summarised. The use of sebum is not limited to just skin diseases or drug monitoring but also demonstrated for other systemic disease. Most of the work carried out for untargeted analysis of metabolites associated with sebum has been in the recent two decades. CONCLUSION: Sebum has a huge potential beyond skin research and understanding how one's physiological state affects or reflects on the skin metabolome via the sebaceous glands itself or by interactions with sebaceous secretion, will open doors for simpler biomonitoring. Sebum acts as a sink to environmental metabolites and has applications awaiting to be explored, such as biosecurity, cross-border migration, localised exposure to harmful substances, and high-throughput population screening. These applications will be possible with rapid advances in volatile headspace and lipidomics method development as well as the ability of the metabolomics community to annotate unknown species better. A key issue with skin surface analysis that remains unsolved is attributing the source of the metabolites found on the skin surface before meaningful biological interpretation.

Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.

Applications of ion mobility mass spectrometry for high throughput, high resolution glycan analysis.

BACKGROUND: Diverse varieties of often heterogeneous glycans are ubiquitous in nature. They play critical roles in recognition events, act as energy stores and provide structural stability at both molecular and cellular levels. Technologies capable of fully elucidating the structures of glycans are far behind the other '-omic' fields. Liquid chromatography (LC) and mass spectrometry (MS) are currently the most useful techniques for high-throughput analysis of glycans. However, these techniques do not provide full unambiguous structural information and instead the gap in full sequence assignment is frequently filled by a priori knowledge of the biosynthetic pathways and the assumption that these pathways are highly conserved. SCOPE OF THE REVIEW: This comprehensive review details the rise of the emerging analytical technique ion mobility spectrometry (IMS) (coupled to MS) to facilitate the determination of three-dimensional shape: the separation and characterization of isobaric glycans, glyco(peptides/proteins), glycolipids, glycosaminoglycans and other polysaccharides; localization of sites of glycosylation; or interpretation of the conformational change to proteins upon glycan binding. MAJOR CONCLUSIONS: IMS is a highly promising new analytical route, able to provide rapid isomeric separation (ms timescale) of either precursor or product ions facilitating MS characterization. This additional separation also enables the deconvolution of carbohydrate MS(/MS) information from contaminating ions, improving sensitivity and reducing chemical noise. Derivation of collision cross sections (CCS) from IM-MS(/MS) data and subsequent calculations validate putative structures of carbohydrates from ab initio derived candidates. IM-MS has demonstrated that amounts of specific glycan isomers vary between disease states, which would be challenging to detect using standard analytical approaches. GENERAL SIGNIFICANCE: IM-MS is a promising technique that fills an important gap within the Glycomics toolbox, namely identifying and differentiating the three-dimensional structure of chemically similar carbohydrates and glycoconjugates. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

The effect of thermal processing on the behaviour of peanut allergen peptide targets used in multiple reaction monitoring mass spectrometry experiments.

Mass spectrometry-based methods offer an alternative means of determining allergens in foods. Whilst targeted methods are likely to offer the most robust approach for detection and quantification, little is known about how food processing may affect the behaviour of peptide targets. A systematic study has been undertaken to investigate the effects of thermal processing (boiling, roasting, frying) on the behaviour of a suite of peanut peptide targets representing the major clinically-relevant allergens. Initially the effect of thermal processing on protein extractability was investigated and a mass spectrometry-compatible buffer identified comprising 50 mM Tris-HCl, pH 8.8 containing 50 mM dithiothreitol and 0.04% (w/v) acid labile detergent which was able to extract 45-100% of protein from raw, boiled, roasted and fried peanuts using sonication at 60 °C. Eight peptide targets were identified including two peptides from each cupin allergen, Ara h1 and Ara h3 and four peptides from the prolamin superfamily allergens Ara h2, 6 and 7. AQUA peptide standards were synthesised and used to undertake multiple-reaction monitoring experiments, giving assay sensitivities of 0.1-30 amoles of peptide on-column (3 : 1 signal : noise), calculated limits of quantification between 96-1343 amoles of peptide on-column and a linear dynamic range of 4-5 orders of magnitude. Absolute quantification of individual peanut allergens in thermally processed samples showed that peptide targets in the cupin allergens were more prone to processing-induced effects than those from Ara h2, 6 and 7. Targets flanked by arginine residues showed greater thermostability. Identification of processing-stable targets, coupled with more efficient extraction procedures and a wide dynamic range, shows that targeted mass spectrometry methods have great potential as an additional method for quantifying peanut allergens in complex food matrices.

Beta-defensin evolution: selection complexity and clues for residues of functional importance.

We have examined the evolution of the genes at the major human beta-defensin locus and the orthologous loci in a range of other primates and mammals. For the first time, these data allow us to examine selective episodes in the more recent evolutionary history of this locus as well as in the ancient past. We have used a combination of maximum-likelihood-based tests and a maximum-parsimony-based sliding window approach to give a detailed view of the varying modes of selection operating at this locus. We provide evidence for strong positive selection soon after the duplication of these genes within an ancestral mammalian genome. During the divergence of primates, however, variable selective pressures have acted on beta-defensin genes in different evolutionary lineages, with episodes of both negative and, more rarely, positive selection. Positive selection appears to have been more common in the rodent lineage, accompanying the birth of novel rodent-specific beta-defensin gene clades. Sites in the second exon have been subject to positive selection and, by implication, are important in functional diversity. A small number of sites in the mature human peptides were found to have undergone repeated episodes of selection in different primate lineages. Particular sites were consistently implicated by multiple methods at positions throughout the mature peptides. These sites are clustered at positions that are predicted to be important for the function of beta-defensins.

DNA sequence analysis of the rat RT1.B alpha gene.

The major histocompatibility complex (MHC) of the rat (RT1 complex) encodes two sets of class II molecules referred to as RT1.B and RT1.D. The RT1.B alpha gene was isolated for a Sprague-Dawley (RT1b) rat genomic library using a rat RT1.B alpha chain cDNA as a hybridization probe. The coding and the majority of the intron DNA sequence was determined. The structure of the RT1.B alpha gene is equivalent to that of H-2 and HLA alpha chain genes. Comparison of the nucleotide and predicted amino acid sequences of the RT1.B alpha gene with those of the H-2 and HLA genes revealed a high degree of overall sequence conservation. However, two regions of the first external domain (alpha 1), residues 19-23 and 45-78, exhibit marked sequence variation. Two blocks of conserved nucleotide sequence were identified in the 5' promoter region of the RT1.B alpha gene that have been described in all MHC class II genes sequenced to date. These conserved sequences may be involved in the coordinate regulation of expression of class II genes. The cloned RT1.B alpha gene was efficiently transcribed when transfected to mouse L cells.

Modification of CD43 and other lymphocyte O-glycoproteins by core 2 N-acetylglucosaminyltransferase.

CD43, the major leukocyte sialoglycoprotein, is expressed on T lymphocytes in two predominant glycoforms. CD43 115 kDa is a pan T cell marker and is specifically recognized by the monoclonal antibody S7. CD43 130 kDa is associated with T cell activation and is specifically recognized by the monoclonal antibody 1B11. The thymoma EL-4 has been identified to express mainly CD43 115 kDa and little or no CD43 130 kDa. Transfection of EL-4 cells with core 2 beta 1-->6N-acetylglucosaminyltransferase (C2GnT), an enzyme in the O-glycan biosynthesis pathway, resulted in an enhanced expression of the 1B11 epitope, CD43 130 kDa, and a loss of expression of the S7 epitope, CD43 115 kDa. Analysis of CD43 by SDS-PAGE revealed that CD43 in C2GnT transfected EL-4 cells has a molecular weight of 125 kDa compared to 115 kDa in nontransfected or control transfected EL-4 cells. SDS-PAGE analysis of three other lymphocyte O-glycoproteins, CD44, CD45, and RPTP alpha, revealed that C2GnT expression resulted in a molecular weight increase of approximately 3-5 kDa for each of these three cell surface glycoproteins. Our data indicate that, while CD43 may be a predominant substrate for C2GnT, other lymphocyte O-glycoproteins are also modified by this glycosyltransferase. Increased reactivity of cells with the monoclonal antibody 1B11, which specifically detects the expression of murine CD43 130 kDa, may thus be a marker of increases in branching of O-linked glycans generally.

In vivo overexpression of Core2 N-acetylglucosaminyltransferase prevents repopulation of the bone marrow with colony forming cells but fails to affect normal T cell development.

UDP-GlcNAc:Galbet1 --> 3GalNAc-R beta1 --> 6N-acetylglucosaminyltransferase (Core2 N-acetyl-glucosaminyltransferase, C2GnT; EC 2.4.1.102) forms beta1 --> 6N-acetyl-glucosaminyl linkages in O-glycoproteins and creates branches for the addition of N-acetyl-lactosamine antennae. Changes in C2GnT activity have been associated with immune disorders, malignancies, and T-cell ontogeny. In this study, we used SCID (severe combined immune deficiency) mice to determine the effects of C2GnT overexpression on hemopoiesis, and in particular, on thymocyte development. BALB/c bone marrow cells transfected with C2GnT using the retroviral murine stem cell vector were used to repopulate SCID mice. Mice were analysed 3 weeks to 3 months after bone marrow transfer. Elevated levels of C2GnT activity in bone marrow, spleen, and thymus from mice repopulated with C2GnT transfected bone marrow cells indicated that C2GnT was overexpressed in recipient mice. In C2GnT repopulated mice, up to 50% of T cells showed an increase in CD43 130-kDa expression, compared with T cells from control animals, indicative of an elevated C2GnT activity in these cells. Furthermore, T-cell subset numbers appeared to be normal, suggesting that C2GnT overexpression did not alter T-cell ontogeny. Interestingly, C2GnT overexpression negatively affected the repopulation of myeloid cells. Only insignificant numbers of interleukin-3/granulocyte-macrophage colony stimulating factor (IL-3/GM-CSF) responsive bone marrow cells were found to be retrovirally transfected in C2GnT repopulated mice, whereas up to 50% of IL-3/GM-CSF responsive bone marrow cells were found to be retrovirally transfected in corresponding controls. These data indicate that in vivo overexpression of C2GnT negatively interferes with the myeloid differentiation pathway but does not affect T-cell development.

Comparisons in the behavior of stable copper(II), silver(II), and gold(II) complexes in the gas phase: are there implications for condensed-phase chemistry?

Experiments conducted in the gas phase have led to the formation of a series of stable gold(II) complexes with nitrogen- and oxygen-containing ligands. Such complexes are very rare in condensed-phase chemistry. However, there is also a significant group of potential ligands, for example, H2O and NH3, for which stable complexes could not be formed. There are strong similarities between these observations and earlier results presented for silver(II), but both metal ions behave markedly different from copper(II). As a group the majority of successful gold(II) ligands are characterized by being good sigma donor-pi acceptor molecules; however, it is also possible to understand the ability of individual ligands to stabilize the metal ion in terms of a simple electrostatic model. Application of the latter reveals a semiquantitative trend between the physical properties of a ligand, e.g. ionization energy, dipole moment, and polarizability, and the ligand's ability to stabilize either Cu(II), Ag(II), or Au(II). The model successfully accounts for the preference of Cu(II) for aqueous chemistry, in comparison to the complete absence of such behavior on the part of Ag(II) and Au(II). Ligands from recent examples of stable condensed-phase gold(II) complexes appear to meet at least one of the criteria identified from the model.