RESUMO
We have previously shown that precursors of odorous components characteristic of axillary sweat are hardly detectable or undetectable in individuals carrying the 538G > A SNP in the ABCC11 transporter gene. However, it is unclear, whether ABCC11 is directly involved in the transport of these compounds. To approach this question, transport of peptide-conjugated potential precursors of 3-methyl-3-sulfanylhexanol (3M3SH), a key determinant of axillary malodour, was measured using membrane vesicles of Sf9 insect cells overexpressing human ABCC11. Whilst no ABCC11-mediated transport was detected for the dipeptide precursor Cys-Gly-3M3SH, the glutathione conjugate of 3M3SH (SG-3M3SH) was robustly taken up by ABCC11 at a transport rate of 0.47 pmol/mg/min. Collectively, these results illuminate SG-3M3SH as a putative precursor of 3M3SH, which then may undergo intra-vesicular maturation to generate Cys-Gly-3M3SH. Critically, the apocrine sweat gland was demonstrated to express γ-glutamyl transferase 1 (GGT1) protein, which is known to catalyse the deglutamylation of glutathionyl conjugates. Additionally, we provide evidence that recombinant and isolated hepatic human GGT1 is capable of transforming SG-3M3SH to Cys-Gly-3M3SH in vitro. To sum up, we demonstrate that the functionality of ABCC11 is likely to play an important role in the generation of axillary malodour. Furthermore, we identify GGT1 as a key enzyme involved in the biosynthesis of Cys-Gly-3M3SH.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Glândulas Apócrinas/metabolismo , Hexanóis/metabolismo , Ácidos Sulfanílicos/metabolismo , gama-Glutamiltransferase/metabolismo , Animais , Linhagem Celular , Humanos , OdorantesRESUMO
RATIONALE: The fast and univocal identification of different species in mixtures of pollen grains is still a challenge. Apart from microscopic evaluation and Raman spectroscopy, no other techniques are available. METHODS: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry was applied to the analysis of extracts of single pollen grains and pollen mixtures. Pollen grains were fixed, treated and covered with matrix directly on the MALDI target. RESULTS: Clearly resolved MALDI ion intensity images could be obtained enabling the identification of single pollen grains in a mixture. CONCLUSIONS: Our results demonstrate the potential and the suitability of MALDI imaging mass spectrometry as an additional method for the identification of pollen mixtures.
Assuntos
Pólen/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Magnoliopsida/química , Imagem Molecular/métodos , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
RATIONALE: For the matrix-assisted laser desorption/ionization (MALDI) imaging of proteins and tryptic peptides it is recommendable to remove salts, lipids, and phospholipids prior to analysis. However, thorough investigations of the influence of commonly used washing protocols on the entire protein content and the spectral quality have not been carried out. METHODS: After the application of various on-tissue washing protocols, proteins and peptides were eluted by use of different solvents. Subsequently, protein quantities of the eluates were determined by a bicinchoninic acid assay. The spectral quality of the tryptic peptide eluates was investigated based upon peak picking. A MALDI time-of-flight (TOF) mass spectrometer was used to generate mass spectra. Skin tissue samples were prepared by embedding them either in carboxymethyl cellulose or in a cutting medium containing polyethylene glycol. RESULTS: Our work shows the numerical decrease in protein content after applying different on-tissue washing protocols. Protein losses in a range of 17-38% were observed. From evaluating the spectral quality, two washing protocols were shown to be beneficial, enabling the detection of a high number of tryptic peptides. Procedures to thoroughly remove polyethylene glycol (deriving from special embedding media) were determined. Critically, aqueous washing steps conducted as short dips in two different jars were beneficial in achieving complete removal. CONCLUSIONS: Washing steps have a strong impact on improving the spectral quality, but they may lead to a high decrease in the protein content. Our results show that there is a balancing act between avoiding protein loss and obtaining high spectra quality.
Assuntos
Técnicas Histológicas/métodos , Proteínas/análise , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Humanos , Quinolinas/química , Pele/química , Inclusão do TecidoRESUMO
Matrix-assisted laser/desorption ionization (MALDI) mass-spectrometric imaging (MSI), also known as MALDI imaging, is a powerful technique for mapping biological molecules such as endogenous proteins and peptides in human skin tissue sections. A few groups have endeavored to apply MALDI-MSI to the field of skin research; however, a comprehensive article dealing with skin tissue sections and the application of various matrices and enzymes is not available. Our aim is to present a multiplex method, based on MALDI-MSI, to obtain the maximum information from skin tissue sections. Various matrices were applied to skin tissue sections: (1) 9-aminoacridine for imaging metabolites in negative ion mode; (2) sinapinic acid to obtain protein distributions; (3) α-cyano-4-hydroxycinnamic acid subsequent to on-tissue enzymatic digestion by trypsin, elastase, and pepsin, respectively, to localize the resulting peptides. Notably, substantial amounts of data were generated from the distributions retrieved for all matrices applied. Several primary metabolites, e.g. ATP, were localized and subsequently identified by on-tissue postsource decay measurements. Furthermore, maps of proteins and peptides derived from on-tissue digests were generated. Identification of peptides was achieved by elution with different solvents, mixing with α-cyano-4-hydroxycinnamic acid, and subsequent tandem mass spectrometry (MS/MS) measurements, thereby avoiding on-tissue MS/MS measurements. Highly abundant peptides were identified, allowing their use as internal calibrants in future MALDI-MSI analyses of human skin tissue sections. Elastin as an endogenous skin protein was identified only by use of elastase, showing the high potential of alternative enzymes. The results show the versatility of MALDI-MSI in the field of skin research. This article containing a methodological perspective depicts the basics for a comprehensive comparison of various skin states.
Assuntos
Proteínas/metabolismo , Pele/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Aminacrina/química , Humanos , Processamento de Imagem Assistida por Computador , Elastase Pancreática/metabolismo , Peptídeos/metabolismo , Pele/metabolismo , Coloração e Rotulagem , Tripsina/metabolismoRESUMO
Localization of endogenous and exogenous compounds directly in tissue sections is a challenging task in skin research. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful label-free technique that enables determination of the distribution of a large range of biomolecules directly in tissue sections. Nevertheless, its application in this field is limited in large part by the low adhesion of skin tissue sections to indium-tin oxide-coated (ITO) glass slides. For the first time corona discharge (CD) treatment was used to modify the glass slide surface for improved adhesion. Localization of endogenous cholesterol sulfate was performed directly in human skin tissue sections. A spatial resolution of approximately 30 µm was sufficient for assignment of mass signals to skin structure morphology. Furthermore, imaging of an exogenous model compound, Nile red, was performed directly in skin tissue sections after ex-vivo penetration into porcine skin, enabling determination of the pathway and depth of penetration. Finally, the ion density map of Nile red was compared with its high resolution fluorescence micrograph. This work provides new insights into the application of MALDI-MSI in skin research.
Assuntos
Ésteres do Colesterol/análise , Pele/ultraestrutura , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Ésteres do Colesterol/metabolismo , Vidro/química , Humanos , Microscopia de Fluorescência , Oxazinas/análise , Pele/metabolismo , Coloração e Rotulagem , Suínos , Compostos de Estanho/químicaRESUMO
Identification of endogenous skin surface compounds is an intriguing challenge in comparative skin investigations. Notably, this short communication is focused on the analysis of small molecules, e.g. natural moisturizing factor (NMF) components and lipids, using a novel sampling method with DIP-it samplers for non-invasive examination of the human skin surface. As a result, extraction of analytes directly from the skin surface by use of various solvents can be replaced with the mentioned procedure. Screening of measureable compounds is achieved by direct analysis in real time mass spectrometry (DART-MS) without further sample preparation. Results are supplemented by dissolving analytes from the DIP-it samplers by use of different solvents, and subsequent matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) measurements. An interesting comparison of the mentioned MS techniques for determination of skin surface compounds in the mass range of 50-1000 Da is presented.