Julia Pruns

MALDI imaging in human skin tissue sections: focus on various matrices and enzymes.

AbstractMatrix-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. FigureMatrix-assisted laser/desorption ionization (MALDI) mass-spectrometric imaging (MSI), also known as MALDI imaging, is a powerful technique for mapping biological molecules in human skin tissue sections. In this body of work, a multiplex method, based on MALDI-MSI, is presented to obtain maximum information from skin tissue sections. Therefore, various matrices were applied to skin tissue sections: (1) 9-aminoacridine (9-AA) for imaging small molecules in negative ion mode; (2) sinapinic acid (SA) to obtain protein distributions; (3) α-cyano-4-hydroxycinnamic acid (α-HCHA) subsequent to on-tissue enzymatic digestion by trypsin, elastase, and pepsin, respectively, to localize the resulting peptides. Of note, identification of metabolites was achieved by post-source decay (PSD) MALDI, and proteins were identified subsequent to enzymatic digestion via the resulting peptides which were eluted from the skin tissue section and afterwards analyzed with use of a tandem time-of-flight (ToF) mass spectrometer. The application of alternative enzymes, such as pepsin and elastase, is highlighted within this article

A novel sampling method for identification of endogenous skin surface compounds by use of DART-MS and MALDI-MS

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.

Influence of various on-tissue washing procedures on the entire protein quantity and the quality of matrix-assisted laser desorption/ionization spectra

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.