Issey Osaka

Interlocked peptide molecules exhibit peptidase resistance as well as enzyme inhibitory activity, making them promising candidates for peptide‐based drugs. Lasso peptides possess a [1]rotaxane structure, in which a peptide chain threads through the cavity of the cyclic peptide moiety. However, the construction of such peptide‐based interlocked structures remains highly challenging, and the total chemical synthesis of natural lasso peptides has not yet been achieved. Here, we aim to develop a synthetic method for a peptide‐based rotaxane using a metal‐template strategy. A macrocyclic complex bearing a Pd(II) ion fixed by a pincer ligand is employed to examine the threading of Val‐8mer in DMSO. Pseudorotaxane formation is assessed by characteristic ¹ H NMR shifts and DOSY measurements, and is confirmed by electrospray ionization mass spectrometry (ESI‐MS). Titration and variable‐temperature (VT) NMR experiments are also performed to elucidate the dynamic behavior of the rotaxanation. These results demonstrate the feasibility of creating lasso‐peptide‐like architectures via metal‐templated rotaxanation, providing a new strategy for the synthesis of natural and artificial lasso peptides.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) imaging enables spatially resolved molecular analysis; however, its sensitivity depends strongly on matrix uniformity and ionization efficiency, which pose challenges for low-abundance compounds such as steroid hormones. In this study, we developed a platinum (Pt) thin film–based surface-assisted laser desorption/ionization mass spectrometry (SALDI/MS) and matrix-enhanced SALDI/MS (ME-SALDI/MS) method to increase ion yields and improve steroid imaging with minimal sample pretreatment. Using sputter-deposited Pt films, 20 endogenous steroids were comprehensively detected without chemical derivatization. Furthermore, derivatized steroids analyzed by ME-SALDI/MS exhibited significantly higher ion yields than those obtained by conventional MALDI/MS or SALDI/MS, demonstrating a synergistic enhancement of desorption and ionization efficiency. ME-SALDI/MS imaging of porcine adrenal gland tissue enabled visualization of multiple derivatized steroids with high ion yields—including progesterone, corticosterone, and cortisol—reflecting their biosynthetic localization across cortical zones. Collectively, these results highlight the potential of Pt thin film–based SALDI and ME-SALDI/MS as robust tools that provide high ion yields for imaging of steroids and other biomolecules with inherently low desorption and ionization efficiency. Graphical abstract

Matrix-assisted laser desorption/ionization (MALDI), surface-assisted laser desorption/ionization (SALDI), and time-of-flight mass spectrometry (TOFMS) imaging are used for visualizing the spatial distribution of analytes. Mass spectrometry (MS) imaging of a sample with a rough surface with a uniform distribution of an analyte does not provide uniform ion intensities in the image. A shift in the m/z value of the analyte ions is also observed. To resolve these issues, we proposed a normalization method for obtaining images based on the actual localization of an analyte. A platinum (Pt) film was used to facilitate ionization in SALDI. The Pt ions obtained from the film and copper(II)-phthalocyanine from a commercial blue pigment were used to evaluate the shift in the m/z values and ion intensities originating from the sample surface placed diagonally. The relationship between the shift in the m/z value and peak area in mass spectra was determined by analyzing the relationship between the shift and irradiated height position, as well as between the peak area and irradiated height position on the sample. The SALDI image can be obtained based on the actual location of the analyte to normalize the raw image data. Thus, the normalization of the ion intensities enables SALDI-MS imaging of samples with rough surfaces using high-resolution TOFMS. The proposed normalization method for SALDI-MS imaging enhances the accuracy and resolution of analyte localization on rough surfaces, benefiting applications in material science, biomedical research, forensic analysis, and industrial quality control.

Matrix-assisted laser desorption ionization (MALDI) and surface-assisted laser desorption ionization (SALDI) mass spectrometry (MS), which can detect biomolecules and polymers, are widely used in biochemistry and materials science. Some compounds that are difficult to ionize using MALDI can be ionized using SALDI. However, it is difficult to obtain high ion yields using SALDI/MS. In this study, a fabricated platinum (Pt) film with nanostructures on the sample surface using a sputtering method was evaluated to determine the optimal metal film for ion yield in SALDI. The surface morphology of the Pt film was analyzed using scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), X-ray reflectivity (XRR), and ultraviolet–visible–near-infrared (UV-Vis-NIR) reflectance spectroscopy. The SEM, AFM, and TEM images of the Pt film showed the deposited metal film giving high ion yields of polyethylene glycol (PEG) in SALDI/MS with a Pt film (Pt-SALDI) that had a rough surface. The densities and reflectivity of films were analyzed by XRR and UV-Vis-NIR. The higher ion yields of PEG were obtained by Pt-SALDI with the Pt films with lower densities and reflectivity. This indicates that the deposition conditions for the Pt films significantly improved the ion yield in Pt-SALDI/MS. The Pt-SALDI has ionization capabilities different from those of MALDI. Therefore, optimization of Pt film for SALDI/MS and the MS imaging allows more compounds to be detected with higher sensitivity.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been widely used for analyses of biomolecules and industrial materials. Surface-assisted laser desorption/ionization (SALDI) is studied to complement the ionization ability for the MALDI/MS. In this study, lab-made mist chemical vapor deposition (mist CVD) system was used to produce metal films as ionization assistance materials for SALDI/MS. The system could give Ag film from inexpensive silver trifluoroacetate solution rapidly and simply under atmospheric pressure. Phosphatidylcholines could be detected high sensitively and diacylglycerols (DAGs) could not be detected in MALDI/MS. In the SALDI/MS and the MS imaging with Ag film by mist CVD, both the phosphatidylcholines and the DAGs could be detected and the localized images. In the Ag film-SALDI/MS of lipids, not only Ag-adducted ions but also Na- and K-adducted ions were detected. The Ag film formed by the mist CVD to act as an ionization-assistance material and a cationization agent in SALDI would be useful in MS imaging of biological tissue sections.

Peptidoglycan for elongation in Escherichia coli is synthesized by the Rod complex, which includes RodZ. Although various mutant strains of the Rod complex have been isolated, the relationship between the activity of the Rod complex and the overall physical and chemical structures of the peptidoglycan have not been reported. We constructed a RodZ mutant, termed RMR, and analyzed the growth rate, morphology, and other characteristics of cells producing the Rod complexes containing RMR. The growth and morphology of RMR cells were abnormal, and we isolated suppressor mutants from RMR cells. Most of the suppressor mutations were found in components of the Rod complex, suggesting that these suppressor mutations increase the integrity and/or the activity of the Rod complex. We purified peptidoglycan from wild‐type, RMR, and suppressor mutant cells and observed their structures in detail. We found that the peptidoglycan purified from RMR cells had many large holes and different compositions of muropeptides from those of WT cells. The Rod complex may be a determinant not only for the whole shape of peptidoglycan but also for its highly dense structure to support the mechanical strength of the cell wall.

Aldosterone-producing adenomas (APAs) have different steroid profiles in serum, depending on the causative genetic mutation. Ion mobility is a separation technique for gas-phase ions based on their m/z values, shapes, and sizes. Human serum (100 µL) was purified by liquid–liquid extraction using tert-butyl methyl ether/ethyl acetate at 1/1 (v/v) and mixed with deuterium-labeled steroids as the internal standard. The separated supernatant was dried, re-dissolved in water containing 20% methanol, and injected into a liquid chromatography–ion mobility–mass spectrometer (LC/IM/MS). We established a highly sensitive assay system by separating 20 steroids based on their retention time, m/z value, and drift time. Twenty steroids were measured in the serum of patients with primary aldosteronism, essential hypertension, and healthy subjects and were clearly classified using principal component analysis. This method was also able to detect phosphatidylcholine and phosphatidylethanolamine, which were not targeted. LC/IM/MS has a high selectivity for known compounds and has the potential to provide information on unknown compounds. This analytical method has the potential to elucidate the pathogenesis of APA and identify unknown steroids that could serve as biomarkers for APA with different genetic mutations.

Steroids are present in all animals and plants, from mammals to prokaryotes. In the medical field, steroids are commonly classified as glucocorticoids, mineralocorticoids, and gonadal steroid hormones. Monitoring of hormones is useful in clinical and research fields for the assessment of physiological changes associated with aging, disease risk, and the diagnostic and therapeutic effects of various diseases. Since the discovery and isolation of steroid hormones, measurement methods for steroid hormones in biological samples have advanced substantially. Although immunoassays (IAs) are widely used in daily practice, mass spectrometry (MS)-based methods have been reported to be more specific. Steroid hormone measurement based on MS is desirable in clinical practice; however, there are several drawbacks, including the purchase and maintenance costs of the MS instrument and the need for specialized training of technicians. In this review, we discuss IA- and MS-based methods currently in use and briefly present the history of steroid hormone measurement. In addition, we describe recent advances in IA- and MS-based methods and future applications and considerations.

Sample pretreatment is key to obtaining good data in matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI). Although sublimation is one of the best methods for obtaining homogenously fine organic matrix crystals, its sensitivity can be low due to the lack of a solvent extraction effect. We investigated the effect of incorporating a thin film of metal formed by zirconium (Zr) sputtering into the sublimation process for MALDI matrix deposition for improving the detection sensitivity in mouse liver tissue sections treated with olanzapine. The matrix‐enhanced surface‐assisted laser desorption/ionization (ME‐SALDI) method, where a matrix was formed by sputtering Zr to form a thin nanoparticle layer before depositing MALDI organic matrix comprising α‐cyano‐4‐hydroxycinnamic acid (CHCA) by sublimation, resulted in a significant improvement in sensitivity, with the ion intensity of olanzapine being about 1800 times that observed using the MALDI method, comprising CHCA sublimation alone. When Zr sputtering was performed after CHCA deposition, however, no such enhancement in sensitivity was observed. The enhanced sensitivity due to Zr sputtering was also observed when the CHCA solution was applied by spraying, being about twice as high as that observed by CHCA spraying alone. In addition, the detection sensitivity of these various pretreatment methods was similar for endogenous glutathione. Given that sample preparation using the ME‐SALDI‐MSI method, which combines Zr sputtering with the sublimation method for depositing an organic matrix, does not involve a solvent, delocalization problems such as migration of analytes observed after matrix spraying and washing with aqueous solutions as sample pretreatment are not expected. Therefore, ME‐Zr‐SALDI‐MSI is a novel sample pretreatment method that can improve the sensitivity of analytes while maintaining high spatial resolution in MALDI‐MSI.