Monitoring lipid alterations in Drosophila heads in an amyotrophic lateral sclerosis model with time-of-flight secondary ion mass spectrometry.

Lipid alterations in the brain are well-documented in disease and aging, but our understanding of their pathogenic implications remains incomplete. Recent technological advances in assessing lipid profiles have enabled us to intricately examine the spatiotemporal variations in lipid compositions within the complex brain characterized by diverse cell types and intricate neural networks. In this study, we coupled time-of-flight secondary ion mass spectrometry (ToF-SIMS) to an amyotrophic lateral sclerosis (ALS) Drosophila model, for the first time, to elucidate changes in the lipid landscape and investigate their potential role in the disease process, serving as a methodological and analytical complement to our prior approach that utilized matrix-assisted laser desorption/ionization mass spectrometry. The expansion of G4C2 repeats in the C9orf72 gene is the most prevalent genetic factor in ALS. Our findings indicate that expressing these repeats in fly brains elevates the levels of fatty acids, diacylglycerols, and ceramides during the early stages (day 5) of disease progression, preceding motor dysfunction. Using RNAi-based genetic screening targeting lipid regulators, we found that reducing fatty acid transport protein 1 (FATP1) and Acyl-CoA-binding protein (ACBP) alleviates the retinal degeneration caused by G4C2 repeat expression and also markedly restores the G4C2-dependent alterations in lipid profiles. Significantly, the expression of FATP1 and ACBP is upregulated in G4C2-expressing flies, suggesting their contribution to lipid dysregulation. Collectively, our novel use of ToF-SIMS with the ALS Drosophila model, alongside methodological and analytical improvements, successfully identifies crucial lipids and related genetic factors in ALS pathogenesis.

Utilization of chromogenic enzyme substrates for signal amplification in multiplexed detection of biomolecules using surface mass spectrometry.

MicroRNAs (miRNAs) are important post-transcriptional gene regulators and can serve as potential biomarkers for many diseases. Most of the current miRNA detection techniques require purification from biological samples, amplification, labeling, or tagging, which makes quantitative analysis of clinically relevant samples challenging. Here we present a new strategy for the detection of miRNAs with uniformity over a large area based on signal amplification using enzymatic reactions and measurements using time-of-flight secondary ion mass spectrometry (ToF-SIMS), a sensitive surface analysis tool. This technique has high sequence specificity through hybridization with a hairpin DNA probe and allows the identification of single-base mismatches that are difficult to distinguish by conventional mass spectrometry. We successfully detected target miRNAs in biological samples without purification, amplification, or labeling of target molecules. In addition, by adopting a well-known chromogenic enzymatic reaction from the field of biotechnology, we extended the use of enzyme-amplified signal enhancement ToF (EASE-ToF) to protein detection. Our strategy has advantages with respect to scope, quantification, and throughput over the currently available methods, and is amenable to multiplexing based on the outstanding molecular specificity of mass spectrometry (MS). Therefore, our technique not only has the potential for use in clinical diagnosis, but also provides evidence that MS can serve as a useful readout for biosensing to perform multiplexed analysis extending beyond the limitations of existing technology.

Role of Chalcogenides in Sensitive Therapeutic Drug Monitoring Using Laser Desorption and Ionization.

This study investigates the applicability of six transition metal dichalcogenides to efficient therapeutic drug monitoring of ten antiepileptic drugs using laser desorption/ionization-mass spectrometry. We found that molybdenum ditelluride and tungsten ditelluride are suitable for the sensitive quantification of therapeutic drugs. The contribution of tellurium to the enhanced efficiency of laser desorption ionization was validated through theoretical calculations utilizing an integrated model that incorporates transition-metal dichalcogenides and antiepileptic drugs. The results of our theoretical calculations suggest that the relatively low surface electron density for the tellurium-containing transition metal dichalcogenides induces stronger Coulombic interactions, which results in enhanced laser desorption and ionization efficiency. To demonstrate applicability, up to 120 patient samples were analyzed to determine drug concentrations, and the results were compared with those of immunoassay and liquid chromatography-tandem mass spectrometry. Agreements among these methods were statistically evaluated using the Passing-Bablok regression and Bland-Altman analysis. Furthermore, our method has been shown to be applicable to the simultaneous detection and multiplexed quantification of antiepileptic drugs.

Quantitative Analysis of Immunosuppressive Drugs Using Tungsten Disulfide Nanosheet-Assisted Laser Desorption Ionization Mass Spectrometry.

For organ transplantation patients, the therapeutic drug monitoring (TDM) of immunosuppressive drugs is essential to prevent the toxicity or rejection of the organ. Currently, TDM is done by immunoassays or liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods; however, these methods lack specificity or are expensive, require high levels of skill, and offer limited sample throughput. Although matrix-assisted (MA) laser desorption ionization (LDI) mass spectrometry (MS) can provide enhanced throughput and cost-effectiveness, its application in TDM is limited due to the limitations of the matrixes such as a lack of sensitivity and reproducibility. Here, we present an alternative quantification method for the TDM of the immunosuppressive drugs in the blood of organ transplant patients by utilizing laser desorption ionization mass spectrometry (LDI-MS) based on a tungsten disulfide nanosheet, which is well-known for its excellent physicochemical properties such as a strong UV absorbance and high electron mobility. By adopting a microliquid inkjet printing system, a high-throughput analysis of the blood samples with enhanced sensitivity and reproducibility was achieved. Furthermore, up to 80 cases of patient samples were analyzed and the results were compared with those of LC-MS/MS by using Passing-Bablok regression and Bland-Altman analysis to demonstrate that our LDI-MS platform is suitable to replace current TDM techniques. Our approach will facilitate the rapid and accurate analysis of blood samples from a large number of patients for immunosuppressive drug prescriptions.

Comparative study on formation of protein coronas under three different serum origins.

Nanomaterials form a complex called "protein corona" by contacting with protein-containing biological fluids such as serum when they are exposed to physiological environments. The characteristics of these proteins, which are one of the substantial factors in cellular response, are affected by the interactions between the nanomaterials and the biological systems. Many studies have investigated the biological behaviors of nanomaterials by conducting experiments in vitro and in vivo; however, the origin of the biological materials used is rather inconsistent. This is due to the fact that the composition of the protein coronas may differ depending on the animal origin, not on the composition or size of the nanoparticles. The resulting differences in the composition of the protein coronas can lead to different conclusions. To identify the differences in protein corona formation among sera of different species, we investigated protein coronas of gold and silica nanoparticles in serum obtained from various species. Using comparative proteomic analysis, common proteins adsorbed onto each nanoparticle among the three different sera were identified as highly abundant proteins in the serum. These findings indicate that protein corona formation is dependent on the serum population rather than the size or type of the nanoparticles. Additionally, in the physiological classification of protein coronas, human serum (HS) was found to be rich in apolipoproteins. In conclusion, our data indicate that HS components are different from those of bovine or mouse, indicating that the serum species origin should be carefully considered when selecting a biological fluid.

Numerical evaluation of polyethylene glycol ligand conjugation to gold nanoparticle surface using ToF-SIMS and statistical analysis.

Nanoparticles (NPs) are substances between 1 and 100 nm in size. They have been the subject of numerous studies because of their potential applications in a wide range of fields such as cosmetics, electronics, medicine, and food. For biological applications of nanoparticles, they are usually coated with a substance capable of preventing agglomeration of the nanoparticles and nonspecific binding and exhibiting water-solubility characteristics with specific immobilized (bio)molecules. In order to evaluate the chemical properties of the surface-modified nanoparticles for bioapplications, including drug delivery, a simple and reliable method for the analysis of the presence of the surface chemicals and the ligand states of the nanoparticles is necessary. In this study, the authors numerically evaluated the extent of polyethylene glycol (PEG) ligand conjugation on AuNPs by concurrently adopting a microliquid inkjet printing system for sampling of the PEGylated AuNPs solution and ToF-SIMS imaging together with statistical analysis. The statistical correlation values calculated from the signals of PEG and Au measured by ToF-SIMS imaging on the sample spots made by a microliquid inkjet printing system showed better reproducibility and improved correlation values compared to the pipet spotting. Their improved method will be useful to evaluate ligand-conjugated nanoparticles for quality control of each conjugation process.

Ar-gas cluster ion beam in ToF-SIMS for peptide and protein analysis.

Since Ar-gas cluster ion beams (Ar-GCIBs) have been introduced into time-of-flight secondary ion mass spectrometry (ToF-SIMS), there have been various attempts to analyze organic materials and biomolecules that require low-damage analysis and high sensitivity, because Ar-GCIBs allow soft ionization of large molecules such as peptides and proteins due to the low energy per atom. Here, the authors adopted the Ar-GCIB as a primary beam to detect proteins including human insulin, ubiquitin, and cytochrome C (molecular weights are 5808, 8564, and 12 327 Da, respectively). They have confirmed that the detection of the intact proteins was possible when the Ar-GCIB was used as a primary ion beam. In addition, they successfully identified each protein by analyzing the trypsin-digested peptides in myoglobin, cytochrome C, and bovine serum albumin. They also attempted on-surface enzymatic digestion to identify proteins on the surface of the Si wafer and obtained results identical to those of in-solution digestion. It is expected that the authors' on-surface digestion method can enable the application of ToF-SIMS for the analysis of proteins present in biological tissues.