Study of the influence of hyperglycemia on the abundance of amino acids, fatty acids, and selected lipids in extracellular vesicles using TOF-SIMS.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) with the Bi3+ liquid metal ion gun was used to investigate the content of lipids and amino acids (AAs) in extracellular vesicles (EVs). We induced metabolic changes in human pancreatic ß-cells by stimulation with high glucose concentrations (35 mM) and tested the hypothesis of hyperglycemia (HG) has a detrimental effect on lipids and AAs in released EV subpopulations: ectosomes and exosomes. As a result of HG treatment, selected fatty acids (FAs) such as arachidonic, myristic and palmitic acids, changed their abundance in ectosomes and exosomes. Also, intensities of the characteristic peaks for cholesterol (m/z 95.09; 147.07; 161.11; 369.45) along with the molecular ion m/z 386.37 [C27H46O+] under HG conditions, both for ectosomes and exosomes, have changed significantly. Comparative analysis of HG EVs and normoglycemic (NG) ones showed statistically significant differences in the signal intensities of four AAs: valine (m/z 72.08 and 83.05), isoleucine (m/z 86.10), phenylalanine (m/z 120.08 and 132.05) and tyrosine (m/z 107.05 and 136.09). We confirmed that ToF-SIMS is a useful technique to study selected AAs and lipid profiles in various EV subpopulations. Our study is the first demonstration of changes in FAs and AAs in exosomes and ectosomes derived from ß-cells under the influence of HG.

Design and Optimization of a Biosensor Surface Functionalization to Effectively Capture Urinary Extracellular Vesicles.

For this study, we tested and optimized silicon surface functionalization procedures for capturing urinary extracellular vesicles (uEVs). The influence of the silane type (APTES or GOPS) and protein concentration on the efficiency of uEVs binding was investigated. Human lactadherin protein (LACT) was used to capture uEVs. We applied surface characterization techniques, including ellipsometry, atomic force microscopy, and time-of-flight secondary ion mass spectrometry, to observe changes in the biosensor surface after each functionalization step. uEVs were purified by a low-vacuum filtration method and concentrated by ultracentrifugation. The physical parameters of uEVs after the isolation procedure, such as morphology and size distribution, were determined using transmission electron microscopy and tunable resistive pulse sensing methods. We observed a gradual growth of the molecular layer after subsequent stages of modification of the silicon surface. The ToF-SIMS results showed no changes in the mean intensities for the characteristic peaks of amino acids and lipids in positive and negative polarization, in terms of the surface-modifying silane (APTES or GOPS) used. The most optimal concentration of LACT for the tested system was 25 µg/mL.

Using a lactadherin-immobilized silicon surface for capturing and monitoring plasma microvesicles as a foundation for diagnostic device development.

Microvesicles (MVs) are found in several types of body fluids and are promising disease biomarkers and therapeutic targets. This study aimed to develop a novel biofunctionalized surface for binding plasma microvesicles (PMVs) based on a lab-on-a-chip (LOC) approach. A new lactadherin (LACT)-functionalized surface was prepared and examined for monitoring PMVs. Moreover, two different strategies of LACT immobilization on a silicon surface were applied to compare different LACT orientations. A higher PMV to LACT binding efficiency was observed for LACT bonded to an αvß3 integrin-functionalized surface compared with that for LACT directly bonded to a glutaraldehyde-modified surface. Effective binding of PMVs and its components for both LACT immobilization strategies was confirmed using spectral ellipsometry and time-of-flight secondary ion mass spectrometry methods. The proposed PMV capturing system can be used as a foundation to design novel point-of-care (POC) diagnostic devices to detect and characterize PMVs in clinical samples. Graphical Abstract.

Relevance of the poly(ethylene glycol) linkers in peptide surfaces for proteases assays.

Poly(ethylene glycol)s (PEGs) with different lengths were used as linkers during the preparation of peptide surfaces for protease detection. In the first approach, the PEG monolayers were prepared using a "grafting to" method on 3-aminopropyltrietoxysilane (APTES)-modified silicon wafers. Protected peptides with a fluorescent marker were synthesized by Fmoc solid phase synthesis. The protected peptide structures enabled their site-specific immobilization onto the PEG surfaces. Alternatively, the PEG-peptide surface was obtained by immobilizing a PEG-peptide conjugate directly onto the modified silicon wafer. The surfaces (composition, grafting density, hydrophilicity, and roughness) were characterized by time-of-flight-secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS), contact angle (CA), and atomic force microscopy (AFM). Introducing the PEG linker between the peptide and surface increased their resistance toward nonspecific protein adsorption. The peptide surfaces were examined as analytical platforms to study the action of trypsin as a representative protease. The products of the enzymatic hydrolysis were analyzed by fluorescence spectroscopy, electrospray ionization-mass spectrometry (ESI-MS), and ToF-SIMS. Conclusions about the optimal length of the PEG linker for the analytical application of PEG-peptide surfaces were drawn. This work demonstrates an effective synthetic procedure to obtain PEG-peptide surfaces as attractive platforms for the development of peptide microarrays.

ToF-SIMS spectrometry to observe fatty acid profiles of breast tissues in broiler chicken subjected to varied vegetable oil diet.

This study is aimed to observe changes in fatty acid profiles by time of flight secondary ion mass spectrometry (ToF-SIMS) in breast muscle tissues of broilers. Four different groups were identified. The source of fat in group I was soy oil (rich in linoleic acid, ω-6), group II received linseed oil (ω-3), and the third group was fed a mixture of the two mentioned oils. Broilers in the control group were fed with beef tallow, used in mass commercial production. The results reveal that the use of vegetable oils in animal nutrition determines the lipid profile of fatty acids. ToF-SIMS measurements showed that the lipid profile of muscle fibers and intramuscular fat reflect the composition of fats used as feed additives. In both structures, the ratio of ω-6/ω-3 fatty acids, which is most favorable for human health, was found in the groups in which a mixture of vegetable oils and a supplement of linseed oil were used.

Study of cholesterol and vitamin E levels in broiler meat from different feeding regimens by TOF-SIMS.

The quality of chicken meat, which is one of the most widely consumed meats in the world, has been the subject of research and studies for many years. There are several ways to improve the quality of this type of meat, including changing the concentrations of individual molecular components. Such important components of meat are inter alia, cholesterol, vitamin E, and some fatty acids such as ω-3 and ω-6. Manipulation of ingredient levels may be achieved by enriching chicken feed with elements of different types such as vegetable oils, garlic, or selenium. Thus far, various biochemical and biophysical methods have been used to study quality of different meat types, especially broiler meat. Here, the authors demonstrate the use of high-resolution time-of-flight secondary ion mass spectrometry (TOF-SIMS) mass spectrometry to assess how variations in animal nutrition affect concentrations of specific lipids in the meat, such as cholesterol and vitamin E. In the presented experiment, there were four different dietary treatments. Feed for animals in the first group was supplemented with soy oil in 50%, the second group's feed was supplemented with linseed oil in 50%, a combination of these two oils in the proportion of 44%:56% was used for the third group, and in the reference group, animals were fed with beef tallow. From each group, four individuals were selected for further analysis. Positive and negative ion mass spectra were generated from the pectoralis superficialis muscle tissue of the left carcass side of each one animal. Using TOF-SIMS with a bismuth cluster ion source (Bi3 (+)), and based on characteristic peaks for cholesterol in the positive mode and vitamin E in the negative mode, the authors have illustrated the relationship of these lipids levels to the various feeding regimens. Simultaneously, the authors characterized the varying dependences on the concentrations of measured lipids in fat and muscle fibers. The cholesterol concentration in muscle fibers was the lowest in the group fed with soybean oil and the highest in reference group IV (tallow feed). In the fatty region, the highest level of cholesterol was found in the third group. The highest concentrations of vitamin E were found in the fibers of the first group and the fat region of the second group. The obtained results show that SIMS imaging is a useful approach for assessing changes in lipid concentrations in the meat tissue from animals on different diets and provides a foundation for future research.