Enzymatic Phosphorylation of Oxidized Tyrosine Residues.

Post-translational modifications (PTMs) alter the function and fate of proteins and cells in almost every conceivable way. Protein modifications can occur as a result of specific regulating actions of enzymes, such as tyrosine kinases phosphorylating tyrosine residues or by nonenzymatic reactions, such as oxidation related to oxidative stress and diseases. While many studies have addressed the multisite, dynamic, and network-like properties of PTMs, only little is known of the interplay of the same site modifications. In this work, we studied the enzymatic phosphorylation of oxidized tyrosine (l-DOPA) residues using synthetic insulin receptor peptides, in which tyrosine residues were replaced with l-DOPA. The phosphorylated peptides were identified by liquid chromatography-high-resolution mass spectrometry and the site of phosphorylation by tandem mass spectrometry. The results clearly show that the oxidized tyrosine residues are phosphorylated, displaying a specific immonium ion peak in the MS2 spectra. Furthermore, we detected this modification in our reanalysis (MassIVE ID: MSV000090106) of published bottom-up phosphoproteomics data. The modification, where both oxidation and phosphorylation take place at the same amino acid, has not yet been published in PTM databases. Our data indicate that there can be multiple PTMs that do not exclude each other at the same modification site.

Mass Spectrometry Imaging of Arabidopsis thaliana Leaves at the Single-Cell Level by Infrared Laser Ablation Atmospheric Pressure Photoionization (LAAPPI).

In this study, we show that infrared laser ablation atmospheric pressure photoionization mass spectrometry (LAAPPI-MS) imaging with 70 µm lateral resolution allows for the analysis of Arabidopsis thaliana (A. thaliana) leaf substructures ranging from single-cell trichomes and the interveinal leaf lamina to primary, secondary, and tertiary veins. The method also showed its potential for depth profiling analysis for the first time by mapping analytes at the different depths of the leaf and spatially resolving the topmost trichomes and cuticular wax layer from the underlying tissues. Negative ion LAAPPI-MS detected many different flavonol glycosides, fatty acids, fatty acid esters, galactolipids, and glycosphingolipids, whose distributions varied significantly between the different substructures of A. thaliana leaves. The results show that LAAPPI-MS provides a highly promising new tool to study the role of metabolites in plants.

Multiplexed analysis of amino acids in mice brain microdialysis samples using isobaric labeling and liquid chromatography-high resolution tandem mass spectrometry.

We developed a new multiplexed reversed phase liquid chromatography-high resolution tandem mass spectrometric (LC-MS/MS) method. The method is based on isobaric labeling with a tandem mass tag (TMT10-plex) and stable isotope-labeled internal standards, and was used to analyze amino acids in mouse brain microdialysis samples. The TMT10-plex labeling of amino acids allowed analysis of ten samples in one LC-MS/MS run, significantly increasing the sample throughput. The method provides good chromatographic performance (peak half-width between 0.04-0.12 min), allowing separation of all TMT-labeled amino acids with acceptable resolution and high sensitivity (limits of detection typically around 10 nM). The use of stable isotope-labeled internal standards, together with TMT10-plex labeling, ensured good repeatability (relative standard deviation ≤ 12.1 %) and linearity (correlation coefficient > 0.994), indicating good quantitative performance of the multiplexed method. The method was applied to study the effect of d-amphetamine microdialysis perfusion on amino acid concentrations in the mouse brain. All amino acids were reliably detected and quantified, indicating that the method is sensitive enough to detect low concentrations of amino acids in brain microdialysis samples.

Soft X-ray Atmospheric Pressure Photoionization in Liquid Chromatography-Mass Spectrometry.

Soft X-ray atmospheric pressure photoionization (soft X-ray APPI) as an ionization method in liquid chromatography-mass spectrometry (LC-MS) is presented. The ionization mechanism was examined with selected test compounds in the negative ion mode, using soft X-ray APPI source emitting 4.9 keV photons. Test compounds with an acidic group were ionized by a proton transfer reaction, producing deprotonated molecules ([M - H]-), whereas compounds having positive electron affinity were ionized by a charge exchange reaction, producing negative molecular ions (M-•). Soft X-ray APPI does not require a dopant to achieve high ionization efficiency, which is an advantage compared with vacuum ultraviolet APPI with 10 eV photons, in which a dopant is needed to improve ionization efficiency. The energy of the soft X-ray photons is in the keV range, which is high enough to displace a valence electron and often also inner shell electrons from LC eluents and atmospheric gases, initiating an efficient ionization process in the negative ion mode.

Sub-100 µm Spatial Resolution Ambient Mass Spectrometry Imaging of Rodent Brain with Laser Ablation Atmospheric Pressure Photoionization (LAAPPI) and Laser Ablation Electrospray Ionization (LAESI).

In this study, we applied a new IR laser-beam-focusing technique to enable sub-100 µm spatial resolution in laser ablation atmospheric pressure photoionization (LAAPPI) and laser ablation electrospray ionization (LAESI) mass spectrometry imaging (MSI). After optimization of operational parameters, both LAAPPI- and LAESI-MSI with a spatial resolution of 70 µm produced high-quality MS images, which allowed accurate localization of metabolites and lipids in the mouse and rat brain. Negative and positive ion LAAPPI- and LAESI-MS detected many of the same metabolites and lipids in the brain. Many compounds were also detected either by LAAPPI- or LAESI-MS, indicating that LAAPPI and LAESI are more complementary than alternative methods.

Molecular profile of the rat peri-infarct region four days after stroke: Study with MANF.

The peri-infarct region after ischemic stroke is the anatomical location for many of the endogenous recovery processes; however, -the molecular events in the peri-infarct region remain poorly characterized. In this study, we examine the molecular profile of the peri-infarct region on post-stroke day four, a time when reparative processes are ongoing. We used a multiomics approach, involving RNA sequencing, and mass spectrometry-based proteomics and metabolomics to characterize molecular changes in the peri-infarct region. We also took advantage of our previously developed method to express transgenes in the peri-infarct region where self-complementary adeno-associated virus (AAV) vectors were injected into the brain parenchyma on post-stroke day 2. We have previously used this method to show that mesencephalic astrocyte-derived neurotrophic factor (MANF) enhances functional recovery from stroke and recruits phagocytic cells to the peri-infarct region. Here, we first analyzed the effects of stroke to the peri-infarct region on post-stroke day 4 in comparison to sham-operated animals, finding that strokeinduced changes in 3345 transcripts, 341 proteins, and 88 metabolites. We found that after stroke, genes related to inflammation, proliferation, apoptosis, and regeneration were upregulated, whereas genes encoding neuroactive ligand receptors and calcium-binding proteins were downregulated. In proteomics, we detected upregulation of proteins related to protein synthesis and downregulation of neuronal proteins. Metabolomic studies indicated that in after stroke tissue there is an increase in saccharides, sugar phosphates, ceramides and free fatty acids and a decrease of adenine, hypoxantine, adenosine and guanosine. We then compared the effects of post-stroke delivery of AAV1-MANF to AAV1-eGFP (enhanced green fluorescent protein). MANF administration increased the expression of 77 genes, most of which were related to immune response. In proteomics, MANF administration reduced S100A8 and S100A9 protein levels. In metabolomics, no significant differences between MANF and eGFP treatment were detected, but relative to sham surgery group, most of the changes in lipids were significant in the AAV-eGFP group only. This work describes the molecular profile of the peri-infarct region during recovery from ischemic stroke, and establishes a resource for further stroke studies. These results provide further support for parenchymal MANF as a modulator of phagocytic function.

Targeted Clinical Metabolite Profiling Platform for the Stratification of Diabetic Patients.

Several small molecule biomarkers have been reported in the literature for prediction and diagnosis of (pre)diabetes, its co-morbidities, and complications. Here, we report the development and validation of a novel, quantitative method for the determination of a selected panel of 34 metabolite biomarkers from human plasma. We selected a panel of metabolites indicative of various clinically-relevant pathogenic stages of diabetes. We combined these candidate biomarkers into a single ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method and optimized it, prioritizing simplicity of sample preparation and time needed for analysis, enabling high-throughput analysis in clinical laboratory settings. We validated the method in terms of limits of detection (LOD) and quantitation (LOQ), linearity (R2), and intra- and inter-day repeatability of each metabolite. The method's performance was demonstrated in the analysis of selected samples from a diabetes cohort study. Metabolite levels were associated with clinical measurements and kidney complications in type 1 diabetes (T1D) patients. Specifically, both amino acids and amino acid-related analytes, as well as specific bile acids, were associated with macro-albuminuria. Additionally, specific bile acids were associated with glycemic control, anti-hypertensive medication, statin medication, and clinical lipid measurements. The developed analytical method is suitable for robust determination of selected plasma metabolites in the diabetes clinic.

Comparison of liquid chromatography-mass spectrometry and direct infusion microchip electrospray ionization mass spectrometry in global metabolomics of cell samples.

In this study, the feasibility of direct infusion electrospray ionization microchip mass spectrometry (chip-MS) was compared to the commonly used liquid chromatography-mass spectrometry (LC-MS) in non-targeted metabolomics analysis of human foreskin fibroblasts (HFF) and human induced pluripotent stem cells (hiPSC) reprogrammed from HFF. The total number of the detected features with chip-MS and LC-MS were 619 and 1959, respectively. Approximately 25% of detected features showed statistically significant changes between the cell lines with both analytical methods. The results show that chip-MS is a rapid and simple method that allows high sample throughput from small sample volumes and can detect the main metabolites and classify cells based on their metabolic profiles. However, the selectivity of chip-MS is limited compared to LC-MS and chip-MS may suffer from ion suppression.

Analysis of steroids in urine by gas chromatography-capillary photoionization-tandem mass spectrometry.

A new heated capillary photoionization (CPI) ion source design was developed to photoionize analytes inside a transfer capillary between a gas chromatograph (GC) and a mass spectrometer (MS). The CPI setup included a wide, oval-shaped vacuum-ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to maximize photoionization efficiency and thus sensitivity. The source contained a nitrogen housing around the ionization chamber inlet to avoid undesirable hydrolysis and oxidation reactions with ambient air and to maximize the proportion of formed molecular radical cations of analytes. The feasibility of the ion source was studied by analyzing 18 endogenous steroids in urine as their trimethylsilyl (TMS) derivatives with gas chromatography-tandem mass spectrometry (GC-MS/MS). The method was validated and applied to human urine samples. To our best knowledge, this is the first time that a capillary photoionization ion source has been applied for quantitative analysis of biological samples. The GC-CPI-MS/MS method showed good chromatographic resolution (peak half-widths between 3.1 to 5.3 s), acceptable linearity (coefficient of determination between 0.981 to 0.996), and repeatability (relative standard deviation (RSD%) between 5 to 18%). Limits of detection (LOD) were between 2 to 100 pg mL-1 and limits of quantitation (LOQ) were between 0.05 to 2 ng mL-1. In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the urine of volunteers. The new CPI source design showed excellent sensitivity for analysis of steroids in complex biological samples.

Rapid analysis of intraperitoneally administered morphine in mouse plasma and brain by microchip electrophoresis-electrochemical detection.

Animal studies remain an essential part of drug discovery since in vitro models are not capable of describing the complete living organism. We developed and qualified a microchip electrophoresis-electrochemical detection (MCE-EC) method for rapid analysis of morphine in mouse plasma using a commercial MCE-EC device. Following liquid-liquid extraction (LLE), we achieved within-run precision of 3.7 and 4.5% (coefficient of variation, CV, n = 6) and accuracy of 106.9% and 100.7% at biologically relevant morphine concentrations of 5 and 20 µM in plasma, respectively. The same method was further challenged by morphine detection in mouse brain homogenates with equally good within-run precision (7.8% CV, n = 5) at 1 µM concentration. The qualified method was applied to analyze a set of plasma and brain homogenate samples derived from a behavioral animal study. After intraperitoneal administration of 20 mg/kg morphine hydrochloride, the detected morphine concentrations in plasma were between 6.7 and 17 µM. As expected, the morphine concentrations in the brain were significantly lower, ca. 80-125 nM (280-410 pg morphine/mg dissected brain), and could only be detected after preconcentration achieved during LLE. In all, the microchip-based separation system is proven feasible for rapid analysis of morphine to provide supplementary chemical information to behavioral animal studies.

Capillary photoionization: interface for low flow rate liquid chromatography-mass spectrometry.

This is the first report on capillary photoionization (CPI) interfacing a liquid chromatograph (LC) and mass spectrometer (MS). A new heated CPI ion source was developed, including a heated transfer capillary, a wide oval-shaped and low-depth ionization chamber with a vacuum ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to increase the photoionization efficiency and thus the sensitivity. As both analytes and eluent are first vaporized and then photoionized inside the CPI ion source between the atmosphere and the vacuum of MS, the ion transfer efficiency into the MS and thus the sensitivity is improved. The effect of the most important operation parameters, the eluent flow rate and temperature of the CPI source, on the signal intensity was studied with selected steroids. The feasibility of LC-CPI-MS/MS for the quantitative analysis of steroids was also studied in terms of linearity, repeatability, and limits of detection. The method showed good quantitative performance and sensitivity down to the low femto-mole level.

TiO2 Photocatalyzed Oxidation of Drugs Studied by Laser Ablation Electrospray Ionization Mass Spectrometry.

In drug discovery, it is important to identify phase I metabolic modifications as early as possible to screen for inactivation of drugs and/or activation of prodrugs. As the major class of reactions in phase I metabolism is oxidation reactions, oxidation of drugs with TiO2 photocatalysis can be used as a simple non-biological method to initially eliminate (pro)drug candidates with an undesired phase I oxidation metabolism. Analysis of reaction products is commonly achieved with mass spectrometry coupled to chromatography. However, sample throughput can be substantially increased by eliminating pretreatment steps and exploiting the potential of ambient ionization mass spectrometry (MS). Furthermore, online monitoring of reactions in a time-resolved way would identify sequential modification steps. Here, we introduce a novel (time-resolved) TiO2-photocatalysis laser ablation electrospray ionization (LAESI) MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO2-coated glass slides as well as solutions containing suspended TiO2 nanoparticles, and the results were in excellent agreement with studies on biological oxidation of verapamil, buspirone, testosterone, andarine, and ostarine. Finally, a time-resolved LAESI MS setup was developed and initial results for verapamil showed excellent analytical stability for online photocatalyzed oxidation reactions within the set-up up to at least 1 h. Graphical Abstract.

Molecular Atlas of Postnatal Mouse Heart Development.

Background The molecular mechanisms mediating postnatal loss of cardiac regeneration in mammals are not fully understood. We aimed to provide an integrated resource of mRNA , protein, and metabolite changes in the neonatal heart for identification of metabolism-related mechanisms associated with cardiac regeneration. Methods and Results Mouse ventricular tissue samples taken on postnatal day 1 (P01), P04, P09, and P23 were analyzed with RNA sequencing and global proteomics and metabolomics. Gene ontology analysis, KEGG pathway analysis, and fuzzy c-means clustering were used to identify up- or downregulated biological processes and metabolic pathways on all 3 levels, and Ingenuity pathway analysis (Qiagen) was used to identify upstream regulators. Differential expression was observed for 8547 mRNA s and for 1199 of 2285 quantified proteins. Furthermore, 151 metabolites with significant changes were identified. Differentially regulated metabolic pathways include branched chain amino acid degradation (upregulated at P23), fatty acid metabolism (upregulated at P04 and P09; downregulated at P23) as well as the HMGCS ( HMG -CoA [hydroxymethylglutaryl-coenzyme A] synthase)-mediated mevalonate pathway and ketogenesis (transiently activated). Pharmacological inhibition of HMGCS in primary neonatal cardiomyocytes reduced the percentage of BrdU-positive cardiomyocytes, providing evidence that the mevalonate and ketogenesis routes may participate in regulating the cardiomyocyte cell cycle. Conclusions This study is the first systems-level resource combining data from genomewide transcriptomics with global quantitative proteomics and untargeted metabolomics analyses in the mouse heart throughout the early postnatal period. These integrated data of molecular changes associated with the loss of cardiac regeneration may open up new possibilities for the development of regenerative therapies.

Impact of Pore Size and Surface Chemistry of Porous Silicon Particles and Structure of Phospholipids on Their Interactions.

By exploiting its porous structure and high loading capacity, porous silicon (PSi) is a promising biomaterial to fabricate protocells and biomimetic reactors. Here, we have evaluated the impact of physicochemical properties of PSi particles [thermally oxidized PSi, TOPSi; annealed TOPSi, AnnTOPSi; (3-aminopropyl) triethoxysilane functionalized thermally carbonized PSi, APTES-TCPSi; and thermally hydrocarbonized PSi, THCPSi] on their surface interactions with different phospholipids. All of the four phospholipids were similarly adsorbed by the surface of PSi particles, except for TOPSi. Among four PSi particles, TOPSi with hydrophilic surface and smaller pore size showed the weakest adsorption toward phosphatidylcholines. By increasing the pore size from roughly 12.5 to 18.0 nm (TOPSi vs AnnTOPSi), the quantity of phosphatidylcholines adsorbed by TOPSi was enhanced to the same level of hydrophilic APTES-TCPSi and hydrophobic THCPSi. The 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibited the highest release ratio of phospholipids from all four PSi particles, and phosphatidylserine (DPPS) showed the lowest release ratio of phospholipids from PSi particles, except for TOPSi, which adsorbed less phospholipids due to the small pore size. There is consistency in the release extent of phospholipids from PSi particles and the isosteric heat of adsorption. Overall, our study demonstrates the importance of pore size and surface chemistry of PSi particles as well as the structure of phospholipids on their interactions. The obtained information can be employed to guide the selection of PSi particles and phospholipids to fabricate highly ordered structures, for example, protocells, or biomimetic reactors.

TiO2 Photocatalysis-DESI-MS Rotating Array Platform for High-Throughput Investigation of Oxidation Reactions.

We present a new high-throughput platform for studying titanium dioxide (TiO2) photocatalytic oxidation reactions by performing reactions on a TiO2-coated surface, followed by direct analysis of oxidation products from the surface by desorption electrospray ionization mass spectrometry (DESI-MS). For this purpose, we coated a round glass wafer with photocatalytically active anatase-phase TiO2 using atomic layer deposition. Approximately 70 aqueous 1 µL samples can be injected onto the rim of the TiO2-coated glass wafer, before the entire wafer is exposed to UV irradiation. After evaporation of water, the oxidation products can be directly analyzed from the sample spots by DESI-MS, using a commercial rotating sample platform. The method was shown to provide fast photocatalytic oxidation reactions and analysis with throughput of about four samples per minute. The feasibility of the method was examined for mimicking phase I metabolism reactions of amodiaquine, buspirone and verapamil. Their main photocatalytic reaction products were mostly similar to the products observed earlier in TiO2 photocatalysis and in in vitro phase I metabolism assays performed using human liver microsomes.

Feasibility of ultra-performance liquid chromatography-ion mobility-time-of-flight mass spectrometry in analyzing oxysterols.

Oxysterols are oxygenated cholesterols that are important in many cell functions and they may also be indicative of certain diseases. The purpose of this work was to study the feasibility of ultra-performance liquid chromatography-ion mobility-time-of-flight mass spectrometry (UPLC-IM-TOFMS) using traveling wave cell in analyzing oxysterols and especially their isomers in biological samples. Oxysterols were analyzed as their p-toluenesulfonyl isocyanate derivatives, which improved the separation of isomeric oxysterols by ion mobility and ionization efficiency in the electrospray ionization step. The UPLC-IM-TOFMS method was shown to be fast and to provide good quantitative performance. The feasibility of the method was demonstrated in the analyses of oxysterols in fibroblast cell samples.

Oxidation of Tyrosine-Phosphopeptides by Titanium Dioxide Photocatalysis.

Protein phosphorylation has a key role in cell regulation. Oxidation of proteins, in turn, is related to many diseases and to aging, but the effects of phosphorylation on the oxidation of proteins and peptides have been rarely studied. The aim of this study was to examine the mechanistic effect of phosphorylation on peptide oxidation induced by titanium dioxide photocatalysis. The effect of phosphorylation was compared between nonphosphorylated and tyrosine phosphorylated peptides using electrospray tandem mass spectrometry. We observed that tyrosine was the most preferentially oxidized amino acid, but the oxidation reaction was significantly inhibited by its phosphorylation. The study also shows that titanium dioxide photocatalysis provides a fast and easy method to study oxidation reactions of biomolecules, such as peptides.

Charge Exchange Reaction in Dopant-Assisted Atmospheric Pressure Chemical Ionization and Atmospheric Pressure Photoionization.

The efficiencies of charge exchange reaction in dopant-assisted atmospheric pressure chemical ionization (DA-APCI) and dopant-assisted atmospheric pressure photoionization (DA-APPI) mass spectrometry (MS) were compared by flow injection analysis. Fourteen individual compounds and a commercial mixture of 16 polycyclic aromatic hydrocarbons were chosen as model analytes to cover a wide range of polarities, gas-phase ionization energies, and proton affinities. Chlorobenzene was used as the dopant, and methanol/water (80/20) as the solvent. In both techniques, analytes formed the same ions (radical cations, protonated molecules, and/or fragments). However, in DA-APCI, the relative efficiency of charge exchange versus proton transfer was lower than in DA-APPI. This is suggested to be because in DA-APCI both dopant and solvent clusters can be ionized, and the formed reagent ions can react with the analytes via competing charge exchange and proton transfer reactions. In DA-APPI, on the other hand, the main reagents are dopant-derived radical cations, which favor ionization of analytes via charge exchange. The efficiency of charge exchange in both DA-APPI and DA-APCI was shown to depend heavily on the solvent flow rate, with best efficiency seen at lowest flow rates studied (0.05 and 0.1 mL/min). Both DA-APCI and DA-APPI showed the radical cation of chlorobenzene at 0.05-0.1 mL/min flow rate, but at increasing flow rate, the abundance of chlorobenzene M(+.) decreased and reagent ion populations deriving from different gas-phase chemistry were recorded. The formation of these reagent ions explains the decreasing ionization efficiency and the differences in charge exchange between the techniques. Graphical Abstract ᅟ.

Rapid separation of phosphopeptides by microchip electrophoresis-electrospray ionization mass spectrometry.

Protein phosphorylation is a significant biological process, but separation of phosphorylated peptide isomers is often challenging for many analytical techniques. We developed a microchip electrophoresis (MCE) method for rapid separation of phosphopeptides with on-chip electrospray ionization (ESI) facilitating online sample introduction to the mass spectrometer (MS). With the method, two monophosphorylated positional isomers of insulin receptor peptide (IR1A and IR1B) and a triply phosphorylated insulin receptor peptide (IR3), all with the same amino acid sequence, were separated from the nonphosphorylated peptide (IR0) in less than one minute. For efficient separation of the positional peptide isomers from each other derivatization with 9-fluorenylmethyl reagents (either chloroformate, Fmoc-Cl, or N-succinimidyl carbonate, Fmoc-OSu) was required before the analysis. The derivatization improved not only the separation of the monophosphorylated positional peptide isomers in MCE, but also identification of the phosphorylation site based on MS/MS.