Tissue imaging and serum lipidomic profiling for screening potential biomarkers of thyroid tumors by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry

Changes in serum lipidome and in tissue lipidome are associated with cancer. In this study, tissue mass spectrometry imaging (MSI) and serum lipid profiling by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) were performed to investigate significantly changed lipids in both tumor (malignant thyroid cancer (MTC) and benign thyroid tumor (BTT)) tissues and sera. Y-scatterplots of variable importance in the projection (VIP) values vs. fold change values indicate that change trends in the levels of ten lipids (i.e., phosphatidylcholine (PC)(34:1), PC(36:1), PC(38:6), phosphatidic acid (PA) (36:2), PA(36:3), PA(38:3), PA(38:4), PA(38:5), PA(40:5), and sphingomyelin (SM)(34:1)) in both tissues and sera from MTC patients, BTT patients, and normal individuals are significantly associated with these three types of pathophysiological status. In order to examine their diagnostic ability, 289 serum samples from 124 MTC patients, 43 BTT patients, and 122 normal controls were randomly divided into the training set and validation set. A biomarker of PC(34:1) exhibited excellent diagnostic ability to differentiate both MTC and BTT patients from normal individuals, with an area under the receiver operating characteristic (ROC) curve value of 0.984, a sensitivity of 96.4 %, and a specificity of 92.7 %. A panel which included PA(36:3) and SM(34:1) could distinguish between MTC and BTT, with an area under receiver operating characteristic curve (AUC) of 0.961, a sensitivity of 87.8 %, and a specificity of 92.9 %. It is worth noting that a panel consisting of PC(34:1), PA(36:3), and SM(34:1) could differentiate MTC patients from both BTT patients and normal individuals, with an AUC of 0.841, a sensitivity of 86.6 %, and a specificity of 75.5 %.

Analysis of the Lipidome of Xenografts Using MALDI-IMS and UHPLC-ESI-QTOF

Human tumor xenografts in immunodeficient mice are a very popular model to study the development of cancer and to test new drug candidates. Among the parameters analyzed are the variations in the lipid composition, as they are good indicators of changes in the cellular metabolism. Here, we present a study on the distribution of lipids in xenografts of NCI-H1975 human lung cancer cells, using MALDI imaging mass spectrometry and UHPLC-ESI-QTOF. The identification of lipids directly from the tissue by MALDI was aided by the comparison with identification using ESI ionization in lipid extracts from the same xenografts. Lipids belonging to PCs, PIs, SMs, DAG, TAG, PS, PA, and PG classes were identified and their distribution over the xenograft was determined. Three areas were identified in the xenograft, corresponding to cells in different metabolic stages and to a layer of adipose tissue that covers the xenograft.

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC–MS with in-source fragmentation

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography–mass spectrometry (LC–MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.

Quantitative bioimaging of trace elements in the human lens by LA-ICP-MS

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for the quantitative imaging of Fe, Cu and Zn in cryostat sections of human eye lenses and for depth profiling analysis in bovine lenses. To ensure a tight temperature control throughout the experiments, a new Peltier-cooled laser ablation cell was employed. For quantification purposes, matrix-matched laboratory standards were prepared from a pool of human lenses from eye donors and spiked with standard solutions containing different concentrations of natural abundance Fe, Cu and Zn. A normalisation strategy was also carried out to correct matrix effects, lack of tissue homogeneity and/or instrumental drifts using a thin gold film deposited on the sample surface. Quantitative images of cryo-sections of human eye lenses analysed by LA-ICP-MS revealed a homogeneous distribution of Fe, Cu and Zn in the nuclear region and a slight increase in Fe concentration in the outer cell layer (i.e. lens epithelium) at the anterior pole. These results were assessed also by isotope dilution mass spectrometry, and Fe, Cu and Zn concentrations determined by ID-ICP-MS in digested samples of lenses and lens capsules.

On-line product analysis of pine wood pyrolysis using synchrotron vacuum ultraviolet photoionization mass spectrometry

The pyrolysis process of pine wood, a promising biofuel feedstock, has been studied with tunable synchrotron vacuum ultraviolet photoionization mass spectrometry. The mass spectra at different photon energies and temperatures as well as time-dependent profiles of several selected species during pine wood pyrolysis process were measured. Based on the relative contents of three lignin subunits, the data indicate that pine wood is typical of softwood. As pyrolysis temperature increased from 300 to 700 °C, some more details of pyrolysis chemistry were observed, including the decrease of oxygen content in high molecular weight species, the observation of high molecular weight products from cellulose chain and lignin polymer, and potential pyrolysis mechanisms for some key species. The formation of polycyclic aromatic hydrocarbons (PAHs) was also observed, as well as three series of pyrolysis products derived from PAHs with mass difference of 14 amu. The time-dependent profiles show that the earliest products are formed from lignin, followed by hemicellulose products, and then species from cellulose.

Direct Analysis of Triacylglycerols from Crude Lipid Mixtures by Gold Nanoparticle-Assisted Laser Desorption/Ionization Mass Spectrometry

Triacylglycerols (TAGs), essential energy storage lipids, are easily detected by conventional MALDI MS when occurring on their own. However, their signals are easily overwhelmed by other lipids, mainly phosphatidylcholines (PCs) and, therefore, require purification. In order to profile TAGs from crude lipid mixtures without prefractionation, we investigated alternative matrixes that can suppress phospholipid ion signals and enhance cationization of TAGs. We found that an aqueous solution of citrate-capped gold nanoparticles (AuNPs) with a diameter of 12 nm is a superior matrix for the laser desorption/ionization mass spectrometry (LDI MS) of TAGs in crude lipid mixtures. The AuNP matrix effectively suppressed other lipid signals such as phospholipids and also provided 100 times lower detection limit for TAGs than 2,5-dihydroxybenzoic acid (DHB), the best conventional MALDI matrix for TAGs. The AuNP-assisted LDI MS enabled us to obtain detailed TAG profiles including minor species directly from crude beef lipid extracts without phospholipid interference. In addition, we could detect TAGs at a trace level from a total brain lipid extract.

Radical polymerization of methyl methacrylate with 2,2,3-triphenylpropanoic acid as an initiator

An unsymmetrical compound, 2,2,3-triphenylpropanoic acid (TPPA), was successfully prepared from phenyllithium, 1,1-diphenylethylene (DPE), gas carbon dioxide (CO₂), and aqueous standard solution of hydrochloric acid with LiCl deprivation. Characterization of the compound was performed by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The polymerization of methyl methacrylate (MMA) was performed in the presence of TPPA at 95 °C. The free radicals obtained were characterized by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). Gel permeation chromatography (GPC) traces of the average molecular weight of poly(MMA) (PMMA) showed a series of translations with increasing time. The average molecular weight of PMMA indicated narrow polydispersity, and an approximately linear relationship was found between ln ([M]₀/[M]) and polymerization time.

Mass spectrometric profiling of Bacillus cereus strains and quantitation of the emetic toxin cereulide by means of stable isotope dilution analysis and HEp-2 bioassay

A fast and robust high-throughput ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC–TOF MS) profiling method was developed and successfully applied to discriminate a total of 78 Bacillus cereus strains into no/low, medium and high producers of the emetic toxin cereulide. The data obtained by UPLC–TOF MS profiling were confirmed by absolute quantitation of cereulide in selected samples by means of high-performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) and stable isotope dilution assay (SIDA). Interestingly, the B. cereus strains isolated from four vomit samples and five faeces samples from patients showing symptoms of intoxication were among the group of medium or high producers. Comparison of HEp-2 bioassay data with those determined by means of mass spectrometry showed differences, most likely because the HEp-2 bioassay is based on the toxic action of cereulide towards mitochondria of eukaryotic cells rather than on a direct measurement of the toxin. In conclusion, the UPLC–electrospray ionization (ESI)–TOF MS and the HPLC–ESI–MS/MS–SIDA analyses seem to be promising tools for the robust high-throughput analysis of cereulide in B. cereus cultures, foods and other biological samples.

Qualitative Gas Chromatography–Mass Spectrometry Analyses Using Amines as Chemical Ionization Reagent Gases

Ammonia is a very useful chemical ionization (CI) reagent gas for the qualitative analyses of compounds by positive ion gas chromatography–mass spectrometry (GCMS). The gas is readily available, inexpensive, and leaves no carbon contamination in the MS source. Compounds of interest to our laboratory typically yield abundant protonated or ammoniated species, which are indicative of a compound’s molecular weight. Nevertheless, some labile compounds fragment extensively by substitution and elimination reactions and yield no molecular weight information. In these cases, a CI reagent gas mixture of methylamine in methane prepared dynamically was found to be very useful in obtaining molecular weight data. Likewise, deuterated ammonia and deuterated methylamine are useful CI reagent gases for determining the exchangeable protons in organic compounds. Deuterated methylamine CI reagent gas is conveniently prepared by dynamically mixing small amounts of methylamine with excess deuterated ammonia.

Metabolomic profiling of mice urine and serum associated with trans-trans 2, 4-decadienal induced lung lesions by liquid chromatography-mass spectrometry

Metabolomics has become an important tool in clinical research and the diagnosis of human disease. Intratracheal instillation of trans-trans 2,4-decadienal (tt-DDE), a major component in cooking oil fumes, has been demonstrated to cause lung lesions in mice at 8 weeks after treatment. The objective of this study was to identify any changes in metabolite profiles associated with the development of tt-DDE-induced lung lesions. Using a metabolomics strategy involving a liquid chromatography–mass spectrometry-based approach in conjunction with principal component analysis and confirmation by liquid chromatography triple quadrupole tandem mass spectrometry, we have demonstrated that the amino acid profiles of the urine and serum of tt-DDE-treated mice are changed. Ten amino acids were significantly reduced in serum of tt-DDE-treated mice at 8 weeks after treatment. Our results suggest that amino acid profiles may be useful as an early indicator of the presence of tt-DDE-induced lung lesions.

Advantages of Monodisperse and Chemically Robust “SpheriCal” Polyester Dendrimers as a “Universal” MS Calibrant

The utilization of dendrimer calibrants as an alternative to peptides and proteins for high mass calibration is explored. These synthetic macromolecules exhibited a number of attractive advantages, including exceptional shelf-lives, broad compatibility with a wide range of matrices and solvents, and evenly spaced calibration masses across the mass range examined, 700–30,000 u. The exceptional purity of these dendrimers and the technical simplicity of this calibration platform validate their broad relevance for high molecular weight mass spectrometry.

Progress in the determination of metalloids and non-metals by means of high-resolution continuum source atomic or molecular absorption spectrometry. A critical review

This work examines the new possibilities introduced with the arrival of commercially available high-resolution continuum source atomic absorption spectrometers for the determination of metalloids (B, Si, Ge, As, Se, Sb and Te) and non-metals (P, S, F, Cl, Br, I and N-based species), such as the improved potential to detect and correct for spectral overlaps and the strategies available to correct for matrix effects. In particular, and considering the increasing number of papers reporting on the use of molecular absorption spectrometry using graphite furnaces and flames as vaporizers, the work discusses in detail the advantages and limitations derived from the monitoring of molecular spectra from a practical point of view, in an attempt to guide future users of the technique.

Least-Squares Fitting of Time-Domain Signals for Fourier Transform Mass Spectrometry

To advance Fourier transform mass spectrometry (FTMS)-based molecular structure analysis, corresponding development of the FTMS signal processing methods and instrumentation is required. Here, we demonstrate utility of a least-squares fitting (LSF) method for analysis of FTMS time-domain (transient) signals. We evaluate the LSF method in the analysis of single- and multiple-component experimental and simulated ion cyclotron resonance (ICR) and Orbitrap FTMS transient signals. Overall, the LSF method allows one to estimate the analytical limits of the conventional instrumentation and signal processing methods in FTMS. Particularly, LSF provides accurate information on initial phases of sinusoidal components in a given transient. For instance, the phase distribution obtained for a statistical set of experimental transients reveals the effect of the first data-point problem in FT-ICR MS. Additionally, LSF might be useful to improve the implementation of the absorption-mode FT spectral representation for FTMS applications. Finally, LSF can find utility in characterization and development of filter-diagonalization method (FDM) MS.

Comparison and optimization of strategies for a more profound profiling of the sialylated N-glycoproteomics in human plasma using metal oxide enrichment

Glycosylation is an important posttranslational modification of proteins and plays a crucial role in both cellular functions and secretory pathways. Sialic acids (SAs), a family of nine-carbon-containing acidic monosaccharides, often terminate the glycan structures of cell surface molecules and secreted glycoproteins and perform an important role in many biological processes. Hence, a more profound profiling of the sialylated glycoproteomics may improve our knowledge of this modification and its effects on protein functions. Here, we systematically investigated different strategies to enrich the SA proteins in human plasma using a newly developed technology that utilizes titanium dioxide for sialylated N-glycoproteomics profiling by mass spectrometry. Our results showed that using a combination of a filter-aided sample preparation method, TiO₂ chromatography, multiple enzyme digestion, and two-dimensional reversed-phase peptide fractionation led to a more profound profiling of the SA proteome. In total, 982 glycosylation sites in 413 proteins were identified, among which 37.8 % were newly identified, to establish the largest database of sialic acid containing proteins from human plasma.

Model system for targeted drug release triggered by immune-specific signals

A new sense-and-act system was realized by integrating a biocatalytic/bioaffinity electrode responding to immune signals represented by an antibody and a polymer-modified electrode loaded with drug-mimicking species. The release of the drug-mimicking species was achieved specifically in response to a signal antibody, thus demonstrating for the first time an immune-induced drug-releasing process. The present approach promises new options for future applications in controlled drug release and personalized medicine.

SEC ICP MS and CZE ICP MS investigation of medium and high molecular weight complexes formed by cadmium ions with phytochelatins

Size-exclusion chromatography (SEC) and capillary zone electrophoresis (CZE) coupled with inductively coupled plasma mass spectrometry were applied to characterize low, medium, and high molecular weight cadmium complexes with glutathione and phytochelatins (PCs). The dominant stoichiometry of the complexes formed in vitro was established as 1:1 using electrospray ionization mass spectrometry. Calculated molecular masses of Cd₁L₁ complexes were used for calibration of the SEC and CZE methods. The results showed a lower (2 kDa) SEC column exclusion limit for cadmium complexes compared with free peptides (10 kDa), and most of the high molecular weight cadmium species were eluted in the void volume of the column. Moreover, the CZE method based on the semiempirical model of Offord to elucidate peptide migration allowed us to show a high propensity of Cd–PC complexes for polymorphism on complexation, which was also observed for extracts of Arabidopsis thaliana treated with cadmium. All the information presented is vital for understanding the mechanism of metal deactivation in plants.

Direct Electrospray Ionization Mass Spectrometric Profiling of Real-World Samples via a Solid Sampling Probe

This study presents a novel direct analysis strategy for rapid mass spectrometric profiling of biochemicals in real-world samples via a direct sampling probe (DSP) without sample pretreatments. Chemical modification is applied to a disposable stainless steel acupuncture needle to enhance its surface area and hydrophilicity. After insertion into real-world samples, biofluid can be attached on the DSP surface. With the presence of a high DC voltage and solvent vapor condensing on the tip of the DSP, analyte can be dissolved and electrosprayed. The simplicity in design, versatility in application aspects, and other advantages such as low cost and disposability make this new method a competitive tool for direct analysis of real-world samples.

Characterization of the alkaloids in goldenseal (Hydrastis canadensis) root by high resolution Orbitrap LC-MSn

Liquid chromatography coupled to multistage mass spectrometry (LC-MSⁿ) is being used increasingly in pharmaceutical research and for quality control in herbal medicines because of its superior sensitivity and selectivity. In this study, a rapid, high-resolution liquid chromatography-mass spectrometry (LC-MSⁿ) method was developed to separate and identify alkaloids in the root extract of goldenseal, which is one of the 20 most popular herbal supplements used worldwide. In total, 28 alkaloids were separated and characterized including one novel compound and 21 identified, or tentatively identified, for the first time in goldenseal. The current high-resolution LC-MSⁿ method provides a rapid and definitive means of profiling the composition of goldenseal root and will provide a useful tool in understanding the bioactivity of this medicinal plant.

Improved MALDI-TOF Microbial Mass Spectrometry Imaging by Application of a Dispersed Solid Matrix

The key step in high quality microbial matrix-assisted laser desorption/ionization mass spectrometry imaging (microbial MALDI MSI) is the fabrication of a homogeneous matrix coating showing a fine-grained morphology. This application note addresses a novel method to apply solid MALDI matrices onto microbial cultures grown on thin agar media. A suspension of a mixture of 2,5-DHB and α-CHCA is sprayed onto the agar sample surface to form highly homogeneous matrix coatings. As a result, the signal intensities of metabolites secreted by the fungus Aspergillus fumigatus were found to be clearly enhanced.

Aptamer-based molecular recognition for biosensor development

Nucleic acid aptamers are an emerging class of synthetic ligands and have recently attracted significant attention in numerous fields. One is in biosensor development. In principle, nucleic acid aptamers can be discovered to recognize any molecule of interest with high affinity and specificity. In addition, unlike most ligands evolved in nature, synthetic nucleic acid aptamers are usually tolerant of harsh chemical, physical, and biological conditions. These distinguished characteristics make aptamers attractive molecular recognition ligands for biosensing applications. This review first concisely introduces methods for aptamer discovery including upstream selection and downstream truncation, then discusses aptamer-based biosensor development from the viewpoint of signal production.