Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.     

Mass spectral characterization of doxylamine and its rhesus monkey urinary metabolites

This study describes the use of mass spectrometry (MS), high-performance liquid chromatography (HPLC) and chemical derivatization techniques for the identification of doxylamine and five rhesus monkey urinary metabolites. The analyses were performed using chemical ionization mass spectrometry with either methane or ammonia as the reagent gas. The confirmation of the structures of two of these urinary metabolites was aided by the synthesis of doxylamine N-oxide and desmethyldoxylamine and by the use of methylation and acetylation derivatization techniques. Doxylamine N-oxide, desmethyldoxylamine, didesmethyldoxylamine, and two metabolites which resulted from the cleavage of the aliphatic tertiary nitrogen side chain to the subsequent 2-[1-phenyl-1-(2-pyridinyl)ethoxy]acetic acid or 2-[1-phenyl-1-(2-pyridinyl)ethoxy]methanol compounds were isolated and identified from rhesus monkey urine. Additional data concerning the mass spectral analysis of derivatization or reaction products from the three chloroformate reactions with doxylamine, and the synthesis and separation techniques which afforded mass spectral identification of the urinary metabolites are also presented.     

The imaging and quantification of aluminium in the human brain using dynamic secondary ion mass spectrometry (SIMS).

Dynamic secondary ion mass spectrometry (SIMS) has been utilised to study the post-mortem distribution of aluminium in air-dried frozen sections from unfixed, unstained human brain in order to minimise contamination of the tissue and avoid redistribution and extraction of endogenous tissue aluminium. Substrates, sputter-coated with silver, were found to be free of focal aluminum surface contamination and thus minimised substrate induced artefacts in the tissue aluminium ion image. SIMS imaging of aluminium secondary ions at a mass resolution that eliminated the major molecular interferences, combined with a photomontage technique provided a unique strategy for studying aluminium distribution in tissue unrivalled by other spatially resolved microanalytical techniques such as laser microprobe mass spectrometry or X-ray microanalysis. Using this strategy, high densities of focal aluminium accumulations have been demonstrated in the cerebral cortex of the majority of chronic renal dialysis patients studied. In contrast, such aluminium accumulations were absent in control patients. SIMS imaging of aluminium appeared to provide much better discrimination between the dialysis patient group and the control group than one of the most widely used techniques for measuring aluminium in bulk samples, graphite furnace atomic absorption spectrometry. Preliminary studies have shown the feasibility of quantifying focal aluminium SIMS images obtained from brain tissue using aluminium-loaded brain homogenates as reference standards.     

A highly specific and sensitive determination of gamma-aminobutyric acid by gas chromatography mass spectrometry

A procedure for the identification and quantification of picomole quantities of gamma-aminobutyric acid in tissue samples is given. This procedure combines the chemical specificity of dinitrophenylation with that of gas chromatography mass spectrometry to eliminate the interferences encountered with other direct derivatization procedures. Only a limited number of dinitrophenyl amino acid ethyl esters and some fatty ethyl esters are detected in the solution used for analysis. Identification is based on retention time and on the relative abundances of the three major ion fragments of the gamma-aminobutyric acid derivative. Quantitation is accomplished using isotope dilution techniques with [2H2]gamma-aminobutyric acid as an internal standard. The procedure has been successfully applied to samples of human cerebrospinal fluid and to extracts of ganglia from the mollusc, Aplysia californica.     

Analysis of low molecular weight compounds by MALDI-FTICR-MS

This review focuses on recent applications of matrix-assisted laser desorption ionization-Fourier-transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) in qualitative and quantitative analysis of low molecular weight compounds. The scope of the work includes amino acids, small peptides, mono and oligosaccharides, lipids, metabolic compounds, small molecule phytochemicals from medicinal herbs and even the volatile organic compounds from tobacco. We discuss both direct analysis and analysis following derivatization. In addition we review sample preparation strategies to reduce interferences in the low m/z range and to improve sensitivities by derivatization with charge tags. We also present coupling of head space techniques with MALDI-FTICR-MS. Furthermore, omics analyses based on MALDI-FTICR-MS were also discussed, including proteomics, metabolomics and lipidomics, as well as the relative MS imaging for bio-active low molecular weight compounds. Finally, we discussed the investigations on dissociation/rearrangement processes of low molecular weight compounds by MALDI-FTICR-MS.     

One-pot microwave derivatization of target compounds relevant to metabolomics with comprehensive two-dimensional gas chromatography

Metabolomics has been defined as the quantitative measurement of all low molecular weight metabolites (sugars, amino acids, organic acids, fatty acids and others) in an organism's cells at a specified time under specific environmental/biological conditions. Currently, there is considerable interest in developing a single method of derivatization and separation that satisfies the needs for metabolite analysis while recognizing the many chemical classes that constitute the metabolome. Chemical derivatization considerably increases the sensitivity and specificity of gas chromatography–mass spectrometry for compounds that are polar and have derivatizable groups. Microwave-assisted derivatization (MAD) of a set of standards spanning a wide range of metabolites of interest demonstrates the potential of MAD for metabolic profiling. A final protocol of 150 W power for 90 s was selected as the derivatization condition, based upon the study of each chemical class. A study of the generation of partially derivatized components established the conditions where this could potentially be a problem; the use of greater volumes of reagent ensured this would not arise. All compounds analyzed by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry in a standard mixture showed good area ratio reproducibility against a naphthalene internal standard (RSD < 10% in all but one case). Concentrations tested ranged from 1 μg/mL to 1000 μg/mL, and the calibration curves for the standard mixtures were satisfactory with regression coefficients generally better than 0.998. The application to gas chromatography–quadrupole mass spectrometry and comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry for a typical reference standard of relevance to metabolomics is demonstrated.     

In Situ Mass Spectrometry Imaging and Ex Vivo Characterization of Renal Crystalline Deposits Induced in Multiple Preclinical Drug Toxicology Studies

Drug toxicity observed in animal studies during drug development accounts for the discontinuation of many drug candidates, with the kidney being a major site of tissue damage. Extensive investigations are often required to reveal the mechanisms underlying such toxicological events and in the case of crystalline deposits the chemical composition can be problematic to determine. In the present study, we have used mass spectrometry imaging combined with a set of advanced analytical techniques to characterize such crystalline deposits in situ. Two potential microsomal prostaglandin E synthase 1 inhibitors, with similar chemical structure, were administered to rats over a seven day period. This resulted in kidney damage with marked tubular degeneration/regeneration and crystal deposits within the tissue that was detected by histopathology. Results from direct tissue section analysis by matrix-assisted laser desorption ionization mass spectrometry imaging were combined with data obtained following manual crystal dissection analyzed by liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy. The chemical composition of the crystal deposits was successfully identified as a common metabolite, bisulphonamide, of the two drug candidates. In addition, an un-targeted analysis revealed molecular changes in the kidney that were specifically associated with the area of the tissue defined as pathologically damaged. In the presented study, we show the usefulness of combining mass spectrometry imaging with an array of powerful analytical tools to solve complex toxicological problems occurring during drug development.     

Application of mass spectrometry technologies for the discovery of low-molecular weight modulators of enzymes and protein-protein interactions

In recent years, mass spectrometry has gained widespread use as an assay and screening technology in drug discovery because it enables sensitive, label-free detection of low-molecular weight modulators of biomolecules as well as sensitive and accurate detection of high-molecular weight modifications of biomolecules. Electrospray and matrix-assisted laser desorption ionization are the most widely used ionization techniques to identify chemical compounds interfering with enzymatic function, receptor-ligand binding or molecules modulating a protein-protein interaction of interest. Mass spectrometry based techniques are no longer restricted to screening in biochemical assay systems but have now become also applicable to imaging of biomolecules and chemical compounds in cell-based assay systems and even in highly complex tissue sections.     

Microwave irradiation for a fast gas chromatography-mass spectrometric analysis of polysaccharide-based plasma volume expanders in human urine

In this contribution we tested the possibility to use microwave irradiation for the screening and confirmation pre-treatment steps of hydroxyethylstarch, with the aim to speed up gas chromatography-mass spectrometric procedures. Acid hydrolysis and derivatization processes were conducted in a temperature-controlled single beam microwave oven for organic synthesis. The kinetics of hydroxyethylstarch chemical hydrolysis and derivatization were investigated at different microwave power, incubation temperature and incubation time. The best hydrolysis conditions were found at a microwave power value of 1200 W (T 100°C) with an incubation time of 2 min; whereas the best derivatization conditions were found at a microwave power value of 1020 W (T 100°C) with an incubation time of 5 min. The effectiveness of this approach was evaluated by gas chromatography-mass spectrometry analyzing more than 20 different pools of blank urine samples spiked with hydroxyethylstarch at a concentration of 1 mg/mL. The results showed that the effect of microwave irradiation on the chemical hydrolysis process was very remarkable: the total sample preparation time can be shortened by 58 min compared to the reference method (2 min instead of 60 min). In addition to this, the time necessary for the derivatization process can also be drastically shortened with respect to the reference procedure (5 min instead of 30 min). The repeatability of the hydrolysis and derivatization recoveries, the limit of detection and the matrix interferences were comparable to the reference method accredited under the ISO 17025 guidelines and presently followed by the accredited sports anti-doping laboratory of Rome.     

Analysis of protein glycosylation by mass spectrometry

We present a detailed protocol for the structural analysis of protein-linked glycans. In this approach, appropriate for glycomics studies, N-linked glycans are released using peptide N-glycosidase F and O-linked glycans are released by reductive alkaline beta-elimination. Using strategies based on mass spectrometry (matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano-electrospray ionization mass spectrometry/mass spectrometry (nano-ESI-MS-MS)), chemical derivatization, sequential exoglycosidase digestions and linkage analysis, the structures of the N- and/or O-glycans are defined. This approach can be used to study the glycosylation of isolated complex glycoproteins or of numerous glycoproteins encountered in a complex biological medium (cells, tissues and physiological fluids).     

Capitalizing on the hydrophobic bias of electrospray ionization through chemical modification in mass spectrometry-based proteomics

Protein chemical derivatization has emerged as an invaluable bioanalytical approach in mass spectrometry-based proteomics with nearly unlimited potential. To date, derivatization strategies in proteomics have primarily focused on improving mass spectral identification and relative quantification of proteins, as well as increasing enrichment yield from complex mixtures. However, there is a great opportunity to develop and exploit front-end chemical processes to enhance the ability to detect low-abundant peptides and proteins for a large number of applications. The content of this article focuses on improvements in targeted, mass spectrometry-based proteomic strategies, achieved by taking advantage of the mechanism of ESI through the use of hydrophobic chemical derivatization.     

Chemical ionization mass spectrometry of trimethylsilylated carbohydrates and organic acids retained in uremic serum

After appropriate sample pretreatment and derivatization, uremic serum was investigated by combined high resolution gas chromatography and mass spectrometry, using both electron impact and chemical ionization methods. Electron impact and chemical ionization spectra of a number of identified (trimethylsilylated) carbohydrates and organic acids are compared. The utilization of chemical ionization mass spectrometry, with isobutane as the reagent gas, is discussed in detail. The influence of the reagent gas pressure on the total ion current and on the spectral appearance was studied. The identification of compounds, based on electron impact mass spectral data, was confirmed and often aided appreciably by using this technique. The chemical ionization spectra of trimethylsilyated alditols and aldonic acids, as well as of other organic acids showed protonated molecular ions, whereas aldoses did not. Differences with electron impact spectra are found mainly in the high mass region. The loss of one or more trimethylsilanol groups becomes the predominating fragmentation route at higher reagent gas pressures.     

Multiple statistical analysis techniques corroborate intratumor heterogeneity in imaging mass spectrometry datasets of myxofibrosarcoma

MALDI mass spectrometry can generate profiles that contain hundreds of biomolecular ions directly from tissue. Spatially-correlated analysis, MALDI imaging MS, can simultaneously reveal how each of these biomolecular ions varies in clinical tissue samples. The use of statistical data analysis tools to identify regions containing correlated mass spectrometry profiles is referred to as imaging MS-based molecular histology because of its ability to annotate tissues solely on the basis of the imaging MS data. Several reports have indicated that imaging MS-based molecular histology may be able to complement established histological and histochemical techniques by distinguishing between pathologies with overlapping/identical morphologies and revealing biomolecular intratumor heterogeneity. A data analysis pipeline that identifies regions of imaging MS datasets with correlated mass spectrometry profiles could lead to the development of novel methods for improved diagnosis (differentiating subgroups within distinct histological groups) and annotating the spatio-chemical makeup of tumors. Here it is demonstrated that highlighting the regions within imaging MS datasets whose mass spectrometry profiles were found to be correlated by five independent multivariate methods provides a consistently accurate summary of the spatio-chemical heterogeneity. The corroboration provided by using multiple multivariate methods, efficiently applied in an automated routine, provides assurance that the identified regions are indeed characterized by distinct mass spectrometry profiles, a crucial requirement for its development as a complementary histological tool. When simultaneously applied to imaging MS datasets from multiple patient samples of intermediate-grade myxofibrosarcoma, a heterogeneous soft tissue sarcoma, nodules with mass spectrometry profiles found to be distinct by five different multivariate methods were detected within morphologically identical regions of all patient tissue samples. To aid the further development of imaging MS based molecular histology as a complementary histological tool the Matlab code of the agreement analysis, instructions and a reduced dataset are included as supporting information.     

Recent methodological advances in the mass spectrometric analysis of free and protein-associated 3-nitrotyrosine in human plasma

L-Tyrosine and L-tyrosine residues in proteins are attacked by various reactive-nitrogen species (RNS) including peroxynitrite to form 3-nitrotyrosine (NO(2)Tyr) and protein-associated 3-nitrotyrosine (NO(2)TyrProt). Circulating NO(2)Tyr and NO(2)TyrProt have been suggested and are widely used as biomarkers of oxidative stress in humans. In this article the mass spectrometry (MS)-based analytical methods recently reported for the quantification of circulating levels of NO(2)Tyr and NO(2)TyrProt are discussed. These methodologies differ in sensitivity, selectivity, specificity and accessibility to interferences with the latter mainly arising from artifactual formation of NO(2)Tyr and NO(2)TyrProt during sample treatment such as acidification and chemical derivatization. Application of these methodologies to healthy normal humans revealed basal circulating levels for NO(2)Tyr which range between 0.7 and 64 nM, i.e. by two orders of magnitude. Application of gas chromatography-tandem mass spectrometry (GC-tandem MS) methods by two independent research groups by using two different protocols to avoid artifactual nitration of L-tyrosine revealed almost identical mean plasma levels of the order of 1.0 nM in healthy humans. The lower limits of quantitation (LOQ) of these methods were 0.125 and 0.3n M, respectively. This order of magnitude for basal NO(2)Tyr is supported by two liquid chromatography-tandem mass spectrometry (LC-tandem MS) methods with LOQ values of 4.4 and 1.4 nM. On the basis of the data provided by GC-tandem MS and LC-tandem MS the use of a range of 0.5-3 nM for NO(2)Tyr and of 0.6 pmol/mg plasma protein or a molar ratio of 3-nitrotyrosine to tyrosine in plasma proteins of the order of 1:10(6) for NO(2)TyrProt in plasma of healthy humans as reference values appear reasonably justified. Recently reported clinical studies involving 3-nitrotyrosine as a biomarker of oxidative stress are discussed in particular from the analytical point of view.     

Matrix-assisted laser desorption/ionization imaging mass spectrometry: a promising technique for reproductive research

Matrix-assisted laser desorption/ionization (MALDI) tissue imaging mass spectrometry is particularly promising among the numerous applications of mass spectrometry. It is used for probing and analyzing the spatial arrangement of a wide range of molecules, including proteins, peptides, lipids, drugs, and metabolites, directly in thin slices of tissue. In the field of proteomics, the technology avoids tedious and time-consuming extraction and fractionation steps classically required for sample analysis. MALDI imaging mass spectrometry is increasingly recognized as a powerful method for clinical proteomics, particularly in cancer research. The technology has particular potential for the discovery of new tissue biomarker candidates, classification of tumors, early diagnosis or prognosis, elucidating pathogenesis pathways, and therapy monitoring. Over recent years, MALDI imaging mass spectrometry has been used for molecular profiling and imaging directly in male and female reproductive tissues. This review will consider some of the recent publications in the field, addressing a range of issues covering embryo development, gene expression product profiling during gametogenesis, and seeking and identifying biomarkers of reproductive cancers. The wealth of advances in mass spectrometry imaging will inevitably attract biologists and clinicians as the advantages and power of this technology become more widely known. This review will also discuss bottlenecks and the many technical issues that remain to be resolved before laboratories in the field can adopt the technology. We foresee that MALDI imaging mass spectrometry will have a major impact in reproductive research by opening new avenues to the understanding of various molecular mechanisms and the diagnosis of reproductive pathologies.     

An improved chemical approach toward the C-terminal sequence analysis of proteins containing all natural amino acids.

An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N-acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H-NMR. This chemical method allows, for the first time, complete derivatization of N-acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C-terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C-terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C-terminal amino acids including proline is a major step forward in the development of a general chemical C-terminal sequencing method that permits the C-terminal sequence analysis of proteins of any amino acid composition.     

Pancreatic Cancer Surgical Resection Margins: Molecular Assessment by Mass Spectrometry Imaging

BackgroundSurgical resection with microscopically negative margins remains the main curative option for pancreatic cancer; however, in practice intraoperative delineation of resection margins is challenging. Ambient mass spectrometry imaging has emerged as a powerful technique for chemical imaging and real-time diagnosis of tissue samples. We applied an approach combining desorption electrospray ionization mass spectrometry imaging (DESI-MSI) with the least absolute shrinkage and selection operator (Lasso) statistical method to diagnose pancreatic tissue sections and prospectively evaluate surgical resection margins from pancreatic cancer surgery.ConclusionsOur findings provide evidence that the molecular information obtained by DESI-MSI/Lasso from pancreatic tissue samples has the potential to transform the evaluation of surgical specimens. With further development, we believe the described methodology could be routinely used for intraoperative surgical margin assessment of pancreatic cancer.     

Quantitative mass spectrometry imaging of propranolol and olanzapine using tissue extinction calculation as normalization factor

In order to quantify small molecules at the early stage of drug discovery, we developed a quantitation approach based on mass spectrometry imaging (MSI) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) without the use of a labeled compound. We describe a method intended to respond to the main challenges encountered in quantification through MALDI imaging dedicated to whole-body or single heterogeneous organ samples (brain, eye, liver). These include the high dependence of the detected signal on the matrix deposition, the MALDI ionization yield of specific target molecules, and lastly, the ion suppression effect on the tissue. To address these challenges, we based our approach on the use of a normalization factor called the TEC (Tissue Extinction Coefficient). This factor takes into account the ion suppression effect that is both tissue- and drug-specific. Through this protocol, the amount of drug per gram of tissue was determined, which in turn, was compared with other analytical techniques such as Liquid Chromatography-Mass spectrometry (LC-MS/MS).     

Mass spectrometry image correlation: quantifying colocalization

A typical imaging mass spectrometry data set can contain 100+ images, each describing the distribution of a specific biomolecule. Multivariate and hierarchical clustering techniques have been developed to investigate the correlations within a data set, and have revealed the differential patterns associated with different organs/anatomical features. These methods do not quantify the correlations between the hundreds of molecular distributions produced in an imaging mass spectrometry experiment, and are extremely difficult to apply to multiple tissue section investigations. This latter aspect includes quantifying the correlation between the results of repeat imaging mass spectrometry experiments, a crucial aspect for determining the significance of any measured changes in distribution. To date, the large chemical background and pixel-to-pixel variation in the images has limited the quantification of correlation between imaging mass spectrometry results. Here, we demonstrate how to quantify the correlations between imaging mass spectrometry images, both within a data set and between data sets.     

A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)

Stable isotope labeling by amino acids in cell culture (SILAC) is a simple, robust, yet powerful approach in mass spectrometry (MS)-based quantitative proteomics. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable isotope-containing amino acids in newly synthesized proteins. Growth medium is prepared where natural ("light") amino acids are replaced by "heavy" SILAC amino acids. Cells grown in this medium incorporate the heavy amino acids after five cell doublings and SILAC amino acids have no effect on cell morphology or growth rates. When light and heavy cell populations are mixed, they remain distinguishable by MS, and protein abundances are determined from the relative MS signal intensities. SILAC provides accurate relative quantification without any chemical derivatization or manipulation and enables development of elegant functional assays in proteomics. In this protocol, we describe how to apply SILAC and the use of nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry for protein identification and quantification. This procedure can be completed in 8 days.