Imaging of metabolites using secondary ion mass spectrometry

This article provides an overview of the technique of secondary ion mass spectrometry imaging and highlights some current and future areas of application relevant to the field of metabolomics. The approach benefits from label-free analysis of molecular species up to ~1500 Da with minimal sample preparation. Offering the highest spatial resolution of current mass spectrometry imaging methodologies, the technique is well-suited to metabolite imaging in both biological tissue and cells, in both 2D and 3D.     

Molecular mass spectrometry imaging in biomedical and life science research

This review describes the current state of mass spectrometry imaging (MSI) in life sciences. A brief overview of mass spectrometry principles is presented followed by a thorough introduction to the MSI workflows, principles and areas of application. Three major desorption-ionization techniques used in MSI, namely, secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption ionization (MALDI), and desorption electrospray ionization (DESI) are described, and biomedical and life science imaging applications of each ionization technique are reviewed. A separate section is devoted to data handling and current challenges and future perspectives are briefly discussed at the end.     

Recent advances in applying mass spectrometry and systems biology to determine brain dynamics

Introduction: Neurological disorders encompass various pathologies which disrupt normal brain physiology and function. Poor understanding of their underlying molecular mechanisms and their societal burden argues for the necessity of novel prevention strategies, early diagnostic techniques and alternative treatment options to reduce the scale of their expected increase.

Areas covered: This review scrutinizes mass spectrometry based approaches used to investigate brain dynamics in various conditions, including neurodegenerative and neuropsychiatric disorders. Different proteomics workflows for isolation/enrichment of specific cell populations or brain regions, sample processing; mass spectrometry technologies, for differential proteome quantitation, analysis of post-translational modifications and imaging approaches in the brain are critically deliberated. Future directions, including analysis of cellular sub-compartments, targeted MS platforms (selected/parallel reaction monitoring) and use of mass cytometry are also discussed.

Expert commentary: Here, we summarize and evaluate current mass spectrometry based approaches for determining brain dynamics in health and diseases states, with a focus on neurological disorders. Furthermore, we provide insight on current trends and new MS technologies with potential to improve this analysis.     

Natural products in Glycyrrhiza glabra (licorice) rhizome imaged at the cellular level by atmospheric pressure matrix‐assisted laser desorption/ionization tandem mass spectrometry imaging

The rhizome of Glycyrrhiza glabra (licorice) was analyzed by high‐resolution mass spectrometry imaging and tandem mass spectrometry imaging. An atmospheric pressure matrix‐assisted laser desorption/ionization imaging ion source was combined with an orbital trapping mass spectrometer in order to obtain high‐resolution imaging in mass and space. Sections of the rhizome were imaged with a spatial resolution of 10 μm in the positive ion mode, and a large number of secondary metabolites were localized and identified based on their accurate mass and MS/MS fragmentation patterns. Major tissue‐specific metabolites, including free flavonoids, flavonoid glycosides and saponins, were successfully detected and visualized in images, showing their distributions at the cellular level. The analytical power of the technique was tested in the imaging of two isobaric licorice saponins with a mass difference of only 0.02 Da. With a mass resolving power of 140 000 and a bin width of 5 ppm in the image processing, the two compounds were well resolved in full‐scan mode, and appeared with different distributions in the tissue sections. The identities of the compounds and their distributions were validated in a subsequent MS/MS imaging experiment, thereby confirming their identities and excluding possible analyte interference. The use of high spatial resolution, high mass resolution and tandem mass spectrometry in imaging experiments provides significant information about the biosynthetic pathway of flavonoids and saponins in legume species, combing the spatially resolved chemical information with morphological details at the microscopic level. Furthermore, the technique offers a scheme capable of high‐throughput profiling of metabolites in plant tissues.     

Paper spray ionization mass spectrometry: recent advances and clinical applications

ABSTRACT

Introduction: Paper spray mass spectrometry has provided a rapid, quantitative ambient ionization method for xenobiotic and biomolecule analysis. As an alternative to traditional sample preparation and chromatography, paper spray demonstrates the sampling ionization of a wide range of molecules and significant sensitivity from complex biofluids. The amenability of paper spray with dried blood spots and other sampling types shows strong potential for rapid, point-of-care (POC) analysis without time-consuming separation procedures.

Areas covered: This special report summarizes the current state and advances in paper spray mass spectrometry that relate to its applicability for clinical analysis. It also provides our perspectives on the future development of paper spray mass spectrometry and its potential roles in clinical settings.

Expert commentary: Paper spray has provided the fundamental aspects of ambient ionization needed for implementation at the POC. With further clinical management and standardization, paper spray has the potential to replace traditional complex analysis procedure for rapid quantitative detection of illicit drugs, therapeutic drugs and metabolites. Surface and substrate modifications also offer significant improvement in desorption and ionization efficiencies, resulting in enhanced sensitivity. Comprehensive analysis of metabolites and lipids will further extend the implementation of paper spray ionization mass spectrometry into clinical applications.     

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.     

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.     

Fast atom Bombardment Mass Spectrometry of Nucleosides,Nucleotides and Oligonucleotides

Abstract

Fast atom bombardment (FAB) mass spectrometry, a new ionization technique, has been applied to a variety of polar, nonvolatile compounds with considerable success. Current literature regarding the analysis of nucleosides, nucleotides and oligonucleotides using FAB is reviewed.     

Recent Developments in Mass Spectrometry: Rna Sequencing

Abstract

Simplified sequencing of an oligoribonucleotide containing 16 bases is accomplished by matrix assisted laser desorptiodionization time of flight (MALDI-TOF) mass spectrometry. We used delayed ion extraction (DE) technique and kinetic degradation with two exonucleases.     

MALDI-TOF Mass Spectrometry as a Powerful Tool to Study Enzymatic Processing of DNA Lesions Inserted into Oligonucleotides

Abstract

MALDI-TOF mass spectrometry measurements, coupled with either exonuclease or DNA N-glycosylases digestions of lesion-containing oligonucleotides, were used to assess biochemical features of several oxidative DNA damage. The latter analytical approach was shown to be an informative and efficient alternative technique to conventional electrophoresis and chromatographic analyses.     

MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the in situ analysis of tissue sections. MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement and enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. In this review, we focus on the current state of the art of MALDI-IMS, describe basic technological developments for MALDI-IMS of animal and human tissues, and discuss some recent applications in basic research and in clinical settings.     

New developments in profiling and imaging of proteins from tissue sections by MALDI mass spectrometry

Molecular imaging of tissue by MALDI mass spectrometry is a powerful tool for visualizing the spatial distribution of constituent analytes with high molecular specificity. Although the technique is relatively young, it has already contributed to the understanding of many diverse areas of human health. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. The purpose of this review is to highlight some of the more recent technological advances that have improved the efficiency of imaging mass spectrometry for clinical applications. Advances in the way MALDI mass spectrometry is integrated with histology, improved methods for automation, and better tools for data analysis are outlined in this review. Refined top-down strategies for the identification and validation of candidate biomarkers found in tissue sections are discussed. A clinical example highlighting the application of these methods to a cohort of clinical samples is described.     

Ambient desorption ionization mass spectrometry (DART, DESI) and its bioanalytical applications

In recent years, ambient desorption ionization techniques for mass spectrometry were introduced. Among them, the most established techniques are Direct Analysis in Real Time (DART) and Desorption Electrospray Ionization (DESI). Therefore, the current review focuses on the bioanalytical applications of ambient desorption ionization techniques by the example of DART and DESI mass spectrometry. The potential and also limitations of both ambient mass spectrometry (MS) techniques in such areas, as identification and quantitation of small molecules, coupling DART-MS and DESI-MS with planar chromatography, protein/peptide analysis, as well as molecular imaging applications, are discussed.     

MALDI imaging mass spectrometry: molecular snapshots of biochemical systems

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is emerging as a powerful tool for investigating the distribution of molecules within biological systems through the direct analysis of thin tissue sections. Unique among imaging methods, MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement. We discuss the current state of the art of MALDI-IMS along with some recent applications and technological developments that illustrate not only its current capabilities but also the future potential of the technique to provide a better understanding of the underlying molecular mechanisms of biological processes.     

When 2D is not enough,go for an extra dimension

The use of an extra SDS separation in a different buffer system provide a technique for deconvoluting 2D gel spots made of several proteins (Colignon et al. Proteomics, 2013, 13, 2077–2082). This technique keeps the quantitative analysis of the protein amounts and combines it with a strongly improved identification process by mass spectrometry, removing identification ambiguities in most cases. In some favorable cases, posttranslational variants can be separated by this procedure. This versatile and easy to use technique is anticipated to be a very valuable addition to the toolbox used in 2D gel‐based proteomics.     

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.     

Evaluating the challenges associated with time-of-fight secondary ion mass spectrometry for metabolomics using pure and mixed metabolites

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is potentially well placed to contribute to metabolomic analysis while bringing the added benefit of high resolution, label free imaging. The focused ion beams used to desorb species from the sample can be focused below 1 μm allowing chemical imaging on a sub-cellular scale. In this study we test the capability of ToF-SIMS to generate mass spectrometry and MSMS spectra from a set of standard metabolites that can be compared with open access metabolite databases containing ESI-CID MSMS spectra. The influence of the chemical environment, the matrix effect, on the observed mass spectra is assessed using a mixed metabolite sample and the data discussed in terms of compound identification and quantification. Radical ions and small fragment ions seem to be less sensitive to ion suppression or enhancement and may provide a route to quantification. Understanding such parameters will be key for the successful application of the technique for in situ metabolomics with ToF-SIMS.     

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.     

Evaluation of Capillary HPLC/Mass Spectrometry as an Alternative Analysis Method for Gel Electrophoresis of Oligonucleotides

Abstract

A method has been developed to monitor the enzymatic incorporation of nucleotides in DNA by electrospray HPLC mass spectrometry. The main advantages of mass spectrometry over electrophoresis are the ability to directly characterize the reaction products and the shorter analysis time.     

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 μm wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 μm and ~40 μm, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.