Mass spectrometry imaging and profiling of single cells

Mass spectrometry imaging and profiling of individual cells and subcellular structures provide unique analytical capabilities for biological and biomedical research, including determination of the biochemical heterogeneity of cellular populations and intracellular localization of pharmaceuticals. Two mass spectrometry technologies-secondary ion mass spectrometry (SIMS) and matrix assisted laser desorption/ionization mass spectrometry (MALDI MS)-are most often used in micro-bioanalytical investigations. Recent advances in ion probe technologies have increased the dynamic range and sensitivity of analyte detection by SIMS, allowing two- and three-dimensional localization of analytes in a variety of cells. SIMS operating in the mass spectrometry imaging (MSI) mode can routinely reach spatial resolutions at the submicron level; therefore, it is frequently used in studies of the chemical composition of subcellular structures. MALDI MS offers a large mass range and high sensitivity of analyte detection. It has been successfully applied in a variety of single-cell and organelle profiling studies. Innovative instrumentation such as scanning microprobe MALDI and mass microscope spectrometers enables new subcellular MSI measurements. Other approaches for MS-based chemical imaging and profiling include those based on near-field laser ablation and inductively-coupled plasma MS analysis, which offer complementary capabilities for subcellular chemical imaging and profiling.     

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).     

Molecular network strategy in multi-omics and mass spectrometry imaging

Human physiological activities and pathological changes arise from the coordinated interactions of multiple molecules. Mass spectrometry (MS)-based multi-omics and MS imaging (MSI)-based spatial omics are powerful methods used to investigate molecular information related to the phenotype of interest from homogenated or sliced samples, including the qualitative, relative quantitative and spatial distributions. Molecular network strategy provides efficient methods to help us understand and mine the biological patterns behind the phenotypic data. It illustrates and combines various relationships between molecules, and further performs the molecule identification and biological interpretation. Here, we describe the recent advances of network-based analysis and its applications for different biological processes, such as, obesity, central nervous system diseases, and environmental toxicology.     

Inclusive sharing of mass spectrometry imaging data requires a converter for all

With continued efforts towards a single MSI data format, data conversion routines must be made universally available. The benefits of a common imaging format, imzML, are slowly becoming more widely appreciated but the format remains to be used by only a small proportion of imaging groups. Increased awareness amongst researchers and continued support from major MS vendors in providing tools for converting proprietary formats into imzML are likely to result in a rapidly increasing uptake of the format. It is important that this does not lead to the exclusion of researchers using older or unsupported instruments. We describe an open source converter, imzMLConverter, to ensure against this. We propose that proprietary formats should first be converted to mzML using one of the widely available converters, such as msconvert and then use imzMLConverter to convert mzML to imzML. This will allow a wider audience to benefit from the imzML format immediately.     

imzML--a common data format for the flexible exchange and processing of mass spectrometry imaging data

The application of mass spectrometry imaging (MS imaging) is rapidly growing with a constantly increasing number of different instrumental systems and software tools. The data format imzML was developed to allow the flexible and efficient exchange of MS imaging data between different instruments and data analysis software. imzML data is divided in two files which are linked by a universally unique identifier (UUID). Experimental details are stored in an XML file which is based on the HUPO-PSI format mzML. Information is provided in the form of a 'controlled vocabulary' (CV) in order to unequivocally describe the parameters and to avoid redundancy in nomenclature. Mass spectral data are stored in a binary file in order to allow efficient storage. imzML is supported by a growing number of software tools. Users will be no longer limited to proprietary software, but are able to use the processing software best suited for a specific question or application. MS imaging data from different instruments can be converted to imzML and displayed with identical parameters in one software package for easier comparison. All technical details necessary to implement imzML and additional background information is available at www.imzml.org.     

Molecular histology of arteries: mass spectrometry imaging as a novel ex vivo tool to investigate atherosclerosis

Atherosclerosis is usually the underlying cause of a fatal event such as myocardial infarction or ictus. The atherome plaque develops silently and asymptomatically within the arterial intima layer. In this context, the possibility to analyze the molecular content of arterial tissue while preserving each molecule’s specific localization is of great interest as it may reveal further insights into the physiopathological changes taking place. Mass spectrometry imaging (MSI) enables the spatially resolved molecular analysis of proteins, peptides, metabolites, lipids and drugs directly in tissue, with a resolution sufficient to reveal molecular features specific to distinct arterial structures. MSI represents a novel ex vivo imaging tool still underexplored in cardiovascular diseases. This review focuses on the MSI technique applied to cardiovascular disease and covers the main contributions to date, ongoing efforts, the main challenges and current limitations of MSI.     

Surface analysis of lipids by mass spectrometry: More than just imaging

Mass spectrometry is now an indispensable tool for lipid analysis and is arguably the driving force in the renaissance of lipid research. In its various forms, mass spectrometry is uniquely capable of resolving the extensive compositional and structural diversity of lipids in biological systems. Furthermore, it provides the ability to accurately quantify molecular-level changes in lipid populations associated with changes in metabolism and environment; bringing lipid science to the “omics” age. The recent explosion of mass spectrometry-based surface analysis techniques is fuelling further expansion of the lipidomics field. This is evidenced by the numerous papers published on the subject of mass spectrometric imaging of lipids in recent years. While imaging mass spectrometry provides new and exciting possibilities, it is but one of the many opportunities direct surface analysis offers the lipid researcher. In this review we describe the current state-of-the-art in the direct surface analysis of lipids with a focus on tissue sections, intact cells and thin-layer chromatography substrates. The suitability of these different approaches towards analysis of the major lipid classes along with their current and potential applications in the field of lipid analysis are evaluated.     

Quality will determine the future of mass spectrometry imaging in clinical laboratories: the need for standardization

Introduction: Mass Spectrometry Imaging (MSI) provides information about the spatial distributions of different analytes on tissue sections and requires no homogenization techniques or labeling. It has a wide spectrum of applications ranging from biomarker discovery to drug response studies. MSI can be adapted to the clinical settings due to its ability to combine mass spectrometry and histological data.

Areas covered: The major obstacle to adapt MSI into clinical settings arises from the limited standardization in MSI experiments. We aimed to review the multi-site studies in MSI specifically focusing on reproducibility. Additionally, we emphasized the necessity of quality assessments in MSI for its potential usage in clinical settings.

Expert opinion: We discuss how important it is to follow optimized and standardized workflows in MSI and conduct potential quality assessments at important stages in order to adapt MSI into clinical applications.     

In situ metabolomic mass spectrometry imaging: recent advances and difficulties

MS imaging (MSI) is a remarkable new technology that enables us to determine the distribution of biological molecules present in tissue sections by direct ionization and detection. This technique is now widely used for in situ imaging of endogenous or exogenous molecules such as proteins, lipids, drugs and their metabolites, and it is a potential tool for pathological analysis and the investigation of disease mechanisms. MSI is also thought to be a technique that could be used for biomarker discovery with spatial information. The application of MSI to the study of endogenous metabolites has received considerable attention because metabolites are the result of the interactions of a system's genome with its environment and a total set of these metabolites more closely represents the phenotype of an organism under a given set of conditions. Recent studies have suggested the importance of in situ metabolite imaging in biological discovery and biomedical applications, but several issues regarding the technical application limits of MSI still remained to be resolved. In this review, we describe the capabilities of the latest MSI techniques for the imaging of endogenous metabolites in biological samples, and also discuss the technical problems and new challenges that need to be addressed for effective and widespread application of MSI in both preclinical and clinical settings.     

Mass spectrometry in studies of protein thiol chemistry and signaling: Opportunities and caveats

Mass spectrometry (MS) has become a powerful and widely utilized tool in the investigation of protein thiol chemistry, biochemistry, and biology. Very early biochemical studies of metabolic enzymes have brought to light the broad spectrum of reactivity profiles that distinguish cysteine thiols with functions in catalysis and protein stability from other cysteine residues in proteins. The development of MS methods for the analysis of proteins using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) coupled with the emergence of high-resolution mass analyzers has been instrumental in advancing studies of thiol modifications, both in single proteins and within the cellular context. This article reviews MS instrumentation and methods of analysis employed in investigations of thiols and their reactivity toward a range of small biomolecules. A selected number of studies are detailed to highlight the advantages brought about by the MS technologies along with the caveats associated with these analyses.     

Lipidomics analysis in drug discovery and development

Despite being a relatively new addition to the Omics' landscape, lipidomics is increasingly being recognized as an important tool for the identification of druggable targets and biochemical markers. In this review we present recent advances of lipid analysis in drug discovery and development. We cover current state of the art technologies which are constantly evolving to meet demands in terms of sensitivity and selectivity. A careful selection of important examples is then provided, illustrating the versatility of lipidomics analysis in the drug discovery and development process. Integration of lipidomics with other omics’, stem-cell technologies, and metabolic flux analysis will open new avenues for deciphering pathophysiological mechanisms and the discovery of novel targets and biomarkers.     

Mass spectrometry techniques for imaging and detection of metallodrugs

Undoubtedly, metallomic approaches based on mass spectrometry have evolved into essential tools supporting the drug development of novel metal-based anticancer drugs. This article will comment on the state-of-the-art instrumentation and highlight some of the recent analytical advances beyond routine, especially focusing on the latest developments in inductively coupled plasma-mass spectrometry (ICP-MS). Mass spectrometry-based bioimaging and single-cell methods will be presented, paving the way to exciting investigations of metal-based anticancer drugs in heterogeneous and structurally, as well as functionally complex solid tumor tissues.     

Nanostructure Initiator Mass Spectrometry for tissue imaging in metabolomics: future prospects and perspectives

Mass spectrometry-based metabolomics provides a new approach to interrogate mechanistic biochemistry related to natural processes such as health and disease. Physiological and pathological conditions, however, are characterized not only by the identities and concentrations of metabolites present, but also by the location of metabolites within a tissue. Unfortunately, most relevant MS platforms in metabolomics can only measure samples in solution, therefore metabolites are typically extracted by tissue homogenization. Recent developments of imaging-MS technologies, however, have allowed particular metabolites to be spatially localized within biological tissues. In this context, Nanostructure-Initiator Mass Spectrometry (NIMS), a matrix-free technique for surface-based analysis, has proven an alternative approach for tissue imaging of metabolites. Here we review the basic principles of NIMS for tissue imaging and show applications that can complement LC/MS and GC/MS-based metabolomic studies investigating the mechanisms of fundamental biological processes. In addition, the new surface modifications and nanostructured materials herein presented demonstrate the versatility of NIMS surface to expand the range of detectable metabolites.     

Mass spectrometry analysis of the human endosulfatase Hsulf-2

The human 6-O-endosulfatases HSulf-1 and -2 catalyze the region-selective hydrolysis of the 6-O-sulfate group of the glucosamine residues within sulfated domains of heparan sulfate, thereby ensuring a unique and original post-biosynthetic modification of the cell surface proteoglycans. While numerous studies point out the role of HSulf-2 in crucial physiological processes as well as in pathological conditions particularly in cancer, its structural organization in two chains and its functional properties remain poorly understood. In this study, we report the first characterization by mass spectrometry (MS) of HSulf-2. An average molecular weight of 133,115 Da was determined for the whole enzyme by MALDI-TOF MS, i.e. higher than the naked amino acid backbone (98,170 Da), highlighting a significant contribution of post-translational modifications. The HSulf-2 protein sequence was determined by Nano-LC-MS/MS, leading to 63% coverage and indicating at least four N-glycosylation sites at Asn 108, 147, 174 and 217. These results provide a platform for further structural investigations of the HSulf enzymes, aiming at deciphering the role of each chain in the substrate binding and specificities and in the catalytic activities.     

Mass spectrometry imaging is moving toward drug protein co-localization

Mass spectrometry (MS)-based technology provides label-free localization of molecules in tissue samples. Drugs, proteins, lipids and metabolites can easily be monitored in their environment. Resolution can be achieved down to the cellular level (10-20μm) for conventional matrix-assisted laser desorption/ionization (MALDI) imaging, or even to the subcellular level for more complex technologies such as secondary ionization mass spectrometry (SIMS) imaging. One question remains: are we going to be able to investigate functional relationships between drugs and proteins and compare with localized phenomena? This review describes the various spatial levels of investigation offered by mass spectrometry imaging (MSI), and the advantages and disadvantages compared with other labeling technologies.     

Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases

Chemical modifications of substrate peptides are often necessary to monitor the hydrolysis of small bioactive peptides. We developed an electrospray ionization mass spectrometry (ESI–MS) assay for studying substrate distributions in reaction mixtures and determined steady-state kinetic parameters, the Michaelis–Menten constant (K_m), and catalytic turnover rate (V_max/[E]_t) for three metallodipeptidases: two carnosinases (CN1 and CN2) from human and Dug1p from yeast. The turnover rate (V_max/[E]_t) of CN1 and CN2 determined at pH 8.0 (112.3 and 19.5 s⁻¹, respectively) suggested that CN1 is approximately 6-fold more efficient. The turnover rate of Dug1p for Cys-Gly dipeptide at pH 8.0 was found to be slightly lower (73.8 s⁻¹). In addition, we determined kinetic parameters of CN2 at pH 9.2 and found that the turnover rate was increased by 4-fold with no significant change in the K_m. Kinetic parameters obtained by the ESI–MS method are consistent with results of a reverse-phase high-performance liquid chromatography (RP–HPLC)-based assay. Furthermore, we used tandem MS (MS/MS) analyses to characterize carnosine and measured its levels in CHO cell lines in a time-dependent manner. The ESI–MS method developed here obviates the need for substrate modification and provides a less laborious, accurate, and rapid assay for studying kinetic properties of dipeptidases in vitro as well as in vivo.     

Mass spectrometry for nutritional peptidomics: How to analyze food bioactives and their health effects

We describe nutritional peptidomics for discovery and validation of bioactive food peptide and their health effects. Understanding nature and bioactivity of nutritional peptides means comprehending an important level of environmental regulation of the human genome, because diet is the environmental factor with the most profound life-long influence on health. We approach the theme from three angles, namely the analysis, the discovery and the biology perspective. Food peptides derive from parent food proteins via in vitro hydrolysis (processing) or in vivo digestion by various unspecific and specific proteases, as opposed to the tryptic peptides typically generated in biomarker proteomics. A food bioactive peptide may be rare or unique in terms of sequence and modification, and many food genomes are less well annotated than e.g. the human genome. Bioactive peptides can be discovered either empirically or by prediction: we explain both the classical hydrolysis strategy and the bioinformatics-driven reversed genome engineering. In order to exert bioactivity, food peptides must be either ingested and then reach the intestine in their intact form or be liberated in situ from their parent proteins to act locally, that is in the gut, or even systemically, i.e. through the blood stream. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

Mass spectrometry studies of demetallation of haemin by recombinant horse L chain apoferritin and its mutant (E 53,56,57,60 Q)

An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the metal free porphyrin is found in a corresponding pocket to haem in bacterioferritins [Précigoux, G., Yariv, J., Gallois, B., Dautant, A., Courseille, C. and Langlois, d'Estaintot B. (1994) A crystallographic study of haem binding to ferritin. Acta Cryst. D 50, 739-743]. A mechanism of demetallation of haemin by L-chain apoferritin was proposed [Crichton, R.R., Soruco, J.A., Roland, F., Michaux, M.A., Gallois, B., Précigoux, G., Mahy, J.P. and Mansuy. (1997) Remarkable ability of horse spleen apoferritin to demetallate hemin and to metallate protoporphyrin IX as a function of pH. J. P. Biochem. 36, 49, 15049-15054]: this involved four Glu residues (53,56,57,60) situated at the entrance of the hydrophobic pocket and appeared to be favoured by acidic conditions. To verify this mechanism, we have mutated these four Glu to Gln and examined demetallation in both acidic and basic conditions. In this paper, we report the mass spectrometry studies of L-chain apoferritin and its mutant incubated with haemin and analysed after different times of incubation: 15 days, 2 months, 6 months, 9 months and 12 months. These studies show that the recombinant L-chain apoferritin and its mutant are able to demetallate haemin to give a hydroxyethyl protoporphyrin IX derivative in a dimeric form [Macieira, S., Martins, B. M. and Huber, R. (2003) Oxygen-dependent coproporphyrinogen IX oxidase from Escherichia coli: one-step purification and biochemical characterization. FEMS. Microbiology Letters 226, 31-37].     

Mass spectrometry protein tests: ready for prime time (or not)

Introduction: Mass spectrometry has played an important role in protein biomarker discovery. Yet, very few of the candidate biomarkers have been validated, and mass spectrometry-based protein tests have not made a significant inroad into clinical laboratories.

Areas covered: Offered here is a unique perspective on the future of mass spectrometry protein tests, in view of the following determinants: the true demand for such clinical tests, end-users requirements, platforms and systems design, sample preparation bottlenecks, analytical and clinical validation, and regulatory approval.

Expert commentary: Fresh thoughts and attitudes toward MS protein tests are required in order to move them toward clinical utilization and diagnostic use en masse, with critical emphasis on content, simplicity and cost. In its current format and state of the art, they are simply not ready for prime time.     

Web Resources for Mass Spectrometry-based Proteomics

With the development of high-resolution and high-throughput mass spectrometry(MS)technology, a large quantum of proteomic data is continually being generated. Collecting and sharing these data are a challenge that requires immense and sustained human effort. In this report, we provide a classification of important web resources for MS-based proteomics and present rating of these web resources, based on whether raw data are stored, whether data submission is supported,and whether data analysis pipelines are provided. These web resources are important for biologists involved in proteomics research.