Mass Spectrometry Imaging Techniques Research Articles

A deeper peek into living organisms

Multi-isotope imaging mass spectrometry (MIMS) is a new and generally applicable method for the study of DNA replication, lipid and protein turnover and cell fate in animals and humans. In a proof-of-principle study, MIMS was used to test the 'immortal strand hypothesis', which proposes that stem cells maintain a master genetic template that is protected from cancer-causing mutations. The hypothesis remains hotly debated, in part because of the difficulties involved in testing it experimentally. Stable isotope incorporation was viewed and measured by MIMS in mammalian intestinal cell division, Drosophila melanogaster lipid metabolism and human lymphopoiesis. In the auditory system, the mechanosensory hair cells of the inner ear convert sound-induced vibrations into electrical signals. The apical surface of a hair cell consists of stereocilia with a core of actin filaments that function as mechanosensors. It has been suggested that these actin filaments are replaced within two to three days by a treadmilling process. Using the newly developed multi-isotope imaging mass spectrometry (MIMS) technique, Zhang et al. quantify protein turnover in hair-cell stereocilia in vivo and find that turnover is slow throughout the stereocilia, except for the tip region, and does not involve a treadmilling process.

Identification of oligosaccharides from histopathological sections by MALDI imaging mass spectrometry

Direct tissue analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) provides the means for in situ molecular analysis of a wide variety of biomolecules. This technology--known as imaging mass spectrometry (IMS)--allows the measurement of biomolecules in their native biological environments without the need for target-specific reagents such as antibodies. In this study, we applied the IMS technique to formalin-fixed paraffin-embedded samples to identify a substance(s) responsible for the intestinal obstruction caused by an unidentified foreign body. In advance of IMS analysis, some pretreatments were applied. After the deparaffinization of sections, samples were subjected to enzyme digestion. The sections co-crystallized with matrix were desorbed and ionized by a laser pulse with scanning. A combination of α-amylase digestion and the 2,5-dihydroxybenzoic acid matrix gave the best mass spectrum. With the IMS Convolution software which we developed, we could automatically extract meaningful signals from the IMS datasets. The representative peak values were m/z 1,013, 1,175, 1,337, 1,499, 1,661, 1,823, and 1,985. Thus, it was revealed that the material was polymer with a 162-Da unit size, calculated from the even intervals. In comparison with the mass spectra of the histopathological specimen and authentic materials, the main component coincided with amylopectin rather than amylose. Tandem MS analysis proved that the main components were oligosaccharides. Finally, we confirmed the identification of amylopectin by staining with periodic acid-Schiff and iodine. These results for the first time show the advantages of MALDI-IMS in combination with enzyme digestion for the direct analysis of oligosaccharides as a major component of histopathological samples.

Nondestructive, histologically compatible tissue imaging by desorption electrospray ionization mass spectrometry.

Imaging mass spectrometry (MS) is a powerful technique which has been gaining increased attention and broad use in many fields of science. Many biological applications are being pursued using the comprehensive information it provides on the distribution of multiple endogenous and exogenous molecules within animal and plant tissues[1]. Amongst the imaging MS techniques[2], those based on ambient ionization[3] such as desorption electrospray ionization mass spectrometry (DESI-MS)[4], laser ablation electrospray ionization (LAESI)[5] and atmospheric pressure fentosecond laser imaging mass spectrometry (AP fs-LDI IMS)[6] amongst others[7], allow analysis at atmospheric pressure without significant sample preparation. In the past few years, much effort has gone into advancing ambient imaging mass spectrometry, especially in cancer diagnostics[8]. The prospect of improving the accuracy of histopathological cancer evaluation by adding chemical information to morphological microscopic analysis now represents an attainable advance.

Human bone material characterization: integrated imaging surface investigation of male fragility fractures

The interrelation of calcium and phosphorus was evaluated as a function of bone material quality in femoral heads from male fragility fracture patients via surface analytical imaging as well as scanning microscopy techniques. A link between fragility fractures and increased calcium to phosphorus ratio was observed despite normal mineralization density distribution. Bone fragility in men has been recently recognized as a public health issue, but little attention has been devoted to bone material quality and the possible efficacy in fracture risk prevention. Clinical routine fracture risk estimations do not consider the quality of the mineralized matrix and the critical role played by the different chemical components that are present. This study uses a combination of different imaging and analytical techniques to gain insights into both the spatial distribution and the relationship of phosphorus and calcium in bone. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry imaging techniques were used to investigate the relationship between calcium and phosphorus in un-embedded human femoral head specimens from fragility fracture patients and non-fracture age-matched controls. The inclusion of the bone mineral density distribution via backscattered scanning electron microscopy provides information about the mineralization status between the groups. A link between fragility fracture and increased calcium and decreased phosphorus in the femoral head was observed despite normal mineralization density distribution. Results exhibited significantly increased calcium to phosphorus ratio in the fragility fracture group, whereas the non-fracture control group ratio was in agreement with the literature value of 1.66 M ratio in mature bone. Our results highlight the potential importance of the relationship between calcium and phosphorus, especially in areas of new bone formation, when estimating fracture risk of the femoral head. The determination of calcium and phosphorus fractions in bone mineral density measurements may hold the key to better fracture risk assessment as well as more targeted therapies.

Micrometric molecular histology of lipids by mass spectrometry imaging

Time-Of-Flight Secondary Ion Mass Spectrometry is compared to other mass spectrometry imaging techniques, and recent improvements of the experimental methods, driven by biological and biomedical applications, are described and discussed. This review shows that this method that can be considered as a micrometric molecular histology is particularly efficient for obtaining images of various lipid species at the surface of a tissue sample, without sample preparation, and with a routine spatial resolution of 1μm or less.

Metabolomics: Where seeing is believing

Metabolomics: Where seeing is believing

Imaging of complex sulfatides SM3 and SB1a in mouse kidney using MALDI-TOF/TOF mass spectrometry

Sulfatides, a class of acidic glycosphingolipids, are highly expressed in mammalian myelin and in kidney, where they are thought to stabilize neuronal structures and signaling and to influence osmotic stability of renal cells, respectively. Recently, 9-aminoacridine (9-AA) has been introduced as a negative ion matrix that displays high selectivity for low complexity galactosylceramid-I(3)-sulfate sulfatides and that is suitable for quantitative analysis by matrix-assisted desorption/ionization (MALDI) mass spectrometry (MS). Analyzing acidic fractions of lipid extracts and cryosections from kidneys of wild type and arylsulfatase A-deficient (ASA -/-) mice, we demonstrate that 9-AA also enables sensitive on-target analysis as well as imaging of complex lactosylceramide-II(3)-sulfate and gangliotetraosylceramide-II(3), IV(3) bis-sulfate sulfatides by MALDI-TOF/TOF MS. Utilizing the MALDI imaging MS technique, we show differential localization in mouse kidney of (1) sulfatides with identical ceramide anchors, but different glycan-sulfate head groups but also of (2) sulfatides with identical head groups but with different acyl- or sphingoid base moieties. A comparison of MALDI images of renal sulfatides from control and sulfatide storing arylsulfatase A-deficient (ASA -/-) mice revealed relative expression differences, very likely reflecting differences in sulfatide turnover of the various renal cell types. These results establish MALDI imaging MS with 9-AA matrix as a label-free method for spatially resolved ex vivo investigation of the relative turnover of sulfatides in animal models of human glycosphingolipid storage disease.

Binary Matrix for MALDI Imaging Mass Spectrometry of Phospholipids in Both Ion Modes

Phospholipids (PLs) are the major building block molecules of cellular membranes. Their composition varies depending on cell types and cellular compartments. Thus, the information regarding PL distribution in tissue has important physiological and pathological significance. Recent developments in imaging mass spectrometry (IMS) have allowed complete mapping of the PL species on tissue. The IMS technique can detect different classes of PLs as well as their location information directly from tissue sections. PL head groups carry either positive and/or negative charges; therefore, IMS experiments must be conducted in both positive- and negative-ion mode to detect all types of phospholipids. Several conventional matrixes were applied on tissue for better identification. This study was conducted to enable appropriate matrix selection and optimized matrix preparation for IMS experiments in both ion modes that maximize PL identification from a single brain tissue section. The optimized matrix 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) with a mixture of trifluoroacetic acid (TFA) and piperidine as ion pairing agents showed improved stability and consistency during both ion mode experiments and successfully identified >100 peaks of PLs determined by parent ion m/z value. Further tandem mass spectrometric analysis (MS/MS) was performed to those PLs that are anatomically important according to their distribution on rat brain tissue section.

Advances in analytical mass spectrometry

This special issue of Analytical and Bioanalytical Chemistry contains a selection of 13 manuscripts dealing with the most recent advances in analytical mass spectrometry (MS). Besides updating readers on developments in analytical MS, further aims of this issue are to describe trends in both elemental and molecular MS in bioanalysis and life sciences and to provide a survey on modern applications. The topics covered in these contributions range frommatrixfree methods for laser desorption/ionization (LDI) MS to imaging MS techniques. Reviews on instrumental developments in direct coupling of liquid chromatography (LC) with MS using electron ionization, developments in protein–ligand affinity MS, and the combination of fast LC with MS for the diagnostic assay of metabolites and proteins are included. A review paper on the use of MS as a test bench for medicinal chemistry studies is presented. Trends in analytical atomic and molecularMSwith a focus on biology and life sciences are also reviewed, presenting developments in coupling microfluidics with MS and in miniaturization of mass spectrometers. Matrixfree methods for LDI MS and molecular imaging MS techniques are addressed. Advances in atomic MS concern recent technological developments in MS imaging of elements, making imaging MSmethods indispensable tools for obtaining information relevant to structural biology. The impressive progress shown by inductively coupled plasma (ICP) MS for high-precision trace element analysis and for speciation of elements in biological samples is also highlighted. Pharmaceutical, medical, and bioanalytical fields have strongly pushed MS to new heights, and the significance of MS techniques in those application areas is still growing rapidly. In particular, the development of MS approaches based on LDI and on ion mobility separation coupled with imaging MS brings new perspectives to biological studies. It is therefore not surprising that MS techniques applied to proteomics and metabolomics play a major role in this special issue. Forensic analysis also benefits from technological advances in MS techniques for the purposes of separation and reliable identification of explosives and drugs. Original papers cover an equally wide range of topics, including recent applications of imaging time-of-flight–secondary ion MS in medical research, analytical strategies for drug analysis byUPLC-MS/MS, ionmobilityMS coupledwith planar solid-phase microextraction for analysis of explosives, and the use of ion-exchange matrix separation coupled with ICP-MS for determination of trace element emissions for environmental monitoring. The special issue provides both a status report on recent developments in analytical atomic and molecular MS and current trends and future perspectives in upcoming fields. I would like to thank all the authors for their contributions to this special issue and also the editorial staff of Analytical and Bioanalytical Chemistry for fruitful cooperation.

Multivariate analysis of a 3D mass spectral image for examining tissue heterogeneity.

The tissue microenvironment critically influences the molecular characteristics of a tumor. However, as tumorous tissue is highly heterogeneous it may harbor various sub-populations with different microenvironments, greatly complicating the unambiguous analysis of tumor biology. Mass spectrometry imaging techniques allow for the direct analysis of tumors in the spatial context of their microenvironment. However, discovery of heterogeneous sub-populations often depends on the use of multivariate statistical methods. While this is routinely used for 2D images, multivariate statistical approaches are rarely seen in the context of 3D images. Here we present the automatic alignment of 2D images recorded by nanostructure-initiator mass spectrometry (NIMS) to reconstruct a 3D model of a mouse mammary tumor. Multivariate statistical analysis was applied to the whole 3D reconstruction at once, revealing distinct tumor regions, an observation that would not have been possible in such clarity through the analysis of isolated 2D sections. These sub-structures were confirmed by H&E and Oil Red O stains. This study shows that the combination of 3D imaging and multivariate statistics can be used to define tumor regions.

The study of the distribution of melamine in rat renal tissues by imaging mass spectrometry

The recent tainting events of milk and pet foods with melamine and melamine-related compounds (MRCs), in attempts to elevate the total nitrogen content, have led to many cases of acute renal failure in infants and domestic pets. In this study, we employed matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry-based imaging mass spectrometry (IMS) technique to visualize the distribution of melamine in renal tissue. For rats orally administered with melamine alone, the protonated melamine ion at m/z 127 was detected only in the papilla. No melamine signal was detected in the kidney of rats receiving cyanuric acid alone. Melamine was accumulated in kidney only when the rats received high dose of melamine (100 mg day−1) together with cyanuric acid for 3 days. Under such regimen, the molecular signal was visible in both cortex and medulla, with an accumulation in renal papilla. Our IMS results for rat renal tissue are consistent with previous reports and histological observations. Nonetheless, there was no difference between the lipid distributions on the renal tissues of the control rats and those administered with melamine/cyanuric acid.

Imaging mass spectrometry of gastric carcinoma in formalin‐fixed paraffin‐embedded tissue microarray

The popularity of imaging mass spectrometry (IMS) of tissue samples, which enables the direct scanning of tissue sections within a short time-period, has been considerably increasing in cancer proteomics. Most pathological specimens stored in medical institutes are formalin-fixed; thus, they had been regarded to be unsuitable for proteomic analyses, including IMS, until recently. Here, we report an easy-to-use screening method that enables the analysis of multiple samples in one experiment without extractions and purifications of proteins. We scanned, with an IMS technique, a tissue microarray (TMA) of formalin-fixed paraffin-embedded (FFPE) specimens. We detected a large amount of signals from trypsin-treated FFPE-TMA samples of gastric carcinoma tissues of different histological types. Of the signals detected, 54 were classified as signals specific to cancer with statistically significant differences between adenocarcinomas and normal tissues. We detected a total of 14 of the 54 signals as histological type-specific with the support of statistical analyses. Tandem MS revealed that a signal specific to poorly differentiated cancer tissue corresponded to histone H4. Finally, we verified the IMS-based finding by immunohistochemical analysis of more than 300 specimens spotted on TMAs; the immunoreactivity of histone H4 was remarkably strong in poorly differentiated cancer tissues. Thus, the application of IMS to FFPE-TMA can enable high-throughput analysis in cancer proteomics to aid in the understanding of molecular mechanisms underlying carcinogenesis, invasiveness, metastasis, and prognosis. Further, results obtained from the IMS of FFPE-TMA can be readily confirmed by commonly used immunohistochemical analyses.

Clioquinol Decreases Amyloid-β Burden and Reduces Working Memory Impairment in a Transgenic Mouse Model of Alzheimer's Disease

Clioquinol (CQ) is a "metal protein attenuating compound" that crosses the blood-brain barrier and binds, with high affinity, copper(II) and zinc(II), two metal ions critically involved in amyloid-beta aggregation and toxicity. CQ was recently proposed for the treatment of Alzheimer's disease, but controversial data have been reported so far concerning its real therapeutic advantages. We describe here results of chronic CQ treatment in the TgCRND8 mouse model of Alzheimer's disease. Remarkably, based on classical behavioral tests, CQ treatment was found to reverse, to a large extent, the working memory impairments that are characteristic of this mouse model. Pairwise, a significant reduction of amyloid-beta plaque burden, both in the cortex and in the hippocampus, was detected as well as an attenuation of astrogliosis. MALDI Mass Spectrometry Imaging technique revealed a specific localization of CQ in the above mentioned brain areas. Modest but significant effects on the absolute and relative brain concentrations of the three most important biometals (i.e., copper, zinc, and iron) were highlighted following CQ treatment. The pharmacological and mechanistic implications of the above findings are thoroughly discussed.

MALDI mass spectrometry imaging of gangliosides in mouse brain using ionic liquid matrix

Mass spectrometry imaging has emerged as a powerful tool for the direct detection of biomolecules, mainly phospholipids, proteins and peptides, in tissue samples. To date, there is very little information available on the direct analysis of gangliosides in brain tissue. One major hurdle for imaging gangliosides in tissue using mass spectrometry is that sialic acid residues can be dissociated in ionization process. In this report, we investigated an ionic liquid matrix for mass spectrometry imaging of gangliosides. This ionic liquid matrix offered excellent sensitivity for detection gangliosides without significant loss of sialic acid residues. Thus, it can be used to study the abundance and anatomical localization of gangliosides in mouse brain using mass spectrometry imaging technique. Mass spectrometry image analyses of the mouse brain tissue sections demonstrated that the N-fatty acyl chains of gangliosides were differentially distributed in mouse hippocampal regions, whereby the gangliosides with N-C 18 acyl chain were enriched in CA1 region, while gangliosides with N-C 20 acyl chain were enriched in dentate gyrus. In addition, this observation is true for mono-, di- and tri-sialylated gangliosides. Although the linkage information was not determined, the mass spectrometry imaging technique was capable of spatial tissue mapping of ceramide structures in gangliosides.

Surface analysis by imaging mass spectrometry

A review of four MS-based techniques available for molecular surface imaging is presented. The main focus is on the commercially available mass spectrometry imaging techniques: secondary ion mass spectrometry (SIMS), matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), desorption electrospray ionization mass spectrometry (DESI-MS) and laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS). A short historical perspective is presented and traditional desorption ionization techniques are also briefly described. The four techniques are compared mainly with respect to their usage for imaging of biological surfaces. MALDI is evaluated as the most successful in life sciences and the only technique usable for imaging of large biopolymers. SIMS is less common but offers superior spatial lateral resolution and DESI is considered to be an emerging alternative approach in mass spectrometry imaging. LA-ICP ionization is unbeatable in terms of limits of detection but does not provide structural information. All techniques are considered extremely useful, representing a new wave of expansion of mass spectrometry into surface science and bioanalysis. A minireview with 121 references.

Identification of Glioma Neovascularization-related Proteins by Using MALDI-FTMS and Nano-LC Fractionation to Microdissected Tumor Vessels

The identification of angiogenesis-related proteins is important for the development of new antiangiogenic therapies, and such proteins are potential new biomarkers for gliomas. The aim of this study was to identify proteins that are exclusively present in glioma neovasculature and not in the vasculature of normal brain. We combined advanced proteomics techniques to compare the expression profiles of microdissected blood vessels from glioma with blood vessels of normal control brain samples. We measured the enzymatic generated peptide profiles from these microdissected samples by MALDI-FTMS. Subsequently, the samples were fractionated by nano-LC prior to MALDI-TOF/TOF. This combined approach enabled us to identify four proteins that appeared to be exclusively expressed in the glioma blood vessels. Two of these proteins, fibronectin and colligin 2, were validated on tissue sections using specific antibodies. We found that both proteins are present in active angiogenesis in glioma, other neoplasms, and reactive conditions in which neoangiogenesis takes place. This work proves that gel-free mass spectrometric techniques can be used on relatively small numbers of cells generated by microdissection procedures to successfully identify differentially expressed proteins.

Imaging mass spectrometry of neuropeptides in decapod crustacean neuronal tissues.

Imaging mass spectrometry (IMS) of neuropeptides in crustacean neuronal tissues was performed on a MALDI-TOF/TOF instrument. Sample preparation protocols were developed for the sensitive detection of these highly complex endogenous signaling molecules. The neuromodulatory complements of the pericardial organ (PO) and brain of the Jonah crab, Cancer borealis, were mapped. Distributions of peptide isoforms belonging to 10 neuropeptide families were investigated using the IMS technique. Often, neuropeptides of high sequence homology were similarly located. However, two RFamide-family peptides and a truncated orcokinin peptide were mapped to locations distinct from other members of their respective families. Over 30 previously sequenced neuropeptides were identified based on mass measurement. For increased confidence of identification, select peptides were fragmented by post-source decay (PSD) and collisional-induced dissociation (CID). Collectively, this organ-level IMS study elucidates the spatial relationships between multiple neuropeptide isoforms of the same family as well as the relative distributions of neuropeptide families.

Photodissociation dynamics of CBr4 at 267nm by means of ion velocity imaging

The photodissociation dynamics of CBr4 at 267 nm has been studied using time of flight (TOF) mass spectrometry and ion velocity imaging techniques. The photochemical products are detected with resonance enhanced multiphoton ionization (REMPI) as well as single-photon vacuum ultraviolet ionization at 118 nm. REMPI at 266.65 and 266.71 nm was used to detect the ground Br(2P32) and spin-orbit excited Br(2P12) atoms, respectively. The translational energy and angular distributions are consistent with direct dissociation from an excited triplet state and indirect dissociation from high vibrational levels on the singlet ground state surface. Br2+ ions are also observed in the TOF spectra with a focused 267 nm laser. The counter fragment, CBr2+, is observed when this photolysis laser is unfocused, and photons at 118 nm are used to ionize the radical products. The translational energy distributions of the CBr2+ and Br2+ products can be momentum matched, which indicates that molecular Br2 elimination is one of the primary dissociation channels.

Novel dermaseptins from Phyllomedusa hypochondrialis (Amphibia)

Six new antimicrobial peptides structurally related to the dermaseptin family have been isolated from the skin secretion of the amphibian Phyllomedusa hypochondrialis. The primary structures of these molecules named as DShypo 01, 02, 03, 04, 06, and 07 were determined by de novo MS/MS experiments, Edman degradation, and cDNA sequencing. The fifth peptide was found to be precisely the same DS 01 from Phyllomedusa oreades previously described by our group. The majority of the peptides purified from the crude skin secretion could be directly localized and mapped onto a freshly dissected dorsal skin fragment using mass spectrometry-imaging techniques. Comparisons between peptides and commercial drugs on their antibacterial and anti-Leishmania amazonensis efficiencies, associated with peptide lytic effects on mammalian blood cells and surface plasmon resonance interaction studies on immobilized DMPC vesicles, were also performed.

Matrix‐assisted laser desorption/ionization imaging mass spectrometry for direct measurement of clozapine in rat brain tissue

Matrix-assisted laser desorption/ionization hyphenated with quadrupole time-of-flight (QTOF) mass spectrometry (MS) has been used to directly determine the distribution of pharmaceuticals in rat brain tissue slices which might unravel their disposition for new drug development. Clozapine, an antipsychotic drug, and norclozapine were used as model compounds to investigate fundamental parameters such as matrix and solvent effects and irradiance dependence on MALDI intensity but also to address the issues with direct tissue imaging MS technique such as (1) uniform coating by the matrix, (2) linearity of MALDI signals, and (3) redistribution of surface analytes. The tissue sections were coated with various matrices on MALDI plates by airspray deposition prior to MS detection. MALDI signals of analytes were detected by monitoring the dissociation of the individual protonated molecules to their predominant MS/MS product ions. The matrices were chosen for tissue applications based on their ability to form a homogeneous coating of dense crystals and to yield greater sensitivity. Images revealing the spatial localization in tissue sections using MALDI-QTOF following a direct infusion of (3)H-clozapine into rat brain were found to be in good correlation with those using a radioautographic approach. The density of clozapine and its major metabolites from whole brain homogenates was further confirmed using fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) procedures.