Layer-specific sulfatide localization in rat hippocampus middle molecular layer is revealed by nanoparticle-assisted laser desorption/ionization imaging mass spectrometry

Lipids are major structural component of the brain and play key roles in signaling functions in the central nervous system (CNS), such as the hippocampus. In particular, sulfatide is an abundant glycosphingolipid component of both the central and the peripheral nervous system and is an essential lipid component of myelin membranes. Lack of sulfatide is observed in myelin deformation and neurological deficits. Previous studies with antisulfatide antibody have investigated distribution of sulfatide expression in neurons; however, this method cannot distinguish the differences of sulfatide lipid species raised by difference of carbon-chain length in the ceramide portion in addition to the differences of sulfatide and seminolipid. In this study, we solved the problem by our recently developed nanoparticle-assisted laser desorption/ionization (nano-PALDI)-based imaging mass spectrometry (IMS). We revealed that the level of sulfatide in the middle molecular layer was significantly higher than that in granule cell layers and the inner molecular layer in the dentate gyrus of rat hippocampus. H. Ageta and S. Asai contributed equally to this work.     

De novo discovery of phenotypic intratumour heterogeneity using imaging mass spectrometry

An essential and so far unresolved factor influencing the evolution of cancer and the clinical management of patients is intratumour clonal and phenotypic heterogeneity. However, the de novo identification of tumour subpopulations is so far both a challenging and an unresolved task. Here we present the first systematic approach for the de novo discovery of clinically detrimental molecular tumour subpopulations. In this proof‐of‐principle study, spatially resolved, tumour‐specific mass spectra were acquired, using matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry, from tissues of 63 gastric carcinoma and 32 breast carcinoma patients. The mass spectra, representing the proteomic heterogeneity within tumour areas, were grouped by a corroborated statistical clustering algorithm in order to obtain segmentation maps of molecularly distinct regions. These regions were presumed to represent different phenotypic tumour subpopulations. This was confirmed by linking the presence of these tumour subpopulations to the patients' clinical data. This revealed several of the detected tumour subpopulations to be associated with a different overall survival of the gastric cancer patients (p = 0.025) and the presence of locoregional metastases in patients with breast cancer (p = 0.036). The procedure presented is generic and opens novel options in cancer research, as it reveals microscopically indistinct tumour subpopulations that have an adverse impact on clinical outcome. This enables their further molecular characterization for deeper insights into the biological processes of cancer, which may finally lead to new targeted therapies. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

High-resolution magnetic resonance and mass spectrometry imaging of the human larynx

High-resolution, noninvasive and nondestructive imaging of the subepithelial structures of the larynx would enhance microanatomic tissue assessment and clinical decision making; similarly, in situ molecular profiling of laryngeal tissue would enhance biomarker discovery and pathology readout. Towards these goals, we assessed the capabilities of high-resolution magnetic resonance imaging (MRI) and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) imaging of rarely reported paediatric and adult cadaveric larynges that contained pathologies. The donors were a 13-month-old male, a 10-year-old female with an infraglottic mucus retention cyst and a 74-year-old female with advanced polypoid degeneration and a mucus retention cyst. MR and molecular imaging data were corroborated using whole-organ histology. Our MR protocols imaged the larynges at 45–117 μm² in-plane resolution and capably resolved microanatomic structures that have not been previously reported radiographically—such as the vocal fold superficial lamina propria, vocal ligament and macula flavae; age-related tissue features—such as intramuscular fat deposition and cartilage ossification; and the lesions. Diffusion tensor imaging characterised differences in water diffusivity, primary tissue fibre orientation, and fractional anisotropy between the intrinsic laryngeal muscles, mucosae and lesions. MALDI-MS imaging revealed peptide signatures and putative protein assignments for the polypoid degeneration lesion and the N-glycan constituents of one mucus retention cyst. These imaging approaches have immediate application in experimental research and, with ongoing technology development, potential for future clinical application.     

飞行质谱法测定血清脑硫苷脂

目的建立基质辅助的激光解吸附离子化-飞行时间质谱法（MALDI-TOF—MS）测定血清脑硫苷脂的方法,并探讨人血清参考范围。方法经去蛋白、各种条件的高温皂化、萃取、干燥后的血清,用不同浓度的过滤溶剂在MonoTip C18洗脱管中过滤杂质．以桂皮酸为基质,在飞行质谱仪上点样测定。在优化后的条件下测定方法的线性范围、回收率与人血清参考范围。结果用90％甲醇配制的浓度0．1mol／LNaOH在150℃皂化25min、用体积比6：4的蒸馏水与甲醇在MonoTip C18管过滤杂质效果最好。方法线性范围为浓度0—20nmol／ml,回收率为73．7％-75．5％,40例健康人血清参考范围：浓度（8．2±1．5）nmol／ml,男女间差异无统计学意义（P〉0．05）。结论通过飞行质谱仪测定血清脑硫苷脂的含量,具有较高的准确性和线性范围。     

Nanostructure-initiator mass spectrometry (NIMS) imaging of brain cholesterol metabolites in Smith-Lemli-Opitz syndrome

Cholesterol is an essential component of cellular membranes that is required for normal lipid organization and cell signaling. While the mechanisms associated with maintaining cholesterol homeostasis in the plasma and peripheral tissues have been well studied, the role and regulation of cholesterol biosynthesis in normal brain function and development have proven much more challenging to investigate. Smith–Lemli–Opitz syndrome (SLOS) is a disorder of cholesterol synthesis characterized by mutations of 7-dehydrocholesterol reductase (DHCR7) that impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol and lead to neurocognitive deficits, including cerebellar hypoplasia and austism behaviors. Here we have used a novel mass spectrometry-based imaging technique called cation-enhanced nanostructure-initiator mass spectrometry (NIMS) for the in situ detection of intact cholesterol molecules from biological tissues. We provide the first images of brain sterol localization in a mouse model for SLOS (Dhcr7^−/−). In SLOS mice, there is a striking localization of both 7DHC and residual cholesterol in the abnormally developing cerebellum and brainstem. In contrast, the distribution of cholesterol in 1-day old healthy pups was diffuse throughout the cerebrum and comparable to that of adult mice. This study represents the first application of NIMS to localize perturbations in metabolism within pathological tissues and demonstrates that abnormal cholesterol biosynthesis may be particularly important for the development of these brain regions.     

MALDI质谱成像技术在狂犬病病毒小鼠组织内定位研究中的应用

目的:建立基于MALDI质谱的狂犬病病毒小鼠脑组织内质谱成像实验方法,寻找狂犬病病毒脑组织内定位标记物。方法:制备冰冻切片,进行组织上原位酶解及基质覆盖,利用MALDI质谱扫描成像,以及FlexImaging 2.1软件分析,对差异肽段进行二级质谱鉴定。结果:初步鉴定出了四段狂犬病病毒肽段,可作为组织内狂犬病病毒定位标志物。结论:为进一步提高狂犬病病毒组织内定位的精确度奠定了研究基础。     

Monoisotopic silver nanoparticles-based mass spectrometry imaging of human bladder cancer tissue: Biomarker discovery

PurposeBladder cancer (BC) is the 10th most common form of cancer worldwide and the 2nd most common cancer of the urinary tract after prostate cancer, taking into account both incidence and prevalence.Materials/methodsTissues from patients with BC and also tissue extracts were analyzed by laser desorption/ionization mass spectrometry imaging (LDI-MSI) with monoisotopic silver-109 nanoparticles-enhanced target (¹⁰⁹AgNPET).ResultsUnivariate and multivariate statistical analyses revealed 10 metabolites that differentiated between tumor and normal tissues from six patients with diagnosed BC. Selected metabolites are discussed in detail in relation to their mass spectrometry (MS) imaging results. The pathway analysis enabled us to link these compounds with 17 metabolic pathways.ConclusionsAccording to receiver operating characteristic (ROC) analysis of biomarkers, 10 known metabolites were identified as the new potential biomarkers with areas under the curve (AUC) higher than >0.99. In both univariate and multivariate analysis, it was predicted that these compounds could serve as useful discriminators of cancerous versus normal tissue in patients diagnosed with BC.     

Validation of DNA sequences using mass spectrometry coupled with nucleoside mass tagging

We present a mass spectrometry (MS)-based nucleoside-specific mass-tagging method to validate genomic DNA sequences containing ambiguities not resolved by gel electrophoresis. Selected types of (13)C/(15)N-labeled dNTPs are used in PCR amplification of target regions followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-MS analysis. From the mass difference between the PCR products generated with unlabeled nucleosides and products containing (13)C/(15)N-labeled nucleosides, we determined the base composition of the genomic regions of interest. Two approaches were used to verify the target regions: The first approach used nucleosides partially enriched with stable isotopes to identify a single uncalled base in a gel electrophoresis-sequenced region. The second approach used mass tags with 100% heavy nucleosides to examine a GC-rich region of a polycytidine string with an unknown number of cytidines. By use of selected (13)C/(15)N-labeled dNTPs (dCTPs) in PCR amplification of the target region in tandem with MALDI-TOF-MS, we determined precisely that this string contains 11 cytidines. Both approaches show the ability of our MS-based mass-tagging strategy to solve critical questions of sequence identities that might be essential in determining the proper reading frames of the targeted regions.     

基质辅助激光解析质谱成像技术的样品制备及其应用

基质辅助激光解析质谱成像技术(matrix-assisted laser desorption mass spectrometry imaging,MALDI-MSI)已成为目前蛋白质组学研究中的经典技术.在该技术的成功应用过程中,合适的样品制备方法起着首要和关键的作用.只有将合适的样品制备方法与专业的仪器分析方法结合起来,才能实现对多肽和蛋白质等生物大分子的准确鉴定.MALDI-MSI是一项新的并迅速成熟的技术领域,本文针对MALDI-MSI在多肽和蛋白质中的应用,以哺乳动物组织为例,对MALDI-MSI的成像原理、样品制备及该技术的应用进行介绍.     

Medical imaging strategies

Computed tomography (CT), intravenous digital subtraction angiography (IV-DSA), digital radiographic image processing and dual energy subtraction are four examples of medical imaging strategies that have met with various degrees of success as judged by diagnostic performance. The success of CT has been spectacular; IV-DSA has provided modest benefits; digital image processing of chest radiographs has been singularly disappointing; and the verdict on dual energy subtraction is undecided. The degree of success of each of these techniques can be understood by considering the degree to which each simplifies image interpretation or isolates a fingerprint of disease.     

质谱技术研究进展

质谱技术具有高灵敏度、高精确度等特点,已广泛应用于生物学、生物医学、生物化学等学科的研究,特别是在蛋白质等生物大分子的研究中作用越来越重要。对质谱技术的研究现状及新技术的研究进展作一综述,并对未来予以展望。     

质谱技术在蛋白质组学中的应用

质谱技术是通过测定样品离子的不同质荷比（m/z）及其分布来进行结构和成分分析的一种分析方法.自从20世纪中期商品化的离子仪出现以后,由于其电离技术和分析技术的不断发展与完善,质谱在环境化学、有机化学、地质化学及制药等多个领域都有着较为广泛的应用.     

Surface-MALDI mass spectrometry in biomaterials research

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used for over a decade for the determination of purity and accurate molecular masses of macromolecular analytes, such as proteins, in solution. In the last few years the technique has been adapted to become a new surface analysis method with unique capabilities that complement established biomaterial surface analysis methods such as XPS and ToF-SSIMS. These new MALDI variant methods, which we shall collectively summarize as Surface-MALDI-MS, are capable of desorbing adsorbed macromolecules from biomaterial surfaces and detecting their molecular ions with high mass resolution and at levels much below monolayer coverage. Thus, Surface-MALDI-MS offers unique means of addressing biomaterial surface analysis needs, such as identification of the proteins and lipids that adsorb from multicomponent biological solutions in vitro and in vivo, the study of interactions between biomaterial surfaces and biomolecules, and identification of surface-enriched additives and contaminants. Surface-MALDI-MS is rapid, experimentally convenient, overcomes limitations in mass resolution and sensitivity of established biochemical techniques such as SDS-PAGE, and can in some circumstances be used for the quantitative analysis of adsorbed protein amounts. At this early stage of development, however, limitations exist: in some cases proteins are not detectable, which appears to be related to tight surface binding. This review summarizes ways in which Surface-MALDI-MS methods have been applied to the study of a range of issues in biomaterials surfaces research.     

Rapid detection and molecular serotyping of adenovirus by use of PCR followed by electrospray ionization mass spectrometry

We have developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay for the rapid detection, identification, and serotyping of human adenoviruses. The assay employs a high-performance mass spectrometer to “weigh” the amplicons obtained from PCR using primers designed to amplify known human adenoviruses. Masses are converted to base compositions and, by comparison against a database of the genetic sequences, the serotype present in a sample is determined. The performance of the assay was demonstrated with quantified viral standards and environmental and human clinical samples collected from a military training facility. Over 500 samples per day can be analyzed with sensitivities greater than 100 genomes per reaction. This approach can be applied to many other families of infectious agents for rapid and sensitive analysis.     

DNA analysis by MALDI-TOF mass spectrometry

The last decade has seen an increased demand for high-throughput DNA analysis. This is mainly due to the human genome sequencing project that is now completed. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was pinpointed early on as a technology that could be of great use for sequence variation analysis in the post-genome sequencing era. Applications developed first on this platform were for SNP genotyping. Several strategies for allele-discrimination (hybridization, cleavage, ligation, and primer extension) were combined with MALDI-TOF mass spectrometric detection. Nowadays, in practice, only primer extension methods are applied for large-scale SNP genotyping studies with MALDI-TOF detection. Problems surrounding the integration of SNP genotyping by MALDI-TOF mass spectrometry at high throughput are largely mastered now. Mass spectrometry geared presentations at the HUGO Mutation Detection Meeting in Palm Cove, Australia almost exclusively focused on novel applications that go beyond standard SNP genotyping. These applications are more demanding in terms of chemistry and molecular biology. Molecular haplotyping, expression profiling, DNA methylation analysis, and mutation detection are now being demonstrated.