Multi order correction algorithms to remove image distortions from mass spectrometry imaging datasets

Time-of-flight secondary ion mass spectrometry imaging is a rapidly evolving technology. Its main application is the study of the distribution of small molecules on biological tissues. The sequential image acquisition process remains susceptible to measurement distortions that can render imaging data less analytically useful. Most of these artifacts show a repetitive nature from tile to tile. Here we statistically describe these distortions and derive two different algorithms to correct them. Both, a generalized linear model approach and the linear discriminant analysis approach are able to increase image quality for negative and positive ion mode datasets. Additionally, performing simulation studies with repetitive and non-repetitive tiling error we show that both algorithms are only removing repetitive distortions. It is further shown that the spectral component of the dataset is not altered by the use of these correction methods. Both algorithms presented in this work greatly increase the image quality and improve the analytical usefulness of distorted images dramatically

An Orbitrap/Time-of-Flight Mass Spectrometer for Photofragment Ion Imaging and High-Resolution Mass Analysis of Native Macromolecular Assemblies

We discuss the design, development, and evaluation of an Orbitrap/time-of-flight (TOF) mass spectrometry (MS)-based instrument with integrated UV photodissociation (UVPD) and time/mass-to-charge ratio ( m/ z)-resolved imaging for the comprehensive study of the higher-order molecular structure of macromolecular assemblies (MMAs). A bespoke TOF analyzer has been coupled to the higher-energy collisional dissociation cell of an ultrahigh mass range hybrid quadrupole-Orbitrap MS. A 193 nm excimer laser was employed to photofragment MMA ions. A combination of microchannel plates (MCPs)-Timepix (TPX) quad and MCPs-phosphor screen-TPX3CAM assemblies have been used as axial and orthogonal imaging detectors, respectively. The instrument can operate in four different modes, where the UVPD-generated fragment ions from the native MMA ions can be measured with high-mass resolution or imaged in a mass-resolved manner to reveal the relative positions of the UVPD fragments postdissociation. This information is intended to be utilized for retrieving higher-order molecular structural details that include the conformation, subunit stoichiometry, and molecular interactions as well as to understand the dissociation dynamics of the MMAs in the gas phase

Hypertension Is Associated with Marked Alterations in Sphingolipid Biology: A Potential Role for Ceramide

Background Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function. Methods and Findings In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p Conclusions Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone

Protein classification and distribution in osteoarthritic human synovial tissue by matrix-assisted laser desorption ionization mass spectrometry imaging

The remodeling of the synovial membrane, which normally lubricates the joints by producing synovial fluid, is one of the most characteristic events in the pathology of osteoarthritis (OA). The heterogeneity and spatial distribution of proteins in the synovial membrane are poorly studied and we hypothesized that they constitute excellent molecular disease classifiers for the accurate diagnosis of the disease. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) allows for the study of the localization and identification of hundreds of different molecules with high sensitivity in very thin tissue sections. In this work, we employed MALDI-MSI in combination with principal component analysis and discriminant analysis to reveal the specific profile and distribution of digested proteins in human normal and OA synovial membranes. Proteins such as hemoglobin subunit alpha 2, hemoglobin subunit beta, actin aortic smooth muscle, biglycan, and fibronectin have been directly identified from human synovial biopsies. In addition, we have determined the location of disease-specific OA markers. Some of them which are located in areas of low inflammation provide valuable information on tissue heterogeneity. Finally, we described the OA molecular protein signatures common to synovial and other articular tissues such as cartilage. For the first time, normal and OA human synovial membranes have been classified by MALDI-MSI, thus offering a new sensitive tool for the study of rheumatic pathologies

"Afterlife Experiment": Use of MALDI-MS and SIMS Imaging for the Study of the Nitrogen Cycle within Plants

As part of a project to demonstrate the science of decay, a series of mass spectrometry imaging experiments were performed. The aim was to demonstrate that decay and decomposition are only part of the story and to show pictorially that atoms and molecules from dead plants and animals are incorporated into new life. Radish plants (Raphanus sativus) were grown hydroponically using a nutrient system containing (15)N KNO3 (98% labeled) as the only source of nitrogen. Plants were cropped and left to ferment in water for 2 weeks to create a radish "tea", which was used as a source of nitrogen for radish grown in a second hydroponics experiment. After 5 weeks of growth, the radish plants were harvested and cryosectioned, and sections were imaged by positive-ion MALDI and SIMS mass spectrometry imaging. The presence of labeled species in the plants grown using (15)N KNO3 as nutrient and those grown from the radish "tea" was readily discernible. The uptake of (15)N into a number of identifiable metabolites has been studied by MALDI-MS and SIMS imaging

Oxygen regulates lipid profiles in human primary chondrocyte cultures

Purpose: Articular cartilage is generally exposed to a finely regulated gradient of relatively low oxygen percentages (from 8% at the surface to 1% in the deepest layers). While most cartilage research is performed in supraphysiological oxygen levels (19-21%), culturing chondrocytes under hypoxic oxygen levels (≤8%) promotes the chondrogenic phenotype and cartilage-specific matrix formation. Although hypoxiainducible factor-1α is identified as a key mediator of these beneficial effects on chondrogenesis, the underlying mechanisms remain unclear. A recent study in primary chondrocytes and chondrogenically differentiating mesenchymal stem cells showed that the cholesterol metabolism is altered in hypoxic conditions. Moreover the lipid metabolism is changed in osteoarthritic cartilage and intracellular lipid accumulation is correlated with osteoarthritis (OA) severity. In this study we assessed whether healthy and OA chondrocytes have distinct responses in normoxia or hypoxia with respect to their lipid composition. Methods: Human primary chondrocytes were isolated from cartilage knee biopsies of patients (n = 5) undergoing total knee replacement. From each donor cells were isolated from macroscopically healthy and OA damaged areas. Cells were expanded in monolayer in normoxia (21% oxygen) or hypoxia (2.5% oxygen) and subsequently cultured in 3D pellets in normoxia or hypoxia for 7 days. Lipid profiles were assessed with Matrix Assisted Laser Desorption Ionization mass spectrometry imaging (MALDI MSI). Cell pellets were cryosectioned (10 μm section) and sprayed with α-Cyano-4-hydroxycinnamic acid (CHCA) matrix 5 mg/ml in methanol/water/trifluoroacetic acid (70:30:0.01) using the Suncollect (Sunchrom). A Synapt HDMS MALDI-Q-TOF (Waters) instrument was used to perform the MSI experiments with a spatial raster size of 100 μm. Principal component analysis (PCA) and discriminant analysis (DA) were used to search for spectral similarities and differences between the conditions. Biomap software was used to visualize molecular distributions. The Lipid Maps database was used for lipid assignment and tandem MS for the identification. Results: Chondrocyte cells cultured under different oxygen tensions were discriminated by MALDI MSI followed by discriminant analysis. Discriminant function 1 (DF1) described the lipid profiles specific to each oxygen tension (Figure 1). In hypoxic pellets the intensity of phosphatidylcholine (PC) 16:0/18:1 (m/z 798.5) and sphingomyelin (SM) d18:1/16:0 (m/z 741.5 and 725.5) (Figure 1A)was higher compared to normoxic pellets. Moreover, while various phosphatidylinositol (PI) were present at both oxygen tensions, the intensity of the PI's in normoxia (m/z 865.5 and 885.5) was much higher (Figure 1B). The molecular distribution of SM d18:1/16:0 at m/z 741.5 and PC 18:0/18:1 (m/z 810.6) showed a higher expression at the edges of the pellet (Figure 2). Interestingly, we found that the lipid profiles of chondrocytes harvested from either OA or healthy cartilage showed a more pronounced difference when cultured in hypoxia. In both positive and negative ion modes the second DF separated healthy from OA chondrocytes in hypoxic conditions. Conclusions: Our MALDI-MSI data show that oxygen tension modulated the lipid composition of chondrocytes. Furthermore, culturing OA or healthy chondrocytes in hypoxic conditions resulted in more pronounced differences in lipid profiles as compared to culturing in normoxia. Since glycerophospholipids, including PC, SM, PS, and PI, are key components of the lipid bilayer and involved in metabolism and cell signaling, we will next investigate how these lipids influence chondrocyte metabolism and cell signaling. (Figure presented)

Matrix assisted laser desorption ionization mass spectrometry imaging identifies markers of ageing and osteoarthritic cartilage

INTRODUCTION: Cartilage protein distribution and the changes that occur in cartilage ageing and disease are essential in understanding the process of cartilage ageing and age related diseases such as osteoarthritis. The aim of this study was to investigate the peptide profiles in ageing and osteoarthritic (OA) cartilage sections using matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). METHODS: The distribution of proteins in young, old and OA equine cartilage was compared following tryptic digestion of cartilage slices and MALDI-MSI undertaken with a MALDI SYNAPT™ HDMS system. Protein identification was undertaken using database searches following multivariate analysis. Peptide intensity differences between young, ageing and OA cartilage were imaged with Biomap software. Analysis of aggrecanase specific cleavage patterns of a crude cartilage proteoglycan extract were used to validate some of the differences in peptide intensity identified. Immunohistochemistry studies validated the differences in protein abundance. RESULTS: Young, old and OA equine cartilage was discriminated based on their peptide signature using discriminant analysis. Proteins including aggrecan core protein, fibromodulin, and cartilage oligomeric matrix protein were identified and localised. Fibronectin peptides displayed a stronger intensity in OA cartilage. Age-specific protein markers for collectin-43 and cartilage oligomeric matrix protein were identified. In addition potential fibromodulin and biglycan peptides targeted for degradation in OA were detected. CONCLUSIONS: MALDI-MSI provided a novel platform to study cartilage ageing and disease enabling age and disease specific peptides in cartilage to be elucidated and spatially resolved

A micropixelated ion-imaging detector for mass resolution enhancement of a QMS instrument

An in-vacuum position-sensitive micropixelated detector (Timepix) is used to investigate the time-dependent spatial distribution of different charge state (and hence different mass-to-charge (m/z)) ions exiting an electrospray ionization (ESI)-based quadrupole mass spectrometer (QMS) instrument. Ion images obtained from the Timepix detector provide a detailed insight into the positions of stable and unstable ions of the mass peak as they exit the QMS. With the help of image processing algorithms and by selecting areas on the ion images where more stable ions impact the detector, an improvement in mass resolution by a factor of 5 was obtained for certain operating conditions. Moreover, our experimental approach of mass resolution enhancement was confirmed by in-house-developed novel QMS instrument simulation software. Utilizing the imaging-based mass resolution enhancement approach, the software predicts instrument mass resolution of ?1,0000 for a single-filter QMS instrument with a 210-mm long mass filter and a low operating frequency (880?kHz) of the radio frequency (RF) voltage

Mass Spectrometry Imaging of Drug Related Crystal-Like Structures in Formalin-Fixed Frozen and Paraffin-Embedded Rabbit Kidney Tissue Sections

A multimodal mass spectrometry imaging (MSI) based approach was used to characterize the molecular content of crystal-like structures in a frozen and paraffin embedded piece of a formalin-fixed rabbit kidney. Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) imaging and desorption electrospray ionization (DESI) mass spectrometry imaging were combined to analyze the frozen and paraffin embedded sample without further preparation steps to remove the paraffin. The investigated rabbit kidney was part of a study on a drug compound in development, in which severe renal toxicity was observed in dosed rabbits. Histological examination of the kidney showed tubular degeneration with precipitation of crystal-like structures in the cortex, which were assumed to cause the renal toxicity. The MS imaging approach was used to find out whether the crystal-like structures were composed of the drug compound, metabolites, or an endogenous compound as a reaction to the drug administration. The generated MALDI-MSI data were analyzed using principal component analysis. In combination with the MS/MS results, this way of data processing demonstrates that the crystal structures were mainly composed of metabolites and relatively little parent drug. [Figure: see text