Alan M. Race

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.

Liquid Extraction Surface Analysis Mass Spectrometry Coupled with Field Asymmetric Waveform Ion Mobility Spectrometry for Analysis of Intact Proteins from Biological Substrates

Previously we have shown that liquid extraction surface analysis (LESA) mass spectrometry is suitable for the analysis of intact proteins from a range of biological substrates. Here we show that LESA mass spectrometry may be coupled with high field asymmetric waveform ion mobility spectrometry (FAIMS) for top-down protein analysis directly from thin tissue sections (mouse liver, mouse brain) and from bacterial colonies (Escherichia coli) growing on agar. Incorporation of FAIMS results in significant improvements in signal-to-noise and reduced analysis time. Abundant protein signals are observed in single scan mass spectra. In addition, FAIMS enables gas-phase separation of molecular classes, for example, lipids and proteins, enabling improved analysis of both sets of species from a single LESA extraction.

Memory Efficient Principal Component Analysis for the Dimensionality Reduction of Large Mass Spectrometry Imaging Data Sets

A memory efficient algorithm for the computation of principal component analysis (PCA) of large mass spectrometry imaging data sets is presented. Mass spectrometry imaging (MSI) enables two- and three-dimensional overviews of hundreds of unlabeled molecular species in complex samples such as intact tissue. PCA, in combination with data binning or other reduction algorithms, has been widely used in the unsupervised processing of MSI data and as a dimentionality reduction method prior to clustering and spatial segmentation. Standard implementations of PCA require the data to be stored in random access memory. This imposes an upper limit on the amount of data that can be processed, necessitating a compromise between the number of pixels and the number of peaks to include. With increasing interest in multivariate analysis of large 3D multislice data sets and ongoing improvements in instrumentation, the ability to retain all pixels and many more peaks is increasingly important. We present a new method which has no limitation on the number of pixels and allows an increased number of peaks to be retained. The new technique was validated against the MATLAB (The MathWorks Inc., Natick, Massachusetts) implementation of PCA (princomp) and then used to reduce, without discarding peaks or pixels, multiple serial sections acquired from a single mouse brain which was too large to be analyzed with princomp. Then, k-means clustering was performed on the reduced data set. We further demonstrate with simulated data of 83 slices, comprising 20,535 pixels per slice and equaling 44 GB of data, that the new method can be used in combination with existing tools to process an entire organ. MATLAB code implementing the memory efficient PCA algorithm is provided.

LESA FAIMS Mass Spectrometry for the Spatial Profiling of Proteins from Tissue

We have shown previously that coupling of high field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility, with liquid extraction surface analysis (LESA) mass spectrometry of tissue results in significant improvements in the resulting protein mass spectra. Here, we demonstrate LESA FAIMS mass spectrometry imaging of proteins in sections of mouse brain and liver tissue. The results are compared with LESA mass spectrometry images obtained in the absence of FAIMS. The results show that the number of different protein species detected can be significantly increased by incorporating FAIMS into the workflow. A total of 34 proteins were detected by LESA FAIMS mass spectrometry imaging of mouse brain, of which 26 were unique to FAIMS, compared with 15 proteins (7 unique) detected by LESA mass spectrometry imaging. A number of proteins were identified including α-globin, 6.8 kDa mitochondrial proteolipid, macrophage migration inhibitory factor, ubiquitin, β-thymosin 4, and calmodulin. A total of 40 species were detected by LESA FAIMS mass spectrometry imaging of mouse liver, of which 29 were unique to FAIMS, compared with 24 proteins (13 unique) detected by LESA mass spectrometry imaging. The spatial distributions of proteins identified in both LESA mass spectrometry imaging and LESA FAIMS mass spectrometry imaging were in good agreement indicating that FAIMS is a suitable tool for inclusion in mass spectrometry imaging workflows.

Formal lithium fixation improves direct analysis of lipids in tissue by mass spectrometry.

Mass spectrometry imaging is a powerful method for imaging and in situ characterization of lipids in thin tissue sections. Structural elucidation of lipids is often achieved via collision induced dissociation, and lithium-lipid adducts have been widely reported as providing the most structurally informative fragment ions. We present a method for the incorporation of lithium salts into tissue imaging experiments via fixation of samples in formal lithium solutions. The method is suitable for preparation of single tissue sections, or as an immersion fixation method for whole tissue blocks or organs prior to sectioning. We compare lithium adduct detection and MALDI-MSI of murine brain from analysis of tissues prepared in different ways. Tissues prepared in formal solutions containing lithium or sodium salts before coating in matrix via air-spray deposition are compared with fresh samples coated in lithium-doped matrix preparations by either dry-coating or air-spray deposition. Sample preparation via fixation in formal lithium is shown to yield the highest quality images of lithium adducts, resulting in acquisition of more informative product ion spectra in MALDI MS/MS profiling and imaging experiments. Finally, the compatibility of formal lithium solutions with standard histological staining protocols (hemotoxylin and eosin, Van Giessen and Oil Red O) is demonstrated in a study of human liver tissue.

Analysis of urine, oral fluid and fingerprints by liquid extraction surface analysis coupled to high resolution MS and MS/MS – opportunities for forensic and biomedical science

Liquid Extraction Surface Analysis (LESA) is a new, high throughput tool for ambient mass spectrometry. A solvent droplet is deposited from a pipette tip onto a surface and maintains contact with both the surface and the pipette tip for a few seconds before being re-aspirated. The technique is particularly suited to the analysis of trace materials on surfaces due to its high sensitivity and low volume of sample removal. In this work, we assess the suitability of LESA for obtaining detailed chemical profiles of fingerprints, oral fluid and urine, which may be used in future for rapid medical diagnostics or metabolomics studies. We further show how LESA can be used to detect illicit drugs and their metabolites in urine, oral fluid and fingerprints. This makes LESA a potentially useful tool in the growing field of fingerprint chemical analysis, which is relevant not only to forensics but also to medical diagnostics. Finally, we show how LESA can be used to detect the explosive material RDX in contaminated artificial fingermarks.

Optimisation of colour schemes to accurately display mass spectrometry imaging data based on human colour perception

AbstractThe choice of colour scheme used to present data can have a dramatic effect on the perceived structure present within the data. This is of particular significance in mass spectrometry imaging (MSI), where ion images that provide 2D distributions of a wide range of analytes are used to draw conclusions about the observed system. Commonly employed colour schemes are generally suboptimal for providing an accurate representation of the maximum amount of data. Rainbow-based colour schemes are extremely popular within the community, but they introduce well-documented artefacts which can be actively misleading in the interpretation of the data. In this article, we consider the suitability of colour schemes and composite image formation found in MSI literature in the context of human colour perception. We also discuss recommendations of rules for colour scheme selection for ion composites and multivariate analysis techniques such as principal component analysis (PCA). Graphical Abstracta–t Visualisation of the same data (unnormalised m/z 826 from the cerebellum region of a mouse brain) using colour schemes found in the MSI literature. Intensity spans from 0 to 100 counts. a Grayscale, b red, c green, d blue, e green to white, f cyan to white, g blue to white, h red to white, i pink to white, j copper to white, k hot, l pink hot, m green to yellow, n cyan to magenta to yellow, o double scale (blue to green, red to yellow), p temperature-based, q–t rainbow-based

MALDI Imaging of Liquid Extraction Surface Analysis Sampled Tissue

Combined mass spectrometry imaging methods in which two different techniques are executed on the same sample have recently been reported for a number of sample types. Such an approach can be used to examine the sampling effects of the first technique with a second, higher resolution method and also combines the advantages of each technique for a more complete analysis. In this work matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) was used to study the effects of liquid extraction surface analysis (LESA) sampling on mouse brain tissue. Complementary multivariate analysis techniques including principal component analysis, non-negative matrix factorization, and t-distributed stochastic neighbor embedding were applied to MALDI MS images acquired from tissue which had been sampled by LESA to gain a better understanding of localized tissue washing in LESA sampling. It was found that MALDI MS images could be used to visualize regions sampled by LESA. The variability in sampling area, spatial precision, and delocalization of analytes in tissue induced by LESA were assessed using both single-ion images and images provided by multivariate analysis.

A Novel Hybrid Dual Analyzer SIMS Instrument for Improved Surface and 3D-Analysis

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an established, highly sensitive analytical technique for mass spectrometry (MS) imaging applications with a lateral resolution below 100 nm. Elemental and molecular information is obtained by bombarding the surface with a focused primary ion beam and analyzing the generated secondary ions in a TOF mass analyzer. Furthermore 3D imaging is possible by employing a lower energetic quasi DC sputter beam for material removal (sputter cycle) and a short pulsed small spot analysis beam for optimal mass spectral and imaging performance (so-called dual beam mode). Application of this technique for the localization of drugs and their metabolites in drug-doped cells could be used to find regions in which a pharmaceutical compound accumulates. This would be extremely helpful for selection of possible drug candidates in pre-clinical studies, thereby reducing the development costs for new pharmaceutical products. Furthermore surveying biologically relevant molecules, like lipids, in tissue can give valuable information on the molecular fundamentals of diseases and the effects of treatments.

Raster-Mode Continuous-Flow Liquid Microjunction Mass Spectrometry Imaging of Proteins in Thin Tissue Sections

Mass spectrometry imaging by use of continuous-flow liquid microjunction sampling at discrete locations (array mode) has previously been demonstrated. In this Letter, we demonstrate continuous-flow liquid microjunction mass spectrometry imaging of proteins from thin tissue sections in raster mode and discuss advantages (a 10-fold reduction in analysis time) and challenges (suitable solvent systems, data interpretation) of the approach. Visualization of data is nontrivial, requiring correlation of solvent-flow, mass spectral data acquisition rate, data quality, and liquid microjunction sampling area. The latter is particularly important for determining optimum pixel size. The minimum achievable pixel size is related to the scan time of the instrument used. Here we show a minimum achievable pixel size of 50 μm (x-dimension) when using an Orbitrap Elite; however a pixel size of 600 μm is recommended in order to minimize the effects of oversampling on image accuracy.