Marten F. Snel

Matrix-assisted laser desorption/ionization-ion mobility separation-mass spectrometry imaging of vinblastine in whole body tissue sections

During early-stage drug development, drug and metabolite distribution studies are carried out in animal tissues using a range of techniques, particularly whole body autoradiography (WBA). While widely employed, WBA has a number of limitations, including the following: expensive synthesis of radiolabeled drugs and analyte specificity and identification. WBA only images the radiolabel. MALDI MSI has been shown previously to be advantageous for imaging the distribution of a range of drugs and metabolites in whole body sections. Ion mobility separation (IMS) adds a further separation step to imaging experiments; demonstrated here is MALDI-IMS-MS whole body imaging of rats dosed at 6 mg/kg i.v. with an anticancer drug, vinblastine and shown is the distribution of the precursor ion m/z 811.4 and several product ions including m/z 793, 751, 733, 719, 691, 649, 524, and 355. The distribution of vinblastine within the ventricles of the brain is also depicted. Clearly demonstrated in these data are the removal of interfering isobaric ions within the images of m/z 811.4 and also of the transition m/z 811-751, resulting in a higher confidence in the imaging data. Within this work, IMS has shown to be advantageous in both MS and MS/MS imaging experiments by separating vinblastine from an endogenous isobaric lipid.

On-Tissue Protein Identification and Imaging by MALDI-Ion Mobility Mass Spectrometry

MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for the detection and localization of drugs, proteins, and lipids on-tissue. Nevertheless, this approach can only perform identification of low mass molecules as lipids, pharmaceuticals, and peptides. In this article, a combination of approaches for the detection and imaging of proteins and their identification directly on-tissue is described after tryptic digestion. Enzymatic digestion protocols for different kinds of tissues—formalin fixed paraffin embedded (FFPE) and frozen tissues—are combined with MALDI-ion mobility mass spectrometry (IM-MS). This combination enables localization and identification of proteins via their related digested peptides. In a number of cases, ion mobility separates isobaric ions that cannot be identified by conventional MALDI time-of-flight (TOF) mass spectrometry. The amount of detected peaks per measurement increases (versus conventional MALDI-TOF), which enables mass and time selected ion images and the identification of separated ions. These experiments demonstrate the feasibility of direct proteins identification by ion-mobility-TOF IMS from tissue. The tissue digestion combined with MALDI-IM-TOF-IMS approach allows a proteomics “bottom-up” strategy with different kinds of tissue samples, especially FFPE tissues conserved for a long time in hospital sample banks. The combination of IM with IMS marks the development of IMS approaches as real proteomic tools, which brings new perspectives to biological studies.

Novel molecular tumour classification using MALDI–mass spectrometry imaging of tissue micro-array

The development of tissue micro-array (TMA) technologies provides insights into high-throughput analysis of proteomics patterns from a large number of archived tumour samples. In the work reported here, matrix-assisted laser desorption/ionisation–ion mobility separation–mass spectrometry (MALDI–IMS–MS) profiling and imaging methodology has been used to visualise the distribution of several peptides and identify them directly from TMA sections after on-tissue tryptic digestion. A novel approach that combines MALDI–IMS–MSI and principal component analysis–discriminant analysis (PCA–DA) is described, which has the aim of generating tumour classification models based on protein profile patterns. The molecular classification models obtained by PCA–DA have been validated by applying the same statistical analysis to other tissue cores and patient samples. The ability to correlate proteomic information obtained from samples with known and/or unknown clinical outcome by statistical analysis is of great importance, since it may lead to a better understanding of tumour progression and aggressiveness and hence improve diagnosis, prognosis as well as therapeutic treatments. The selectivity, robustness and current limitations of the methodology are discussed.

MALDI-Ion Mobility Separation-Mass Spectrometry Imaging of Glucose-Regulated Protein 78 kDa (Grp78) in Human Formalin-Fixed, Paraffin-Embedded Pancreatic Adenocarcinoma Tissue Sections

MALDI-mass spectrometry imaging (MALDI-MSI) is a technique that allows proteomic information, that is, the spatial distribution and identification of proteins, to be obtained directly from tissue sections. The use of in situ enzymatic digestion as a sample pretreatment prior to MALDI-MSI analysis has been found to be useful for retrieving protein identification directly from formalin-fixed, paraffin-embedded (ffpe) tissue sections. Here, an improved method for the study of the distribution and the identification of peptides obtained after in situ digestion of fppe pancreatic tumor tissue sections by using MALDI-mass spectrometry imaging coupled with ion mobility separation (IMS) is described. MALDI-IMS-MS images of peptide obtained from pancreatic tumor tissue sections allowed the localization of tumor regions within the tissue section, while minimizing the peak interferences which were observed with conventional MALDI-TOF MSI. The use of ion mobility separation coupled with MALDI-MSI improved the selectivity and specificity of the method and, hence, enabled both the localization and in situ identification of glucose regulated protein 78 kDa (Grp78), a tumor biomarker, within pancreatic tumor tissue sections. These findings were validated using immunohistochemical staining.

Detergent addition to tryptic digests and ion mobility separation prior to MS/MS improves peptide yield and protein identification for in situ proteomic investigation of frozen and formalin-fixed paraffin-embedded adenocarcinoma tissue sections

The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI‐mass spectrometry imaging (MALDI‐MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin‐fixed paraffin‐embedded breast cancer tissue sections were used. An improved protocol for on‐tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin‐fixed paraffin‐embedded tumour tissue sections. A novel approach combining MALDI‐MSI and ion mobility separation MALDI‐tandem mass spectrometry imaging for improving the detection of low‐abundance proteins that are difficult to detect by direct MALDI‐MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI‐MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified.

High-Spatial Resolution Matrix-Assisted Laser Desorption Ionization Imaging Analysis of Glucosylceramide in Spleen Sections from a Mouse Model of Gaucher Disease

MALDI mass spectrometric imaging (MSI) enables spatially resolved mass and intensity information to be obtained directly from tissue sections, thereby illustrating how analytes are distributed within these sections. Here we have used an oversampling technique on a commercially available MALDI orthogonal acceleration TOF mass spectrometer with ion mobility separation capability to produce high spatial resolution images of the glycosphingolipid, glucosylceramide (GC). To exemplify the biological application of our approach, GC was imaged in spleen sections from a conditional knockout mouse model of type 1 Gaucher disease in which the catabolism of this glycosphingolipid is impaired. The laser was continually fired at one position until no more ions were observed and then the sample was moved by 15 microm (laser diameter approximately 150 microm). Ions were generated from only the unirradiated surface at each of these positions achieving an effective spacing of 15 microm. At 15 microm laser step-size, it was possible to visualize macrophages enriched in GC, which could be distinguished from other cell types in the spleen. Current MALDI MSI spatial resolution is typically limited by the diameter of the laser spot-size, which is usually between 50 and 100 microm, covering an area equivalent to tens of mammalian cells.

Small molecule MALDI MS imaging: Current technologies and future challenges.

Imaging of specific small molecules is particularly challenging using conventional optical microscopy techniques. This has led to the development of alternative imaging modalities, including mass spectrometry (MS)-based methods. This review aims to provide an overview of the technologies, methods and future directions of laser-based mass spectrometry imaging (MSI) of small molecules. In particular it will focus on matrix-assisted laser desorption/ionization (MALDI) as the ion source, although other laser mass spectrometry methods will also be discussed to provide context, both historical and current. Small molecule MALDI MSI has been performed on a wide variety of instrument platforms: these are reviewed, as are the laser systems that are commonly used in this technique. Instrumentation and methodology cross over in the areas of achieving optimal spatial resolution, a key parameter in obtaining meaningful data. Also discussed is sample preparation, which is pivotal in maintaining sample integrity, providing a true reflection of the distribution of analytes, spatial resolution and sensitivity. Like all developing analytical techniques there are challenges to be overcome. Two of these are dealing with sample complexity and obtaining quantitative information from an imaging experiment. Both of these topics are addressed. Finally, novel experiments including non-MALDI laser ionization techniques are highlighted and a future perspective on the role of MALDI MSI in the small molecule arena is provided.

Delivery of therapeutic protein for prevention of neurodegenerative changes: comparison of different CSF-delivery methods.

Injection of lysosomal enzyme into cisternal or ventricular cerebrospinal fluid (CSF) has been carried out in 11 lysosomal storage disorder models, with each study demonstrating reductions in primary substrate and secondary neuropathological changes, and several reports of improved neurological function. Whilst acute studies in mucopolysaccharidosis (MPS) type II mice revealed that intrathecally-delivered enzyme (into thoraco-lumbar CSF) accesses the brain, the impact of longer-term treatment of affected subjects via this route is unknown. This approach is presently being utilized to treat children with MPS types I, II and III. Our aim was to determine the efficacy of repeated intrathecal injection of recombinant human sulfamidase (rhSGSH) on pathological changes in the MPS IIIA dog brain. The outcomes were compared with those in dogs treated via intra-cisternal or ventricular routes. Control dogs received buffer or no treatment. Significant reductions in primary/secondary substrate levels in brain were observed in dogs treated via all routes, although the extent of the reduction differed regionally. Treatment via all CSF access points resulted in large reductions in microgliosis in superficial cerebral cortex, but only ventricular injection enabled amelioration in deep cerebral cortex. Formation of glutamic acid decarboxylase-positive axonal spheroids in deep cerebellar nuclei was prevented by treatment delivered via any route. Anti-rhSGSH antibodies in the sera of some dogs did not reduce therapeutic efficacy. Our data indicates the capacity of intra-spinal CSF-injected rhSGSH to circulate within CSF-filled spaces, penetrate into brain and mediate a significant reduction in substrate accumulation and secondary pathology in the MPS IIIA dog brain.

A simple method for early age phenotype confirmation using toe tissue from a mouse model of MPS IIIA

RATIONALE Determination of genotype can be difficult, especially during the early stages of developing an animal model, e.g. when PCR primers are not yet available. An increase or decrease in specific metabolites can be used as a surrogate marker for genotype; for instance, in homozygous MPS IIIA mice heparan sulphate (HS) is increased. METHODS A simple method was developed for extracting and depolymerising HS from mouse toe tissue using methanolysis under acidic conditions. The sample was lyophilised and resuspended in methanolic HCl. The reaction products are desulphated disaccharides and readily analysable by liquid chromatography/tandem mass spectrometry (LC/MS/MS) in positive ion multiple reaction monitoring mode. Measurements were normalised to a spiked deuterated HS internal standard and to endogenous chondroitin sulphate (CS). RESULTS HS was measured in toe tissue taken from 30 mice in three groups of 10 (normal controls, MPS IIIA homozygotes and heterozygotes). A significant difference was observed between the MPS IIIA homozygotes and the other two groups, making it possible to identify mice with the MPS IIIA genotype based on the measurement of HS. Normalisation to CS was shown to correct for sample variability and reaction efficiency. CONCLUSIONS Analysis of toe tissue provides a simple and rapid way of determining a storage phenotype at 5 to 7 days of age. Significantly, this method does not require any additional samples to be taken from animals, as it utilises tissue that is a by-product of toe clipping, a method that is routinely used to permanently identify mice.

A parallel approach to post source decay MALDI-TOF analysis.

We present a novel enhancement to matrix-assisted laser desorption ionization (MALDI) post-source decay (PSD) analysis whereby fragment ions from multiple precursor ions are acquired into the same spectrum without employing a timed ion gate to preselect each parent ion. Fragment ions are matched to their corresponding precursor ions by comparing spectra acquired at slightly different reflectron electric fields. By measuring the difference in time-of-flight (TOF) between the two spectra for each fragment, it is possible to calculate the mass of the fragment ion and its parent. This new “parallel PSD” technique reduces analysis time and consumes less sample than conventional PSD, which requires an ion gate for serial preselection of precursor ions.