Practical guidelines for the preparation of tissue sections for direct analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry are presented. Techniques for proper sample handling including tissue storage, sectioning and mounting are described. Emphasis is placed on optimizing matrix parameters such as the type of matrix molecule used, matrix concentration, and solvent composition. Several different techniques for matrix application are illustrated. Optimal instrument parameters and the necessity for advanced data analysis approaches with regards to direct tissue analysis are also discussed.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is emerging as a powerful tool for investigating the distribution of molecules within biological systems through the direct analysis of thin tissue sections. Unique among imaging methods, MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement. We discuss the current state of the art of MALDI-IMS along with some recent applications and technological developments that illustrate not only its current capabilities but also the future potential of the technique to provide a better understanding of the underlying molecular mechanisms of biological processes.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to directly analyze and image pharmaceutical compounds in intact tissue. The anti-tumor drug SCH 226374 was unambiguously determined in mouse tumor tissue using MALDI-QqTOFMS (QSTAR) by monitoring the dissociation of the protonated drug at m/z 695.4 to its predominant fragment at m/z 228.1. A second drug, compound A, was detected in slices of rat brain tissue following oral administration with doses ranging from 1-25 mg/kg. Quantitation of compound A from whole brain homogenates using routine high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) procedures revealed that concentrations of the drug in whole brain varied from a low of 24 ng/g to a high of 1790 ng/g. The drug candidate was successfully detected by MALDI-QqTOF in samples from each dose, covering a range of approximately two orders of magnitude. In addition, good correlation was observed between the MALDI-QqTOFMS intensities at each dose with the HPLC/MS/MS results. Thus the MALDI-MS response is proportional to the amount of drug in tissue. Custom software was developed to facilitate the imaging of small molecules in tissue using the MALDI-QqTOF mass spectrometer. Images revealing the spatial localization of SCH 226374 in tumor tissue and compound A in brain tissue were acquired.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) elucidates molecular distributions in thin tissue sections. Absolute pixel-to-pixel quantitation has remained a challenge, primarily lacking validation of the appropriate analytical methods. In the present work, isotopically labeled internal standards are applied to tissue sections to maximize quantitative reproducibility and yield accurate quantitative results. We have developed a tissue model for rifampicin (RIF), an antibiotic used to treat tuberculosis, and have tested different methods of applying an isotopically labeled internal standard for MALDI IMS analysis. The application of the standard and subsequently the matrix onto tissue sections resulted in quantitation that was not statistically significantly different from results obtained using HPLC-MS/MS of tissue extracts. Quantitative IMS experiments were performed on liver tissue from an animal dosed in vivo. Each microspot in the quantitative images measures the local concentration of RIF in the thin tissue section. Lower concentrations were detected from the blood vessels and around the portal tracts. The quantitative values obtained from these measurements were comparable (>90% similarity) to HPLC-MS/MS results obtained from extracts of the same tissue.

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.