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.

A matrix-assisted laser desorption/ionization time of flight/time of flight tandem mass spectrometer (MALDI TOF/TOF) has been used for high-speed precursor/fragment ion transition image acquisition. High throughput analysis is facilitated by a Nd:YLF solid state laser capable of pulse repetition rates up to 5 kHz, a high digitizer acquisition rate (up to 50 pixels/second), and continuous laser raster sampling. MS/MS experiments are enabled through the use of a precision timed ion selector, second source acceleration, and a dedicated collision cell. Continuous raster sampling is shown here to facilitate rapid MS/MS ion image acquisition from thin tissue sections for the drug rifampicin and of a common kidney lipid, SM4s(d18:1/24:1). The ability to confirm the structural identity of an analyte as part of the MS/MS imaging experiment is an essential part of the analysis. Additionally, the increase in sensitivity and specificity afforded by an MS/MS approach is highly advantageous, especially when interrogating complex chemical environments such as those in biological tissues. Herein, we report continuous laser raster sampling TOF/TOF imaging methodologies which demonstrate 8-14 fold increases in throughput compared to existing MS/MS instrumentation, an important advantage when imaging large areas on tissues.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R2) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%.

In this published Research article, [1] the timed ion selector (TIS) windows were erroneously reported in units of ns on page 706 and in the caption for Fig. 2; these windows should have been reported in arbitrary units.Also, the previously reported structure for the fragment ion of rifampicin, highlighted in green in the structure inset of Fig. 3b, was recently found to be incorrect. We have determined the correct fragment ion structure using accurate mass tandem mass spectrometry on a Fourier transform ion cyclotron resonance instrument. The correct structure is shown below. Reference[1] B. M. Prentice, C. W. Chumbley, R. M. Caprioli. High-speed MALDI MS/MS imaging mass spectrometry using continuous raster sampling. J. Mass Spectrom. 2015, 50, 703-710.

The identification of amyloid-binding compounds is a crucial step in the development of imaging probes and therapeutics for the detection and cure of Alzheimer's disease. Unfortunately, the process typically lags during the translation from in vitro to in vivo studies due to the impenetrable nature of the blood brain barrier (BBB). Here, we integrate fluorescence assay with MALDI imaging mass spectrometry to screen known compounds and repurpose their properties to enable the second function of binding to amyloid plaques. Through this approach, we identified an antihistamine compound, promethazine, that can bind to amyloid plaques. Finally, we demonstrate that promethazine is retained in the amyloid-burdened brain compared to a normal brain and that its distribution within the brain corroborates with that of amyloid plaques.