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In situ Hydrogel Conditioning of Tissue Samples to Enhance the Drug’s Sensitivity in Ambient Mass Spectrometry Imaging Xiaowei Song, Zhigang Luo, Xin Li, Tiegang Li, Zhonghua Wang, Chenglong Sun, Luojiao Huang, Ping Xie, Xiaoyu Liu, Jiuming He, and Zeper Abliz Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 18 May 2017 Downloaded from http://pubs.acs.org on May 21, 2017
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Analytical Chemistry
In situ Hydrogel Conditioning of Tissue Samples to Enhance the Drug’s Sensitivity in Ambient Mass Spectrometry Imaging Xiaowei Song,† Zhigang Luo,† Xin Li,† Tiegang Li,† Zhonghua Wang,‡ Chenglong Sun,† Luojiao Huang,† Ping Xie,† Xiaoyu Liu,† Jiuming He,*,† and Zeper Abliz*,†,§ †State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China ‡College of life and environmental sciences, Minzu University of China, Beijing 100081, China §Center for imaging and systems biology, Minzu University of China, Beijing 100081, China ABSTRACT: Ion suppression from the tissue matrix has a severe effect on the mass spectrometry imaging (MSI) of drugs. This problem hinders further applications of MSI in preclinical drug research and development. In this study, an in situ hydrogel conditioning method was developed to enhance the sensitivity of air-flow-assisted desorption electrospray ionization (AFADESI)-MSI. Instead of the traditional wash-up or digestion treatment in solvent, this method used a solid phase hydrogel to “wash” tissue sections. It was demonstrated that this in situ hydrogel conditioning method improve the drug signal by as much as 2- to 25-folds in MSI, especially for hydrophobic compounds. Furthermore, the obvious dislocation of analytes was not observed. The evaluation of spatial resolution indicated that the amount of dislocation in tissue sections with hydrogel process was less than the resolution of AFADESI-MSI. The underlying reasons for the MSI signal enhancement were initially investigated. The decreased signal intensities of choline, betaine and carnitine and the increased intensities of the [M+H]+/[M+Na]+ and [M+H]+/[M+K]+ ratios for drugs in the mass spectra of pretreated tissues provided evidence that this method can reduce the levels of highly competitive quaternary ammonium and inorganic salts in the tissues. The preformation of a thin liquid film for droplet-pickup would also raise the ionization efficiency of drugs. These results demonstrated that this in situ hydrogel conditioning method provides a rapid and feasible approach to improving the sensitivity of ambient MSI for drug mapping in tissues.
Ambient mass spectrometry imaging (MSI) is a powerful tool that yield deep insight into the distribution of drugs and biomolecules1-5 at the whole-body,6-9 organ,10-15 tissue16-21 or cellular level.22-24 However, a limited sensitivity for drugs and metabolites, due to the severe ion suppression effect, is one of the drawbacks preventing the wide application of MSI in preclinical drug research and development. Because it is unclear whether a negative result for drug detection via MSI is due to the lack of drug perfusion in a certain biological region or a false negative result caused by the annihilation of the ion signal.25 In a MSI experiment, the ion signal intensity of a drug is not only dependent on the content of the drug in tissue but also sensitive to the local chemical environment, which has various suppressive effects.26,27 Chemical species with a quaternary ammonium group that are in high abundance and response have an intrinsic advantage to result in higher ion yields in competition with drug ionization. In addition, the inorganic salts in native tissue will also suppress drug ionization.28-30 Some washing or digestion strategies have been proposed to date, such as methods using organic solvents, pH-controlled buffer solutions or enzymes.31-33 These methods could effectively lessen salt-or lipid-induced ion suppression, thereby allowing the target drug or protein to achieve a higher response. However, these approaches usually require the tissue section to be immersed into a liquid solvent or sprayed by digestive reagents. The water-soluble or hydrophobic endogenous metabolites might be dissolved in the
buffer solution or organic solvent, respectively. These components would be diffused with the mobile solvent and dislocated on the periphery of the section region. There would also be severe loss of some important endogenous metabolites that were strongly related to the drug action or disease progression. To remove interferents from tissues with less loss in chemical composition and spatial changes, it was necessary to introduce an immobilized medium to complete the washing process. Glenn A. Harris et al. used an alginic acid-calcium hydrogel-mediated digestion method followed by MALDI-MSI to simultaneously extract and identify a peptide from a single tissue section.34 Domenico Taverna et al. used polyacrylamide hydrogel to assist proteolytic digestion.35 In the above mentioned works, hydrogel was introduced as an immobilized medium to guarantee the least amount of change in the tissue structure. Because the hydrogel scaffold can tightly hold a large amount of water in a solid phase, it is hypothesized that hydrogel will have an extractive effect on small molecule interferents in tissues. In this study, an in situ hydrogel conditioning method was developed using a water-enriched solid phase “wash” strategy to extract inorganic salts and high-polar substances responsible for the ion suppression effect. This method was designed with agarose as a support substrate for hydrogel to which abundant water molecules were tightly bound in the solid state at room temperature, with a hydrated layer on the outer gel surface (Figure 1).
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Analytical Chemistry
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LXY6006 (Table S1), a new antitumor candidate for several human carcinomas,36 was selected as the model drug with a plan to determine the in vivo drug distribution using ambient MSI. To map the target molecules in tissues, we employed AFADESI-MSI in our research. Drug-spiked model tissue sections, organs and whole-body sections of a dosed mouse were prepared to optimize the hydrogel conditioning protocol and test the efficiency of the ionization enhancement. The influence of the hydrogel conditioning process on component migration and the MSI spatial resolution was also evaluated. Moreover, several types of drugs, including the antitumor drug paclitaxel, were also used to evaluate not only the underlying reasons for in situ conditioning but also the relationship between the enhancement of the molecular signal and the corresponding lipo-hydro partition coefficient. This research demonstrated that the developed in situ cleanup strategy has a relatively wide applicability for drug signal enhancement in ambient MSI.
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AFADESI-MSI experiment. As was illustrated in our previous work,37 the AFADESI system was built to accomplish the direct desorption and ionization of an analyte on the tissue surface. The Q-Orbitrap mass spectrometer (Q Exactive, Thermo Scientific, Bremen, Germany) was employed to perform the MSI experiments in full MS, target-selected ion monitoring (t-SIM), or full MS/t-SIM alternative mode. To keep the scan rate consistent, the AGC and maximum injection time were set at 3E6 and 200 ms, respectively (Figure S1). MSI Data analysis. To identify the ions that had significantly changed with the increase of conditioning period, the change tendency of the drug ion intensity was selected to locate endogenous ions that had a highly positive or negative correlation with the change in the drug response. The calculated lipo-hydro partition coefficient (cLogP) was employed to investigate the relationship between the molecular properties and the enhancement of the ionization efficiency of these molecules.
RESULTS AND DISCUSSION
Sensitivity improvements. To achieve the optimal efficiency for drug signal enhancement, several key parameters were tested (Figure S2). Two duplicates model tissue sections containing serial dilutions of LXY6006 were tested before and after the section pretreatment to determine whether linearity could be maintained during the hydrogel process (Figure 2). The results not only displayed an ideal linearity within the 144-folds’ concentration range (r = 0.997) but also revealed a significant improvement in the limit of detection (LOD) and response slope for the calibration curve (P
