Utilizing a Mini-Humidifier To Deposit Matrix for ... - ACS Publications

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Utilizing A Mini-humidifier To Deposit Matrix For MALDI-Imaging Xi Huang, Lingpeng Zhan, Jie Sun, Jinjuan Xue, Huihui Liu, Caiqiao Xiong, and Zongxiu Nie Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01714 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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Analytical Chemistry

Utilizing A Mini-humidifier To Deposit Matrix For MALDI-Imaging Xi Huang†‡, Lingpeng Zhan†‡, Jie Sun†‡, Jinjuan Xue†‡, Huihui Liu†, Caiqiao Xiong†, Zongxiu Nie*†‡§ †Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡University of Chinese Academy of Sciences, Beijing 100049, China §National Center for Mass Spectrometry in Beijing, Beijing 100190, China

ABSTRACT: MALDI mass spectrometry imaging (MALDI-MSI) is a powerful tool to study endogenous metabolites. The process of matrix deposition is crucial for a high-quality imaging result. Commercialized instruments for matrix deposition are expensive. Low-cost methods like airbrush will generate too large matrix crystals for high spatial resolution imaging. Sublimation may cause some compounds to go undetected because of the lack of solvent. Herein, we utilized a mini-humidifier, cost less than 5 dollars, to deposit matrix for MALDI-MSI. Compared with Imageprep, a commercialized instrument, our device based on the humidifier provided higher sensitivity and much smaller matrix crystals with diameter less than 10 µm. High-quality ion images in 10 µm spatial resolution were obtained using our method. The enhancement of sensitivity by the humidifier could provide sufficient ion to perform tandem mass imaging. We also performed MALDI MS/MS imaging to separate two lipids in mouse brain.

Metabolites like amino acids, fatty acids, oligosaccharides and lipids play an important role in the body. Significant change in the level of those metabolites in tissue have been observed in nervous disease1, ischemia2, and cancer3,4. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful tool to characterizing the spatial distribution of different metabolites simultaneously5. Nowadays, there are lots of work using MALDI-MSI in cancer research6,7, nervous diseases8,9, cardiovascular diseases10, and rheumatology11, etc. In recent year, various organic matrix have been developed to detect metabolites of which mass below 1000 Da. 2,5-dihydroxybenzoic acid (DHB)12,13 and 14,15 α-Cyano-4-hydroxycinnamic acid were commonly used for analysis in positive ionization mode. 9-aminoacridine16, N-(1-naphthyl) ethylenediamine dihydrochloride17 and 1,5-naphthalenediamine18 were developed to detect metabolites in negative mode. 1,5-naphthalenediamine dihydrochloride (DANHCl)9, a modified matrix from 1,5-naphthalenediamine, was found to be sensitive to detect both lipid and other acidic metabolites. However, it is still tricky to get a high-quality MALDI-MSI image. Matrix coating is a crucial matter for a good MALDI-MSI result. Besides laser beam, the spatial resolution of MALDI-MSI will be strongly affected also by the size of matrix crystals, of which diameter should be smaller than the targeted spatial resolution. Several methods like sublimation, spotting19,20 and spraying have been applied for matrix coating. Sublimation21 provides smallest matrix crystal. However, this method without solvent always have to recrystallize the matrix later to enhance detection sensitivity. Matrices have high sublimation temperature also can’t be deposited by this method. Air brush22 and Imageprep7,23,24 (Bruker Daltonics, Billerica, MA)

can generate micro-droplets. These droplets will touch with tissue surface and extract metabolites to form co-crystals. Such methods tend to generate matrix crystal with diameter bigger than 50 µm, which are not compatible with high spatial resolution MALDI-MSI. Some spraying methods with a oscillating capillary nebulizer have been developed to generate smaller matrix crystals. However, commercial devices25,26 based on this principle are expensive (> 50,000 dollars). Some homemade devices27-29 can also reach good results but it need to optimize many spraying parameters like voltage, flow rate of solution and gas, distance between sample and spray nozzle. Herein, we have developed matrix deposition system with a low-cost mini-humidifier (~5 dollars), to generate uniform matrix crystals with diameter below 10 µm, which is compatible with high resolution MALDI-MSI. Compared with Imageprep, our method is able to enhance both sensitivity and spatial resolution in MALDI-MSI. Experimental Section Chemicals and Reagents. 1,5-DAN, DHB for matrix preparation, were purchased from Sigma-Aldrich (St. Louis, MO). Male Kunming mice were provided by the Experimental Animal Center of Peking University. The animal experiments were performed according to the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication, No. 3040-2, revised 1999, Bethesda, MD) and were approved by the Animal Care and Use Committee of the Chinese Academy of Sciences. Tissue Dissecting. Fresh-frozen mouse brain samples were cut into sections of 10 µm thickness using a Leica CM1950 cryostat (Leica Microsystems GmbH, Wetzlar, Germany) at

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-20 ◦C and thaw mounted onto indium tin oxide (ITO) coated glass slides. The glass slides were then placed into a vacuum desiccator for approximately 30 minutes before matrix application. Matrix Deposition. Solutions of DANHCl were prepared according to the methods described in the Supporting Information. For automatic matrix deposition, the matrix solution, DHB (50% methanol/water, 0.1% TFA, 15 mg/mL) or 1,5-DANHCL (30% ethanol/water, 4 mg/ml), was sprayed onto the tissue sections mounted onto ITO coated glass slides by Imageprep (Bruker Daltonics, Billerica, MA). The matrix was sprayed onto the slides with 80 thin layers for DHB and 40 layers for DANHCl. A mini-humidifier (Guangxin Technology Co. Ltd, Shenzhen, China) was used to make a matrix sprayer. The protocol to make the matrix sprayer was in Figure S1. In brief, the matrix sprayer contained an ultrasonic atomizing tablet, a circuit board and a solution container. This matrix sprayer would generate micro-droplets by its ultrasonic atomizing tablet (flow rate= 35 ml/h, diameter of atomizing area= 4 mm). The circuit board would generate high frequency signal (110 kHz) to vibrate the atomizing table. The circuit board also had a USB-interface and a power switch. We used a mobile phone charger to connect the matrix sprayer with an electrical socket. For matrix deposition by the humidifier, slides were placed in the chamber of Imageprep. The atomizing tablet was pointed to the center of slides. The distance between the atomizing tablet and slides was 15 cm. Same DHB or 1,5-DANHCl solution as described above were add in batches into the container of the humidifier until the matrix on the slides reached same weight as matrix deposited by Imageprep. MALDI-MSI An Ultraflextreme MALDI-TOF/TOF MS (Bruker Daltonics, Billerica, MA) equipped with a smartbeam (Nd:YAG 355 nm) laser was utilized for MALDI analysis. The laser was fired at a repetition rate of 2000 Hz, and the analyzer was operated in reflectron mode. Detailed settings were described in the Supporting Information. The mass spectra data were acquired over a mass range of m/z 0-1000 Da. Mass calibration was performed with PEG-600 for positive mode and oligosaccharides for negative mode. For MSI analysis, each spectrum consists of 200 laser shots. MALDI mass spectra in ion image were performed with the total ion current (TIC) normalization. Further Detailed Structural Confirmation MS/MS fragmentations using the LIFT technique on the Ultraflextreme MALDI-TOF/TOF MS together with Fourier transform ion cyclotron resonance (FTICR) MS as well as Q-TOF MS were used for further confirmation of the identified metabolites. Detailed information was described in the Supporting Information. Metabolites were identified or predicted by comparing MS or MS/MS or referring to databases (METLIN, http://metlin.scripps.edu/; MassBank, http://www.hmdb.ca/; and LIPID MAPS, http://www.lipidmaps.org/). Results and Discussion Matrix deposition for MALDI-MSI We firstly used Imageprep to deposit DHB or DANHCl and weighed that there was 7 mg DHB and 1.5 mg 1,5-DANHCl deposited on the ITO slide. After that, we deposited DHB and 1,5-DANHCl by the humidifier in same weight (Figure 1A). The distance between the slide and atomizing tablet was 15 cm. Such

distance could let droplets cover the whole slide (Figure S2A) and avoided tissue becoming overwet. The size of DHB and 1,5-DANHCl crystals formed in both methods were characterized by microscope. DHB crystals formed in Imageprep method were needle like with diameter over than 50 µm (Figure 2B), which were too big for high spatial resolution MALDI-MSI. However, we found that small and dense DHB crystals could be generated by our humidifier method. Those DHB crystals had average diameter around 10 µm (Figure 2E), which meant it might be compatible with high spatial resolution MALDI-MSI. Besides, both humidifier and Imageprep were able to generate needle-like DANHCl crystals below 10 µm (Figure 2D and G). Aggregates of those DANHCl crystals could be observed in optical image (Figure 2C and F), which had darker colour. The diameter of these aggregates was in proportion to the size of solvent droplets, which showed the humidifier would generate smaller droplet than Imageprep. To directly measure the droplet generated by the humidifier, we deposited solvent (30% ethanol/water) on a glass slide and used a CCD camera to record this process in real time. As shown in Figure S2B，the diameter of all droplets deposited on the glass slide was less than 9 micrometers.

Figure 1. (A) Schematic diagram of the matrix-coating device, made by a mini-humidifier. (B) Optical images of DHB crystals obtained by the Imageprep. (C), (D) Optical images of DANHCl crystals obtained by the Imageprep. (E) Optical images of DHB crystals obtained by the humidifier. (F), (G) Optical images of DANHCl crystals obtained by the humidifier. Aggregates of DANHCl crystals were noted by yellow circle. MALDI-MSI in positive ionization mode After matrix deposition with DHB, MALDI-MSI of the whole mouse brain


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Analytical Chemistry in 100 µm spatial resolution were performed. We compared

the results of two matrix deposition method by absolute signal

Figure 2. Results of MALDI-MSI coated with DHB in positive ionization mode. (A) Average mass spectrum of the whole brain, coated matrix by the humidifier or Imageprep. The vertical axis was absolute ion intensity. (B) Optical image of the H&E-stained whole brain. RGB ion images of the whole brain coated matrix by the humidifier (C) and Imageprep (D) in 100 µm spatial resolution. (E) Optical image of H&E-stained cerebellum. RGB ion images of cerebellum coated matrix by the humidifier (F) and Imageprep (G) in 20 µm spatial resolution. For RGB ion images in this figure, red: PI-Cer(d40:0), m/z= 866.6; green: PC(40:6), m/z= 872.6; blue: PC(36:4), m/z= 820.5. The scale bars were 2.5 mm in C, D and 500 µm in F, G.

Figure 3. Results of MALDI imaging coated with DANHCl in negative ionization mode. (A) Average mass spectrum of the whole brain, coated matrix by the humidifier or Imageprep. The vertical axis was absolute ion intensity (B) Optical image of H&E-stained whole brain. RGB ion images of the whole brain coated matrix by the humidifier (C) and Imageprep (D) in 100 µm spatial resolution. Red: PA(36:1), m/z= 701.5; green: PS(36:2), m/z=786.5; blue: FA(20:4), m/z= 303.2. (E) Optical image of H&E-stained cerebellum. RGB ion images of cerebellum coated matrix by the humidifier (F) and Imageprep (G) in 10 µm spatial resolution. Red: ATP, m/z= 506.0; green: PECer(d38:1), m/z= 715.6; blue: PE(38:4), m/z= 766.5. The scale bars were 2.5 mm in C, D and 250 µm in F, G.

intensity in average mass spectrum (Figure 2A). In spectrum of humidifier method, there were abundant signals of lipids. For spectrum of Imageprep, signals of lipids were poor. Most

of lipids had intensity enhancement by 2~15 times using our humidifier (Table S1). Clear and smooth RGB ion image were obtained by humidifier method, which matched well with H＆



E containing results (Figure 2B and C). The ion image obtained by Imageprep was much darker and lose some pixels (Figure 2D). To perform high spatial resolution MALDI imaging (20 µm), we set laser spot as ‘minimum’ with 10% energy. For the tissue treated by the humidifier, craters caused by laser ablation using such condition had diameter smaller than 10 µm (Figure S2C). The molecular layer, granular layer and white matter of the mouse cerebellum were clearly distinguished in the RGB ion image (Figure 2E and F). For the tissue treated by Imageprep, only pattern of DHB crystals could be recognized (Figure 2G). This result suggested that crystal size would strongly affect the quality of MSI result. To obtain a high-quality ion image, the diameter of crystals should be smaller than the spatial resolution set in the MSI sequence. In addition, more brain samples prepared in different time were imaged to test the reproducibility (Figure S3). These MSI results for mouse brains were reproducible in both 100 µm and 20 µm spatial resolution. MALDI-MSI in negative ionization mode We then performed MALDI-MSI in 100 µm spatial resolution for mouse brains coated with 1,5-DANHCl. From the average spectrum we found that both Imageprep and humidifier methods were able to provide strong signals of endogenous metabolites (Figure 3A). There was moderate enhancement by humidifier for fatty acid and sulfatide, but phosphatidic acid, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine had strong enhancement (Table S2 and S3). From the ion images of picked molecules, we found the humidifier still provided clearer images with more uniform pixels (Figure 3B, C, D and Figure S4). This might because the humidifier could generate smaller and more uniform aggregates of DANHCl. Similar phenomenon was more obvious when we performed MALDI imaging in 10 µm spatial resolution (Figure 3E, F, G and Figure S4). A MALDI-MSI in 10 µm spatial resolution of dentate gyrus of the hippocampus was also performed after matrix-deposition by the humidifier. A structure consisted of granular cells with 20 µm width were clearly observed (Figure S5). These results showed not only size of matrix crystals but also size of solvent droplets would influence quality of ion images. Smaller droplets would reduce diffusion of small molecule metabolites, and distortion in ion images caused by aggregates of matrix. It should be noted we do not mean that only the humidifier of a specific brand can deposit matrix. We also used a humidifier of another brand (Youpinku Technology Co. Ltd, Shenzhen, China) to do the same experiment, which could obtain high-quality ion images either (Figure S6). We wrote some suggestions on how to select the appropriate mini-humidifier for matrix deposition (in Supporting information). MALDI MS/MS Imaging Mass spectrum in areas of lipids (600~1000 Da) are difficult to analyze. Despite of isomers, lipids will generate multiple isotopic peaks which have notable signal intensity. Such isotopic peaks may interfere with the detection of another species of lipid because their m/z are too close. For an example, we found the peak m/z=909.6, which should be the position of fourth isotopic peak of C24-OH, had totally different distribution with the first three isotopic peaks of C24-OH (m/z=906.6346) in ion images (Figure S7A). We used MALDI-FTICR to detect different places of brain and found there were two species around m/z=909.6. The ion images of m/z=909.6 would become ambiguous if both of

them appeared in same position (Figure S7B, C and D). It required sufficient parent ions intensity to perform MALDI-MS/MS analysis by LIFT. Thanks to the three times enhancement of sensitivity by humidifier (Figure S7E), it was easy to perform MS/MS imaging to separate these two species. Besides the fourth isotopic peak of C24-OH (m/z=909.6544), we identified another peak PI(18:0/22:6) (m/z=909.5515) by MS/MS spectrum (Figure S8). From MS/MS ion images, we found C24-OH showed high intensity in white matter of cerebellum, inferior colliculus and callosum (Figure 4A). PI(18:0/22:6) were mainly located in granular layer of cerebellum. There were low amount of PI(18:0/22:6) in commissure of inferior colliculus and no detection in callosum (Figure 4B). Compared with MS/MS ion images, MS ion image would show a merged pattern of PI(18:0/22:6) and C24-OH(Figure 4D). It is important to generate an unambiguous image, especially for determination of border of tumors and distribution of drugs. Our matrix deposition method had potential to be used in these fields to provide high-quality ion images without ambiguity. Conclusion The humidifier could coat tissue with small and uniform matrix crystals. Compared with a commercialized instrument, we enhanced both sensitivity and quality of ion images by the humidifier. As we know, it was the lowest-cost method to deposit matrix for high spatial resolution MALDI-MSI so far. Future works will apply the humidifier to other pretreatments of MALDI-MSI, such as digestion, derivatization and recrystallization.

Figure 4. MS/MS ion image of mouse brain obtained by LIFT in 100 µm spatial resolution. The tissue was coated with DANHCl by humidifier. (A) MS/MS ion image of [HOSO3]- (m/z=97.0), a fragment ion of C24-OH sulfatide (m/z=909.6544). (B) MS/MS ion image of [FA(22:6)]- (m/z=327.3), a fragment ion of PI(18:0/22:6) (m/z=909.5515). (C) Optical image of H&E-stained brain. (D) MS ion image of m/z= 909.6.

ASSOCIATED CONTENT


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Analytical Chemistry Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Details about the experiments methods, Suggestions on how to select and use mini-humidifiers, Tables S1−S3, and Figures S1−S8

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ACKNOWLEDGMENT This work was supported by grants from the National Natural Sciences Foundation of China (Grant Nos. 21625504, 21505140, 21621062, 21675160, 21475139 and 21790390/21790392) and Chinese Academy of Sciences.

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