Imaging of Polar and Nonpolar Species Using ... - ACS Publications

Subscriber access provided by EDINBURGH UNIVERSITY LIBRARY | @ http://www.lib.ed.ac.uk

Article

Imaging of polar and nonpolar species using compact desorption electrospray ionization/post-photoionization mass spectrometry Chengyuan Liu, Keke Qi, Lei Yao, Ying Xiong, Xuan Zhang, Jianye Zang, Changlin Tian, Minggao Xu, Jiuzhong Yang, Zhenkun Lin, Yongmei Lv, Wei Xiong, and Yang Pan Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 26, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Imaging of polar and nonpolar species using compact desorption electrospray ionization/post-photoionization mass spectrometry

Chengyuan Liu1, Keke Qi1, Lei Yao2, Ying Xiong2, Xuan Zhang2, Jianye Zang2, Changlin Tian2, Minggao Xu1, Jiuzhong Yang1, Zhenkun Lin3, Yongmei Lv4, Wei Xiong2,*, and Yang Pan1,* 1 National

Synchrotron Radiation Laboratory, University of Science and Technology of China,

Hefei 230029, China 2

Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences,

University of Science and Technology of China, Hefei 230026, China 3

Center of Scientific Research, The Second Affiliated Hospital and Yuying Children’s Hospital of

Wenzhou Medical University, Wenzhou 325027, China 4

Department of Dermatology, The Second Affiliated Hospital of Anhui Medical University, Hefei

230601, China *Address reprint requests to: Wei Xiong, Tel: +86- 551-63601736 Email: [email protected] Yang Pan, Tel: +86-551-63601986 Email: [email protected]

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 20

ABSTRACT Desorption electrospray ionization (DESI) mass spectrometry imaging (MSI) can simultaneously record the 2D distribution of polar biomolecules in tissue slices at ambient condition. However, sensitivity of DESI-MSI for nonpolar compounds is restricted by low ionization efficiency and strong ion suppression. In this study, a compact post-photoionization assembly combined with DESI (DESI/PI) was developed for imaging polar and nonpolar molecules in tissue sections by switching off/on a portable krypton lamp. Compared with DESI, higher signal intensities of nonpolar compounds could be detected with DESI/PI. To further increase the ionization efficiency and transport of charged ions of DESI/PI, the desorption solvent composition and gas flow in the ionization tube were optimized. In mouse brain tissue, more than two orders of magnitude higher signal intensities for certain neutral biomolecules like creatine, cholesterol and GalCer lipids were obtained by DESI/PI in positive ion mode, compared with that of DESI. In negative ion mode, ion yields of DESI/PI for glutamine and some lipids (HexCer, PE and PE-O) were also increased by several-fold. Moreover, nonpolar constituents in plant tissue, such as catechins in leaf shoot of tea could also be visualized by DESI/PI. Our results indicate that DESI/PI can expand the application field of DESI to nonpolar molecules, which is important for comprehensive imaging of biomolecules in biological tissues with moderate spatial resolution at ambient condition.

Keywords: desorption electrospray ionization, post-photoionization, mass spectrometry imaging

2


Page 3 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Desorption electrospray ionization mass spectrometry (DESI) was introduced as an ambient desorption ionization method for the direct analysis of condensed compounds from surface in 2004.1 In DESI, analytes were impacted and ionized by charged droplets of solvent generated from electrospray through a so-called “droplet pick-up” mechanism,2-3 which is similar to the ionization mechanism of electrospray ionization (ESI). In recent years, DESI has become a powerful tool for mass spectrometry imaging (MSI) that can enable the simultaneous visualization of hundreds of lipids and metabolites in mouse brain at ambient conditions,4-6 and can also be utilized to image other biological tissues (e.g. tumor margins,

7-10

infarcted myocardium11 and attacked plant

tissues12-14). Compared with conventional vacuum matrix assisted laser desorption ionization (MALDI) for MSI, the most important features of DESI-MSI are its simple structure, matrix-free characteristic, and ability to operate at atmospheric pressure. However, DESI-MSI also has some shortcomings. One notable drawback of DESI-MSI is its poor ionization efficiency against nonpolar compounds, such as endogenous molecules (e.g. cholesterol) in biological tissues. Moreover, severe ion suppression effect also exists during the ESI-like ionization process.15-17,18 For example, polar lipids in mouse brain (e.g. quaternary ammonium-carrying phosphatidylcholines (PC)) are in extremely high abundances, which will suppress the ionization of other neutral metabolites/lipids (e.g. monoacylglycerols (MAG), galactosylceramide (GalCer)) in positive ion mode. Moreover, signal suppression in DESI-MSI is so pronounced in the tissue section with highly concentrated salt,15, 19-20 which often happens in the microenvironment of biological tissues. In such circumstance, constituents in the tissue are sometimes ionized as sodium and/or potassium adducts whose signal intensities change with the concentration of salt.21 To overcome the limitation of DESI-MSI, attempts have been made by chemical derivatization (e.g. reactive DESI)22-24 or pretreatments of tissue slices. Nonpolar cholesterol, for example, can be ionized by reactive DESI by spraying solvent containing a charge-labeling reagent (betaine aldehyde), which could be reacted with the alcohol group of cholesterol by nucleophilic addition.25 Although reactive DESI can enhance the selectivity and sensitivity of certain compounds with the specific alcohol group, simultaneous analysis of other classes of biomolecules is still difficult. Some pretreatments such as washing or digestion have been proposed to lessen salt- or lipid- induced ion 3



Page 4 of 20

suppression to achieve a higher response to neutral species.26-28 However, in these pretreatments, the tissue section was required to be immersed into a liquid solvent or sprayed by digestive reagents. Thus, many solvent-soluble endogenous metabolites might be diffused in the mobile solvent resulting in analyte loss and delocalization. Another applicable way to improve the ionization efficiency of nonpolar compounds in DESI is to introduce a secondary ionization. To the best of our knowledge, the so-called post-ionization technique in MSI has been proposed and emerged with several combinations of desorption and ionization methods. ESI was commonly used as a post-ionization method for laser desorption, such as laser ablation ESI, electrospray-assisted laser desorption ionization, infrared MALDI ESI and laser desorption/ionization droplet delivery.29-32 Photoionization is another popular post-ionization method33 and has no polarity discrimination. Veryovkin et al. used a laser to ionize the neutral atoms desorbed by secondary ion mass spectrometry (SIMS) and a more than one order of magnitude improvement was obtained.34 Soltwisch et al. applied a wavelength-tunable laser beam, which intersects the expanding analyte-matrix plume, to initiate secondary MALDI-like ionization processes. The ion yields for numerous lipids, vitamins and saccharides in animal and plant tissue were increased by up to two orders of magnitude.35-36 In this work, a compact post-ionization assembly based-on photoionization was constructed to initiate a secondary ionization for the desorbed neutrals by DESI (DESI/PI). Owing to the favorable soft ionization, no polarity discrimination, and reduced ion suppression characteristics37-39, photoionization has been widely used for the analysis of nonpolar compounds, such as steroids and polycyclic aromatic hydrocarbons (PAHs).40-43 Laser and discharge lamp are commonly used photoionization sources. Here, a portable and low-cost krypton lamp used as ionization source in atmospheric pressure photoionization (APPI) was selected. This lamp can produce photon energies of 10.0 and 10.6 eV with moderate photon flux of 1011 photons/second, and can produce abundant ions by direct ionization and ion-molecule reactions. We found that with the aid of secondary ionization, many polar and nonpolar compounds, such as some neurotransmitters, MAGs and GalCers in mouse brain tissue, and catechins in leaf shoot of tea could be easily and simultaneously visualized with high signal-to-noise ratio by our DESI/PI techniques. Even the detection of some polar compounds can also benefit from the post-photoionization process together. We show that DESI/PI is complementary to DESI, and can supply a more complete image of biomolecules 4


Page 5 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


distribution in biological tissues. Experimental Chemicals and Sample Preparation. Methyltestosterone, reserpine and cholesterol were purchased from Sigma-Aldrich (St. Louis, MO). High performance liquid chromatography grade methanol, toluene and formic acid (FA) were obtained from Merck (Muskegon, MI). Purified water was obtained from Wahaha (Hangzhou, China). All these chemicals were used without further purification. Nebulization gas (Nitrogen, 99.9%) and drying gas (refrigerated industrial grade liquid nitrogen, 99.8%) were purchased from Nanjing Special Gas Factory Co., Ltd. (Nanjing, China). To compare the performances of DESI and DESI/PI, a mixed solution of 69 μg/mL methyltestosterone, 62 μg/mL cholesterol and 85 μg/mL reserpine was prepared for analysis. Animal and Tissue preparation The animal experiments were approved by the Animal Care and Use Committee of the Chinese Academy of Sciences. Mice were sacrificed by decollation and the brain was immediately removed from the skull and flash frozen in liquid nitrogen for 15 s prior to storage at -80 C. Before analysis, the frozen mouse brain was transferred from -80 C to the cryostat chamber of a Vibratome (VT 1200S, Leica, Germany) at -25 C. The brain tissue was cut into 16-μm-thick sagittal sections and collected onto clean microscope slides. Fresh leaf shoot tissue of tea was collected from local tea garden and treated the same way to get the tissue slice. Optical images of the cross sections of mouse brains and tea leaf shoot were taken by an optical microscope prior to analysis. DESI/PI MSI system. As shown in Figure1, the DESI/PI assembly consisted of a DESI sprayer, a 2D scanning stage and a post-photoionization interface. Solvent was infused at a flow rate 3 μL/min through a DESI sprayer (50 μm i.d. and 150 μm o.d. inner fused silica capillary, and a 250 μm i.d. and 350 μm o.d. outer fused silica capillary) and directed onto the surface of a tissue slice with 53 angle of incidence with the assistance of the nebulizing N2 gas (120 psi). The flow of the solvent was driven by a syringe pump and the metal needle tip was connected to a high-voltage power supply (3500 V 5



Page 6 of 20

for positive ion mode and -4000 V for negative ion mode). A schematic cross-sectional view of the post-photoionization interface was shown in Figure 1A. The desorbed compounds were sucked in the heated transfer tube (i.d. 0.5 mm, o.d. 1/16 inch.) with a 10 angle of collection and the unionized neutral molecules would be ionized in a ionization tube (i.d. 4 mm, o.d. 10 mm) by a coaxially oriented krypton DC discharge vacuum ultraviolet (VUV) lamp (Fig 1C, model PKS 106, Heraeus, Cambridge, U.K.), which was positioned to shine towards the exit of transfer tube. Then the ionized species was transferred into capillary of mass spectrometer. In order to improve the transfer efficiency, an air-flow assisted transport arrangement44 was added in this interface and a pneumatic diaphragm pump (60 L/min, model GM-1.0A, Jinteng Experimental Equipment Co., Ltd., Tianjin, China) was connected to the side port of ionization tube. In experiments, the transfer tube and ionization tube were kept at 300 C. Noted that the krypton lamp was turned off in DESI mode and turned on in DESI/ PI mode.

Figure 1. A) Schematic experimental setup of DESI/PI (the cross-section view of the post-photoionization interface was enlarged); B) Photograph of DESI/PI setup; C) Krypton lamp. 6


Page 7 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


All imaging data were collected on an Agilent 6224 Accurate-Mass TOF mass spectrometer (Agilent, USA). The flow rate and temperature of drying gas of the mass spectrometer were set at 5 L/min and 325 C, respectively. A programmable motorized X-Y scanning stage (GCD-203050M, Daheng, Beijing, China) was used for tissue imaging and the scanning process was allowed to be synchronized with the Agilent mass spectrometer data acquisition by a customized stage control software. The sample surface was line scanned in the X direction with a stepper motor at a velocity of 370 μm/s while acquiring mass spectra every 0.5 s. The distance between adjacent scan lines in the Y direction was 200 μm. The acquired multiple scan lines were combined in one data file for ion distribution images by using the freely available standalone version of the MSiReader software.45 The identifications for most of these peaks were facilitated by accurate m/z values, comparison of isotope distribution patterns and tandem mass spectrometry. All the compounds were assigned within an error of 10 ppm by a comparison with literature data and spectra in a human metabolome46 and a lipid database.47 Results and discussion Assessment of post-photoionization Prior to tissue imaging, the performance of post-photoionization for ionization enhancement was evaluated with a mixed solution of methyltestosterone, cholesterol and reserpine with different polarities. The selection of solvent was carefully considered, since it is important for the analyte dissolution and microdroplets formation in the droplet pick-up process of DESI spray.48 In addition, solvent is also critical for photoionization especially for dopant-assisted ionization.49 In APPI and low pressure photoionization, dopant such as toluene, acetone and anisole are crucial for increasing the photo-induced ionization through ion-molecule reactions such as the proton transfer and charge exchange between analytes and ionized dopant.49-51 In this work, methanol/FA (v:v=100:1) and methanol/toluene/FA (v:v:v=70:30:1) were used to compare the solvent effect in DESI and DESI/PI.

7



Page 8 of 20

Figure 2. Background subtracted mass spectra of the mixed solution of 69 μg/mL methyltestosterone, 62 μg/mL cholesterol and 85 μg/mL reserpine (A) by DESI with methanol/FA (v:v=100:1) as solvent, (B) by DESI with methanol/toluene/FA (v:v:v=70:30:1) as solvent and (C) by DESI/PI with methanol/toluene/FA (v:v:v=70:30:1) as solvent. In each case, an aliquot (1μL) of solution was spotted onto the PTFE film and each experiments were repeated three times.

DESI was first carried out, where the condensed sample (prepared by depositing 1 μL aliquot of standard solution of 69 μg/mL methyltestosterone, 62 μg/mL cholesterol and 85 μg/mL reserpine onto a PTFE film and dried as condensed phased sample) was picked up and ionized by methanol/FA (v:v=100:1) from a sprayer. As shown in Figure 2A, only reserpine ([M+H]+, m/z 609.28) and negligible methyltestosterone ([M+H]+, m/z 303.23) were detected. When methanol/toluene/FA (v:v:v=70:30:1) was used as spray solvent, the DESI-generated ion intensities decreased (Figure 2B), indicating that toluene hindered the ionization of analytes. Figure 2C demonstrates the mass spectrum of DESI/PI using methanol/toluene/FA (v:v:v=70:30:1) as solvent. With the help of photoionization, non-polar methyltestosterone ([M+H]+, m/z 303.23 and [M+H-H2O]+, m/z 285.22) and cholesterol ([M+H-H2O]+, m/z 369.35) ions were generated with high signal-to-noise ratio. For methyltestosterone, a 124-fold higher ion intensity was obtained compared with that of DESI. The limit of detection (LOD) of cholesterol by DESI/PI was determined to be 0.6 ng, which is a little lower than that obtained by reactive DESI (1 ng). As for 8


Page 9 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


polar reserpine that can easily be ionized by DESI (Figure 2A), a more than ten-fold increase of protonated reserpine was observed when post-photoionization was initiated by the krypton lamp, indicating that post-photoionization can also promote the ionization of polar neutrals desorbed by DESI. There is nearly no sample leftover in the heated transfer tube for DESI/PI experiments (Figure S1). Imaging of mouse brain section The detection sensitivity of conventional DESI for neutral species in mouse brain tissue is poor due to the ion suppression effect from quaternary ammonium-carrying PC lipids and inorganic salts. As noted above, to realize effective photo-assisted DESI imaging, the composition of spray solvent needs further optimization. Figure S2 shows the effects of solvent composition on the signal intensity in DESI/PI imaging of mouse brain sections. As the methanol-to-toluene ratio was tuned from 10:0, 9:1, 8:2 to 7:3, the ion intensities of creatine ([M+H]+, m/z 132.08), glutamic acid ([M+H]+, m/z 148.06), cholesterol ([M+H-H2O]+, m/z 369.35) and GalCer(d18:1/C24:1) ([M+H]+, m/z 810.68) gradually increased, owing to higher post-photoionization efficiency at higher toluene content. Since higher toluene content (> 30%) will result in the increase of background noise, methanol/toluene/FA (v:v:v=70:30:1) was finally selected as the most suitable solvent. In comparison, signal intensities of PC(32:0) ([M+K]+, m/z 772.53) and PC(34:1) ([M+K]+, m/z 798.54) demonstrated an opposite trend. The useless of post-photoionization was speculated to the non-volatile nature of ionic PC lipids and lower DESI ionization efficiencies at higher toluene content, which was confirmed by the parallel solvent effects on DESI (Figure S3). Another critical factor for sensitivity improvement is the effective transport of produced ions.44, 52-53

As presented in Figure S4, with the air-flow assisted design (see Figure 1), the signal intensities

of creatine ([M+H]+, m/z 132.08), glutamic acid ([M+H]+, m/z 148.06), cholesterol ([M+H-H2O]+, m/z 369.35), GalCer

(d18:1/C22:1) ([M+H]+, m/z 782.65) and GalCer (d18:1/C24:1) ([M+H]+,

m/z 810.68) were increased 2~4-fold after the pump connected to the side port of ionization tube was turned on.

9



Page 10 of 20

Figure 3. The averaged positive mass spectra of whole brain tissue section (background subtracted) by DESI/PI with methanol/toluene/FA (v:v:v=70:30:1) as solvent and DESI with methanol/FA (100:1) as solvent respectively, and the imaging results of some representative species. (A-P) MS images of mass peaks at m/z 90.06, 104.07, 132.08, 147.08, 148.06, 268.10, 282.28, 339.29, 341.31, 369.35, 548.54, 651.54, 782.65, 808.67, 810.68 and 828.69 obtained by DESI/PI. (Q-X) MS images of mass peaks at m/z 713.45, 756.55, 772.53, 820.53, 826.57, 844.53, 848.64 and 872.56 obtained by DESI. Scale bars in white correspond to 1 mm. Based on the 20−80% rule31, 54-55, the spatial resolution was estimated to be around 200 μm (Figure S5).

The averaged mass spectra in positive ion mode and MS images of some representative ions acquired by DESI/PI and DESI under optimized conditions are compared and shown in Figure 3. The details of ion assignments for DESI/PI and DESI mass spectra are listed in Table S1 and S2, respectively. DESI produced relative fewer mass peaks, which was dominated by ionic choline (M+, m/z 104.11), phosphocholine (M+, m/z 184.07), and PC lipids located in the range of m/z 700-900. In comparison, mass spectrum of DESI/PI is more informative, where the distributions of some neutrals including neurotransmitters (e.g. GABA ([M+H]+, m/z 104.07), glutamine ([M+H]+, m/z 10


Page 11 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


147.08), glutamic acid ([M+H]+, m/z 148.06)), creatine ([M+H]+, m/z 132.08), and adenosine ([M+H]+, m/z 268.10) were clearly visualized (see Figure 3B-E). As an example, the signal intensity of creatine was increased by up to 200-fold. In addition to neurotransmitters, more than ten nonpolar lipids including abundant cholesterol ([M+H-H2O]+, m/z 369.35 and [M-H]+, m/z 385.35) and GalCer were found to locate in the white matter of cerebellum and corpus callosum (see Figure 3J and N-P), which is consistent with previous reports.27,

56

The peaks marked by fragments in

Figure 3 were also speculated to be derived from various lipids. For instance, the ions at m/z 630.62 can

be

formed

from

GalCer

(d18:1/C24:1)

by

deglycosylation

and

dehydration

([M+H-C6H10O5-H2O]+) which was proved by the DESI/PI analysis of authentic standard GalCer (d18:1/C24:1) (Figure S8C). Cholesterol is a versatile molecule known as an essential membrane component and serves as the precursor of steroid hormones.57-58 GalCer is the principle glycosphingolipid in brain and is also a specific cell-surface antigenic marker for oligodendrocytes.59 Lipids have been indicated to be involved in the modulation of neurotransmitter release in the central nervous system.60 Thus, the simultaneous imaging of neurotransmitters and lipids has a promising application in neuroscience, pharmacology and neurochemistry research. Next, negative ion mode was tested since it can provide valuable complementary information compared with positive ion mode, and is more effective in the MS visualization of malignancies and evaluation of surgical margin.7-8 Figure 4 presents the averaged mass spectra, optical images and MS images of two consecutive brain slices using DESI/PI and DESI with methanol/toluene (v:v=7:3) as solvent. Detailed peak identifications are listed in Table S3. As can be seen in Figure 4A, ion suppression effects were less pronounced in negative ion DESI mode, and several lipid classes including phosphatidylethanolamine (PE), plasmalogens (PE-O), phosphatidylserine (PS), phosphatidylinositols (PI), and sulfatides (ST) could be detected by both DESI and DESI/PI.

11



Page 12 of 20

Figure 4. The averaged mass spectra of two slices of brain tissue section (background subtracted) acquired by DESI/PI and DESI in negative ion mode (A). Methanol/toluene (v:v=7:3) was used as solvent. The optimal images of the brain tissue are shown in B and the MS images of some representative peaks at m/z 145.06, 146.05, 700.53, 726.54, 750.54, 766.54, 790.54, 798.64, 834.53, 885.55 and 888.63 are presented in C-M. Scale bars in white correspond to 1 mm.

The MS images obtained by DESI and DESI/PI are complementary in negative ion mode. For example, some neutral compositions like glutamine ([M-H]-, m/z 145.06, Figure 4B), glutamic acid ([M-H]-, m/z 146.05, Figure 4C) and hexosylceramides (HexCer) ([M-H]-, m/z 798.64, Figure 4I) could only be imaged by DESI/PI. When post-photoionization was carried out, the ion yields of PE-O and PE lipids, such as PE(O-34:1) ([M-H]-, m/z 700.53), PE(O-36:3) ([M-H]-, m/z 726.54), PE(O-38:5) ([M-H]-, m/z 750.54), PE(38:4) ([M-H]-, m/z 766.54) and PE(40:6) ([M-H]-, m/z 790.54) 12


Page 13 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


could be increased by several-fold (2-4 times), and more informative images could be obtained (see Figure 4E-H). In the cases of PI and ST lipids, such as PI(38:4) ([M-H]-, m/z 885.55, Figure 4K) and ST(d18:1/C24:1) ([M-H]-, m/z 888.63, Figure 4L), the ion intensities obtained by DESI were much higher than those of DESI/PI. The decrease in these ions were speculated to be the combination of these negative ions with the positive toluene ions generated by post-photoionization. Nonetheless DESI/PI still provided superior sensitivity for most metabolites and lipid classes. Imaging of tea Another use of DESI/PI is for visualizing the spatial distribution of polar and nonpolar metabolites in plants. The metabolites in plants have been imaged by different MSI technologies.61-62 As the most widely consumed beverage in the world after water, tea is rich in various kinds of bioactive constituents. For example, catechins account for up to 30 % of the dry weight of fresh tea leaves63 and are believed to be responsible for the health benefits of the consumption of tea.64-65 However, due to their low polarity, the ionization efficiency of these catechins by DESI is poor. Two consecutive fresh leaf shoot cross sections of tea plant were analyzed by DESI/PI and DESI respectively. Figure 5 shows the optical image, averaged mass spectra and MS images of some representative metabolites in leaf shoot of tea. The main peak assignments are summarized in Table S4. For DESI experiment, the most prominent peak was phosphocholine at m/z 184.07. Moreover, DESI showed a relatively weak peak at m/z 195.09, which is attributed to protonated caffeine ([M+H]+), the most abundant constituent in tea. However, when DESI/PI was employed, the signal intensity of caffeine was increased by 200-fold, indicating the great contribution of post-photoionization. In addition, neutral catechins including (-)-epicatechin (EC), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate flavan-3-ols (EGCG) could be detected and imaged by DESI/PI. The ECG and EGCG are thermally labile compounds and thus their fragments ([M+H-C7H6O5]+) were detected at m/z 272.07 and 289.07, respectively. The assignments were further proved by the DESI/PI mass spectra of standards of EC, ECG and EGCG (Figure S10). Our results demonstrate that DESI/PI can increase the detection sensitivity of neutral species and also broaden the applicability of DESI for visualizing nonpolar biomolecules in the MSI of plant tissues, and can be considered as an effective and alternative technique for MALDI with moderate lateral resolution. 13



Page 14 of 20

Figure 5. The averaged mass spectra of two consecutive fresh leaf shoot cross sections of tea plant (background subtracted) acquired by DESI/PI with methanol/tolene/FA (v:v:v=70:30:1) as solvent and DESI with methanol/FA (100:1) as solvent respectively in positive ion mode. (A) The optimal images of the leaf shoot tissue of tea. (B-F) MS images of some representative peaks at m/z 184.07, 195.09, 272.07, 289.07 and 291.09 obtained by DESI/PI. (G-H) MS images of two peaks at m/z 184.07 and 195.09 obtained by DESI. Scale bars in white correspond to 1 mm.

Conclusions In this study, a setup combining conventional DESI with a post-photoionization was established for enhancing the ionization and imaging of desorbed neutral molecules in biological tissue sections. This setup can be switched between conventional DESI mode (with lamp off) and 14


Page 15 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DESI/PI mode (with lamp on). The nonpolar lipids including cholesterol, MAGs and GalCers, neurotransmitters and other metabolites in brain tissue could be imaged simultaneously by DESI/PI without the pretreatments like washing or derivatization at ambient condition, and more than two orders of magnitude higher signal intensities for neutral species like creatine, cholesterol and GalCer lipids were obtained relative to DESI. This method is also suitable for the imaging of neutral metabolites in plant tissue. More importantly, the DESI/PI setup was equipped with a low-cost krypton lamp rather than a laser system. This setup is portable and compact, and can be connected to other MS inlets with minor modifications, by which components with different polarities in tissues sections can be visualized simultaneously with moderate lateral resolution. Hence, DESI/PI can act as a complementary technique to existing MSI methods for the comprehensive imaging of small molecules and is promising in the application of neuroscience, pharmacology, clinical tumor margin and neurochemistry research. Acknowledgements This work was supported by grants from the National Key Research and Development Program of China (2016YFA0400903 and 2016YFC1300500-2), National Natural Science Foundation of China (Grants 91849206,

91649121 and 31471014), the Strategic Priority Research

Program of the Chinese Academy of Sciences (XDPB10 and XDB02010000), the Chinese Universities Scientific Fund, and the Major/Innovative Program of Development Foundation of Hefei Center for Physical Science and Technology (2016FXCX008 and 2017FXZY006), the Key Program of Research and Development of Hefei Science Center CAS (2018HSC-KPRD002), the Users with Excellence Project of Hefei Science Center CAS (2018HSC-UE001 and 2018HSC-UE016), and CAS Interdisciplinary Innovation Team (JCTD-2018-20).

15



Page 16 of 20

References (1) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Mass spectrometry sampling under ambient conditions with desorption electrospray ionization Science 2004, 306 (5695), 471-3. (2) Takats, Z.; Wiseman, J. M.; Cooks, R. G. Ambient mass spectrometry using desorption electrospray ionization (DESI): instrumentation, mechanisms and applications in forensics, chemistry, and biology J. Mass Spectrom. 2005, 40 (10), 1261-75. (3) Miao, Z.; Chen, H. Direct analysis of liquid samples by desorption electrospray ionization-mass spectrometry (DESI-MS) J. Am. Soc. Mass Spectrom. 2009, 20 (1), 10-19. (4) Wiseman, J. M.; Ifa, D. R.; Song, Q.; Cooks, R. G. Tissue imaging at atmospheric pressure using desorption electrospray ionization (DESI) mass spectrometry Angew. Chem. Int. Ed. 2006, 45 (43), 7188-7192. (5) Wiseman, J. M.; Ifa, D. R.; Zhu, Y.; Kissinger, C. B.; Manicke, N. E.; Kissinger, P. T.; Cooks, R. G. Desorption electrospray ionization mass spectrometry: Imaging drugs and metabolites in tissues Proc. Natl. Acad. Sci. 2008. (6) Hsu, C.-C.; Chou, P.-T.; Zare, R. N. Imaging of proteins in tissue samples using nanospray desorption electrospray ionization mass spectrometry Anal. Chem. 2015, 87 (22), 11171-11175. (7) Eberlin, L. S.; Margulis, K.; Planell-Mendez, I.; Zare, R. N.; Tibshirani, R.; Longacre, T. A.; Jalali, M.; Norton, J. A.; Poultsides, G. A. Pancreatic Cancer Surgical Resection Margins: Molecular Assessment by Mass Spectrometry Imaging PLoS Med. 2016, 13 (8), e1002108. (8) Banerjee, S.; Zare, R. N.; Tibshirani, R. J.; Kunder, C. A.; Nolley, R.; Fan, R.; Brooks, J. D.; Sonn, G. A. Diagnosis of prostate cancer by desorption electrospray ionization mass spectrometric imaging of small metabolites and lipids Proc. Natl. Acad. Sci. 2017, 114 (13), 3334-3339. (9) Margulis, K.; Chiou, A. S.; Aasi, S. Z.; Tibshirani, R. J.; Tang, J. Y.; Zare, R. N. Distinguishing malignant from benign microscopic skin lesions using desorption electrospray ionization mass spectrometry imaging Proc. Natl. Acad. Sci. 2018, 115 (25), 6347-6352. (10) Banerjee, S. Ambient ionization mass spectrometry imaging for disease diagnosis: Excitements and challenges J. Biosci. 2018, 43 (4), 731-738. (11) Margulis, K.; Zhou, Z.; Fang, Q.; Sievers, R. E.; Lee, R. J.; Zare, R. N. Combining Desorption Electrospray Ionization Mass Spectrometry Imaging and Machine Learning for Molecular Recognition of Myocardial Infarction Anal. Chem. 2018, 90 (20), 12198-12206. (12) Esquenazi, E.; Dorrestein, P. C.; Gerwick, W. H. Probing marine natural product defenses with DESI-imaging mass spectrometry Proc. Natl. Acad. Sci. 2009, 106 (18), 7269-7270. (13) Lane, A. L.; Nyadong, L.; Galhena, A. S.; Shearer, T. L.; Stout, E. P.; Parry, R. M.; Kwasnik, M.; Wang, M. D.; Hay, M. E.; Fernandez, F. M. Desorption electrospray ionization mass spectrometry reveals surface-mediated antifungal chemical defense of a tropical seaweed Proc. Natl. Acad. Sci. 2009, 106 (18), 7314-7319. (14) Hemalatha, R.; Pradeep, T. Understanding the molecular signatures in leaves and flowers by desorption electrospray ionization mass spectrometry (DESI MS) imaging J. Agr. Food Chem. 2013, 61 (31), 7477-7487. (15) Jackson, A. U.; Talaty, N.; Cooks, R. G.; Van Berkel, G. J. Salt tolerance of desorption electrospray ionization (DESI) J. Am. Soc. Mass Spectrom. 2007, 18 (12), 2218-2225. (16) Suni, N. M.; Lindfors, P.; Laine, O.; Ostman, P.; Ojanpera, I.; Kotiaho, T.; Kauppila, T. J.; Kostiainen, R. Matrix effect in the analysis of drugs of abuse from urine with desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS) and desorption electrospray ionization-mass spectrometry (DESI-MS) Anal. Chim. Acta 2011, 699 (1), 73-80. (17) Suni, N. M.; Aalto, H.; Kauppila, T. J.; Kotiaho, T.; Kostiainen, R. Analysis of lipids with desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS) and desorption electrospray ionization-mass spectrometry 16


Page 17 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(DESI-MS) J. Mass Spectrom. 2012, 47 (5), 611-619. (18) Oss, M.; Kruve, A.; Herodes, K.; Leito, I. Electrospray ionization efficiency scale of organic compounds Anal. Chem. 2010, 82 (7), 2865-2872. (19) Ellis, S. R.; Bruinen, A. L.; Heeren, R. M. A critical evaluation of the current state-of-the-art in quantitative imaging mass spectrometry Anal. Bioanal. Chem. 2014, 406 (5), 1275-1289. (20) Wu, C.; Dill, A. L.; Eberlin, L. S.; Cooks, R. G.; Ifa, D. R. Mass spectrometry imaging under ambient conditions Mass Spectrom. Rev. 2013, 32 (3), 218-243. (21) Song, X.; Luo, Z.; Li, X.; Li, T.; Wang, Z.; Sun, C.; Huang, L.; Xie, P.; Liu, X.; He, J.; Abliz, Z. In Situ Hydrogel Conditioning of Tissue Samples To Enhance the Drug's Sensitivity in Ambient Mass Spectrometry Imaging Anal. Chem. 2017, 89 (12), 6318-6323. (22) Huang, G.; Chen, H.; Zhang, X.; Cooks, R. G.; Ouyang, Z. Rapid screening of anabolic steroids in urine by reactive desorption electrospray ionization Anal. Chem. 2007, 79 (21), 8327-8332. (23) Chen, H.; Cotte-Rodríguez, I.; Cooks, R. G. cis-Diol functional group recognition by reactive desorption electrospray ionization (DESI) Chem. Commun. 2006,

(6), 597-599.

(24) Zhang, Y.; Chen, H. Detection of saccharides by reactive desorption electrospray ionization (DESI) using modified phenylboronic acids Int. J. Mass Spectrom. 2010, 289 (2-3), 98-107. (25) Wu, C.; Ifa, D. R.; Manicke, N. E.; Cooks, R. G. Rapid, direct analysis of cholesterol by charge labeling in reactive desorption electrospray ionization Anal. Chem. 2009, 81 (18), 7618-7624. (26) Seeley, E. H.; Oppenheimer, S. R.; Mi, D.; Chaurand, P.; Caprioli, R. M. Enhancement of protein sensitivity for MALDI imaging mass spectrometry after chemical treatment of tissue sections J. Am. Soc. Mass Spectrom. 2008, 19 (8), 1069-1077. (27) Vens-Cappell, S.; Kouzel, I. U.; Kettling, H.; Soltwisch, J.; Bauwens, A.; Porubsky, S.; Müthing, J.; Dreisewerd, K. On-tissue phospholipase C digestion for enhanced MALDI-MS imaging of neutral glycosphingolipids Anal. Chem. 2016, 88 (11), 5595-5599. (28) Schwartz, S. A.; Reyzer, M. L.; Caprioli, R. M. Direct tissue analysis using matrix ‐ assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation J. Mass Spectrom. 2003, 38 (7), 699-708. (29) Nemes, P.; Vertes, A. Laser ablation electrospray ionization for atmospheric pressure, in vivo, and imaging mass spectrometry Anal. Chem. 2007, 79 (21), 8098-8106. (30) Robichaud, G.; Barry, J. A.; Muddiman, D. C. IR-MALDESI mass spectrometry imaging of biological tissue sections using ice as a matrix J. Am. Soc. Mass Spectrom. 2014, 25 (3), 319-328. (31) Lee, J. K.; Jansson, E. T.; Nam, H. G.; Zare, R. N. High-resolution live-cell imaging and analysis by laser desorption/ionization droplet delivery mass spectrometry Anal. Chem. 2016, 88 (10), 5453-5461. (32) Cheng, C.-Y.; Yuan, C.-H.; Cheng, S.-C.; Huang, M.-Z.; Chang, H.-C.; Cheng, T.-L.; Yeh, C.-S.; Shiea, J. Electrospray-assisted laser desorption/ionization mass spectrometry for continuously monitoring the states of ongoing chemical reactions in organic or aqueous solution under ambient conditions Anal. Chem. 2008, 80 (20), 7699-7705. (33) Lu, Q.; Hu, Y.; Chen, J.; Jin, S. Laser Desorption Postionization Mass Spectrometry Imaging of Folic Acid Molecules in Tumor Tissue Anal. Chem. 2017, 89 (16), 8238-8243. (34) Veryovkin, I. V.; Calaway, W. F.; Tripa, C. E.; Moore, J. F.; Wucher, A.; Pellin, M. J. Laser post-ionization secondary neutral mass spectrometry for ultra-trace analysis of samples from space return missions Nucl. Instrum. Methods Phys. Rev., Sect. B 2005, 241 (1-4), 356-360. (35) Soltwisch, J.; Kettling, H.; Vens-Cappell, S.; Wiegelmann, M.; Müthing, J.; Dreisewerd, K. Mass spectrometry imaging with laser-induced postionization Science 2015, 348 (6231), 211-215. (36) Ellis, S.; Soltwisch, J.; Paine, M. R.; Dreisewerd, K.; Heeren, R. Laser Post-Ionisation Combined with a High Resolving Orbitrap Mass Spectrometer for Enhanced MALDI-MS Imaging of Lipids Chem. Commun. 2017. 17



Page 18 of 20

(37) Zimmermann, R. Photo ionisation in mass spectrometry: light, selectivity and molecular ions Anal. Bioanal. Chem. 2013, 405 (22), 6901-5. (38) Kauppila, T. J.; Syage, J. A.; Benter, T. Recent developments in atmospheric pressure photoionization-mass spectrometry Mass Spectrom. Rev. 2015. (39) Kostko, O.; Bandyopadhyay, B.; Ahmed, M. Vacuum Ultraviolet Photoionization of Complex Chemical Systems Annu. Rev. Phys. Chem. 2016, 67, 19-40. (40) Bente, M.; Sklorz, M.; Streibel, T.; Zimmermann, R. Thermal Desorption− Multiphoton Ionization Time-of-Flight Mass Spectrometry of Individual Aerosol Particles: A Simplified Approach for Online Single-Particle Analysis of Polycyclic Aromatic Hydrocarbons and Their Derivatives Anal. Chem. 2009, 81 (7), 2525-2536. (41) Raffaelli, A.; Saba, A. Atmospheric pressure photoionization mass spectrometry Mass Spectrom. Rev. 2003, 22 (5), 318-331. (42) Kalberer, M.; Morrical, B.; Sax, M.; Zenobi, R. Picogram quantitation of polycyclic aromatic hydrocarbons adsorbed on aerosol particles by two-step laser mass spectrometry Anal. Chem. 2002, 74 (14), 3492-3497. (43) Liu, C.; Wen, W.; Shao, J.; Zhao, W.; Qi, K.; Yang, J.; Pan, Y. Fast and comprehensive characterization of chemical ingredients in traditional Chinese herbal medicines by extractive atmospheric pressure photoionization (EAPPI) mass spectrometry Rapid Commun. Mass Spectrom. 2017, 31 (18), 1491-1498. (44) He, J.; Tang, F.; Luo, Z.; Chen, Y.; Xu, J.; Zhang, R.; Wang, X.; Abliz, Z. Air flow assisted ionization for remote sampling of ambient mass spectrometry and its application Rapid Commun. Mass Spectrom. 2011, 25 (7), 843-50. (45) Robichaud, G.; Garrard, K. P.; Barry, J. A.; Muddiman, D. C. MSiReader: an open-source interface to view and analyze high resolving power MS imaging files on Matlab platform J. Am. Soc. Mass Spectrom. 2013, 24 (5), 718-721. (46) Genome Alberta and Genome Canada, Human Metabolome Database, Version 2.0, http://www.hmdb.ca/, October 6, 2018. (47) LIPID MAPS consortium - Nature Publishing Group, LIPID Metabolites And Pathways Strategy, http://www.lipidmaps.org/, October 6, 2018. . (48) Badu-Tawiah, A.; Bland, C.; Campbell, D. I.; Cooks, R. G. Non-aqueous spray solvents and solubility effects in desorption electrospray ionization J. Am. Soc. Mass Spectrom. 2010, 21 (4), 572-579. (49) Kauppila, T. J.; Kuuranne, T.; Meurer, E. C.; Eberlin, M. N.; Kotiaho, T.; Kostiainen, R. Atmospheric pressure photoionization mass spectrometry. Ionization mechanism and the effect of solvent on the ionization of naphthalenes Anal. Chem. 2002, 74 (21), 5470-5479. (50) Liu, C.; Zhu, Y.; Zhou, Z.; Yang, J.; Qi, F.; Pan, Y. Ultrasonic nebulization extraction/low pressure photoionization mass spectrometry for direct analysis of chemicals in matrices Anal. Chim. Acta. 2015, 891, 203-210. (51) Liu, C.; Zhu, Y.; Yang, J.; Zhao, W.; Lu, D.; Pan, Y. Effects of Solvent and Ion Source Pressure on the Analysis of Anabolic Steroids by Low Pressure Photoionization Mass Spectrometry J. Am. Soc. Mass Spectrom. 2017, 28 (4), 724-728. (52) Luo, Z.; He, J.; Chen, Y.; He, J.; Gong, T.; Tang, F.; Wang, X.; Zhang, R.; Huang, L.; Zhang, L. Air flow-assisted ionization imaging mass spectrometry method for easy whole-body molecular imaging under ambient conditions Anal. Chem. 2013, 85 (5), 2977-2982. (53) He, J.; Sun, C.; Li, T.; Luo, Z.; Huang, L.; Song, X.; Li, X.; Abliz, Z. A sensitive and wide coverage ambient mass spectrometry imaging method for functional metabolites based molecular histology Adv. Sci. 2018, 5 (11), 1800250. (54) Jungmann, J. H.; MacAleese, L.; Buijs, R.; Giskes, F.; De Snaijer, A.; Visser, J.; Visschers, J.; Vrakking, M. J.; Heeren, R. M. Fast, high resolution mass spectrometry imaging using a medipix pixelated detector J. Am. Soc. Mass Spectrom. 2010, 21 (12), 2023-2030. (55) Senoner, M.; Unger, W. E. SIMS imaging of the nanoworld: applications in science and technology J. Anal. Atom. Spectrom. 2012, 27 (7), 1050-1068. (56) Bradford, H. F. Chemical Neurobiology; W. H. Freeman and Co: New York, 1986. 18


Page 19 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(57) Payne, A. H.; Hales, D. B. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones Endocr. Rev. 2004, 25 (6), 947-970. (58) Hooper, N. M. Detergent-insoluble glycosphingolipid/cholesterol-rich membrane domains, lipid rafts and caveolae Mol. Membr. Biol. 1999, 16 (2), 145-156. (59) Raff, M. C.; Mirsky, R.; Fields, K.; LISAK, R. P.; DORFMAN, S. H.; SILBERBERG, D. H.; Gregson, N.; LEIBOWITZ, S.; KENNEDY, M. C. Galactocerebroside is a specific cell-surface antigenic marker for oligodendrocytes in culture Nature 1978, 274 (5673), 813. (60) Colombaioni, L.; Garcia-Gil, M. Sphingolipid metabolites in neural signalling and function Brain Res. Rev. 2004, 46 (3), 328-355. (61) Lee, Y. J.; Perdian, D. C.; Song, Z.; Yeung, E. S.; Nikolau, B. J. Use of mass spectrometry for imaging metabolites in plants Plant J. 2012, 70 (1), 81-95. (62) Bjarnholt, N.; Li, B.; D'Alvise, J.; Janfelt, C. Mass spectrometry imaging of plant metabolites–principles and possibilities Nat. Prod. Rep. 2014, 31 (6), 818-837. (63) Ho, H.-Y.; Cheng, M.-L.; Weng, S.-F.; Leu, Y.-L.; Chiu, D. T.-Y. Antiviral effect of epigallocatechin gallate on enterovirus 71 J. Agr. Food Chem. 2009, 57 (14), 6140-6147. (64) Cabrera, C.; Artacho, R.; Giménez, R. Beneficial effects of green tea—a review J. Am. Coll. Nutr. 2006, 25 (2), 79-99. (65) Khan, N.; Mukhtar, H. Tea polyphenols for health promotion Life Sci. 2007, 81 (7), 519-533.

19



Table of Contents graphic

20


Page 20 of 20

Imaging of Polar and Nonpolar Species Using ... - ACS Publications

Recommend Documents