3-Aminophthalhydrazide (Luminol) as A Novel Matrix for Dual-Polarity

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3-Aminophthalhydrazide (Luminol) as A Novel Matrix for Dual-Polarity MALDI MS Imaging Bin Li, Ruiyang Sun, Andrew Gordon, Junyue Ge, Ying Zhang, Ping Li, and Hua Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00803 • Publication Date (Web): 31 May 2019 Downloaded from http://pubs.acs.org on June 1, 2019

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Analytical Chemistry

1

TITLE

2

3-Aminophthalhydrazide (Luminol) as A Novel Matrix for Dual-

3

Polarity MALDI MS Imaging

4 5

Bin Li 1,2,†,*, Ruiyang Sun1,2,†, Andrew Gordon1,2, Junyue Ge1,2, Ying Zhang1,2, Ping

6

Li1,2,*, Hua Yang1,2,*

7 8

1

State Key Laboratory of Natural Medicines, China Pharmaceutical University,

9

Nanjing, 210009, China

10

2

School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing,

11

211198, China

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

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ABSTRACT

13

In many aspects of matrix-assisted laser desorption/ionization mass spectrometry

14

imaging (MALDI MSI) technique, the discovery of new MALDI matrices has been a

15

major task for the improvement of ionization efficiency, signal intensity and molecular

16

coverage. In this work, five analog compounds, including phthalhydrazide, 3-

17

aminophthalhydrazide (3-APH or luminol) and its sodium salt, 4-aminophthalhydrazide

18

(4-APH), and 3-nitrophthalhydrazide (3-NPH) were evaluated as potential matrices for

19

MALDI Fourier-transform ion cyclotron resonance (FT-ICR) MSI of metabolites in

20

mouse brain tissue. The five candidate MALDI matrices were mainly evaluated

21

according to the solid-state ultraviolet absorption, the ion yields and species, and the

22

dual-polarity detection. Among the five candidate matrices, 3-APH and its sodium salt

23

enabled the detection of endogenous metabolites better than the three other

24

candidates in dual polarities. The best results were observed with 3-APH. Compared

25

with commonly used MALDI matrices such as 2,5-dihydroxybenzoic acid, α-cyano-4-

26

hydroxycinnamic acid, and 9-aminoacridine, 3-APH exhibited superior performance in

27

dual polarity MALDI MSI, higher sensitivity, broader molecular coverage, and lower

28

background noise. The use of 3-APH led to on-tissue MALDI FT-ICR MSI of 159 and

29

207 mouse brain metabolites in the positive and negative ion modes, respectively.

30

Among

31

glycerophospholipids, sphingolipids and saccharolipids. 3-APH was further used for

32

MALDI FT-ICR MSI of metabolic responses to ischemia-induced disturbances in

33

mouse brain subjected to transient middle cerebral artery occlusion (MCAO), thus

34

revealing the alteration of 105 metabolites in the ipsilateral hemispheres. This further

35

emphasizes the great potential of 3-APH as matrix for the localization of biomarkers in

36

brain diseases.

these

metabolites

include

nucleotides,


fatty

acids,

glycerolipids,

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37

INTRODUCTION

38

Mass spectrometry-based imaging (MSI) techniques have become an important tissue

39

imaging tool for precise localization of thousands of biological molecules in a single

40

experiment. It is a label-free, in situ, and untargeted spatio-chemical imaging technique

41

with a high degree of specificity. With its unparalleled and unique capabilities, MSI has

42

become a popular visualization tool and has been broadly applied in biology, medicine

43

and pharmacology.1-3 Among various MSI techniques, matrix-assisted laser

44

desorption/ionization (MALDI) imaging is extensively applied to the imaging analysis

45

of various biomolecules such as proteins, peptides, lipids, small metabolites and drug

46

substances.4-7 Unlike the two other popular MSI techniques, i.e. desorption

47

electrospray ionization (DESI) imaging8 and secondary ionization mass spectrometry

48

(SIMS) imaging9, the detection of molecules by UV-MALDI MS imaging is believed to

49

be largely dependent on the choice of matrix.10 Applying an appropriate UV absorbing

50

matrix is one of pivotal factors in obtaining satisfactory signal-to-noise ratio (S/N), high-

51

coverage of molecular species and high-quality ion images.11,12

52

Until now, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid

53

(CHCA) are the most popular positive ion MALDI matrices, which have been

54

extensively used for the profiling and imaging of lipids, peptides and various secondary

55

metabolites. Sinapinic acid (SA) is the first choice for MALDI analysis of protein

56

molecules weight above approximately 4 kDa.12 In negative ion MALDI MS, 9-

57

aminoacridine (9-AA), a moderately strong base, has been successfully used for the

58

analysis of small molecules such as nucleotides and glucose 6-phosphate.13,14

59

However, some limitations of conventional matrices are observed in MALDI MS

60

imaging. These may include high background signals of the matrix ions below m/z 500,

61

one polarity, and a very broad and uneven crystal size distribution.

62

In the past decade, efforts have been made to search for new organic MALDI

63

matrices with good performances such as low background interferences, high salt-

64

tolerance potential, low volatility in the high vacuum, available for dual-polarity

65

detection, and capable of forming uniform matrix crystals at ~10 µm for high spatial

66

resolution MALDI MSI.11 For example, dithranol15, quercertin16 and curcumin17 were

67

investigated for tissue imaging of lipids in positive ion mode. In negative ion mode, 4-

68

phenyl-α-cyanocinnamic

69

(DMAN)19, 1,8-di(piperidinyl)naphthalene (DPN)20, maleic anhydride proton sponge21,

70

1,6-diphenyl-1,3,5-hexatriene (DPH)22 and N-phenyl-2-naphthylamine (PNA)23 proved

71

to be suitable for imaging small molecules such as amino acids, fatty acids and lipids.

72

Additionally, 1,5-diaminonapthalene (1,5-DAN) exhibited high efficiency by sublimation

acid

amide18,

1,8-bis(dimethyl-amino)


naphthalene


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coating for the imaging of lipids in both positive and negative ion modes.24

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Furthermore, chemical synthesis and structure modification are alternatives to develop

75

new matrices with desirable physicochemical properties such as optical absorption,

76

crystallinity

77

desorption/ionization (LDI) MSI is the development of non-organic materials, such as

78

metallic25,26, carbon27 and silicon based nanomaterials28, metal oxides29 and

79

correspondent modified materials30. These provide clearer background with limited

80

interference peaks and are particularly suitable for tissue imaging of small molecules.

81

Although the mechanism of MALDI is still debated, in the positive ion mode cation

82

transfer from matrix such as weak organic acids (e.g. DHB and CHCA) to analyte

83

molecules is found to be the predominant process for gas-phase cationization.31 In the

84

negative ion mode, abstraction of a proton by the matrix such as strong bases (e.g. 9-

85

AA and DMAN) may be mainly associated with the formation of deprotonated anions.13

86

In terms of dual-polarity MALDI MSI, often used matrices for positive ion MALDI MSI

87

such as DHB and CHCA could also be used in negative ion mode. However, significant

88

differences in ion yields and species are generally observed in the two ionization

89

modes. For example, DHB and CHCA generate high ion yields in positive ion mode

90

but low ion yields in negative ion mode. Thus, resulting in the loss of spatial information

91

of molecules readily ionized in the negative ion mode such as fatty acids, nucleotides,

92

cardiolipins and gangliosides. Matrix with both positive and negative polarities have

93

attracted intense interest because they can significantly broaden the molecular

94

coverage and acquire two ion images from a tissue section.

and

vacuum

stability.18,21

Another

significant

trend

in

laser

95

Generally, several criteria need to be considered when evaluating an organic

96

compound as a potential UV-MALDI matrix. Matching the operational laser wavelength

97

337 nm for nitrogen laser or 355 nm frequency-tripled Nd:YAG laser to the absorption

98

of the matrix is one significant property necessary. The abilities to co-crystallize with

99

the analytes and ionization while generating minimal matrix signals are also necessary

100

characteristics of an ideal MALDI matrix. Moreover, since the process of vacuum

101

MALDI MS imaging of tissue sections typically last for hours, high vacuum stability of

102

matrix is therefore essential for consistent acquisition of a series of mass spectra

103

across the tissues.

104

As aforementioned, several reported MALDI matrices such as 1,5-DAN, 9-AA and

105

PNA are nitrogen substituted aromatic compounds. Therefore, in this work, we

106

evaluated phthalhydrazide and its analogs including 3-aminophthalhydrazide (3-APH,

107

commonly known as luminol) and its sodium salt, 4-aminophthalhydrazide (4-APH,

108

commonly known as isoluminol), and 3-nitrophthalhydrazide (3-NPH) for MALDI

109

Fourier-transform

ion

cyclotron

resonance

(FT-ICR)


MS

measurements

of

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endogenous metabolites in sections of mouse brain tissue surrogates. Five candidate

111

matrices were mainly evaluated based on the following characteristics: matrix

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ultraviolet optical absorption, ion yields and tissue imaging in positive and negative ion

113

modes. 3-APH and its sodium salt exhibited great potential for use as dual-polarity

114

MALDI matrices, with the best results obtained using 3-APH matrix. We therefore

115

present 3-APH as a novel MALDI matrix for the analysis and imaging of variation of

116

brain metabolites in mouse models subjected to middle cerebral artery occlusion

117

(MCAO) in both positive and negative ion modes.

118 119

MATERIALS AND METHODS

120

Chemicals.

121

Phthalhydrazide,

122

aminophthalhydrazide

123

dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and 9-

124

aminoacridine (9-AA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). LC-

125

MS grade methanol, acetonitrile and ammonia hydroxide (NH4OH) were purchased

126

from Merck (Darmstadt, Germany). Deionized water was prepared by a Milli-Q water

127

purification system (Millipore, Billerica, MA, USA).

128

UV Spectroscopy of Candidate Matrices.

129

All UV absorption spectra of candidate matrices in solid state were recorded on a UV-

130

Vis spectrophotometer (U-3900 HITACHI, Japan) in the range of 200-400 nm,

131

respectively. Candidate matrices dissolved in pure methanol containing 1.2% NH4OH

132

were deposited on quartz substrates using a home-built automatic spray for solid state

133

absorption measurements.

134

Animals and MCAO Model.

135

Male 8-week-old C57BL/6J mice were purchased from SIPPR-BK (Shanghai, China)

136

and kept in an environmentally controlled breeding room for at least one week before

137

experiment. Animal experiments were carried out in accordance with the Guidelines

138

for Animal Experimentation of China Pharmaceutical University (Nanjing, China), with

139

the protocol approved by the Animal Ethics Committee of the institution. Mice weighing

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21-23 g were used in this study. Experimental stroke was induced using a middle

141

cerebral artery occlusion (MCAO) model as described previously.32 Briefly, the mice

142

were anesthetized with a mixture of chloral hydrate (3%), 6-0 nylon monofilament

143

coated with poly-l-lysine was introduced into the internal carotid artery to block the

144

middle cerebral artery at its origin for 1 h. At 24 h after MCAO, mice were sacrificed by

3-aminophthalhydrazide (4-APH),

(3-APH),

3-APH

3-nitrophthalhydrazide


sodium

salt,

(3-NPH),

42,5-


145

decapitation and the brain quickly dissected, frozen on dry ice, and stored at −80 °C

146

until use. The brain tissue sections were stained with cresyl violet (Nissl staining) after

147

MALDI MS imaging and removal of the matrix with pure ethanol to identify regions of

148

necrosis. A total of 6 mice were used for the creation of MCAO model. Three MCAO

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mice with similar infarct size determined by Nissl staining and three non-MCAO

150

(control) mice were used for the MALDI MS imaging.

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Sample Preparation for the Evaluation of Candidate Matrices.

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To maintain consistency, we applied tissue surrogates to critically evaluate the

153

performance of the different candidate matrices. Brain dissected from normal mouse

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was rinsed in saline. To the rinsed section, 0.2 mL cold saline was added and

155

homogenized with a ball mill (JXFSTPRP-24, Shanghai Jingxin Experimental

156

Technology, Shanghai, China) for 2 min, immediately frozen, and stored at -80 °C.

157

For the evaluation of candidate matrices, 12-μm thick sections of tissue surrogates

158

were prepared at -20 °C using a cryostat (3050S, Leica, Germany), and thaw-mounted

159

onto indium-tin-oxide (ITO)-coated glass slides (Bruker Daltonics, USA). Repeatability

160

measurements were performed on three sections of tissue surrogates coated with the

161

five candidate matrices for three consecutive day. The matrix application system and

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coating procedure was similar to Tissue Sample Preparation for MALDI MS Imaging.

163

Tissue Sample Preparation for MALDI MS Imaging.

164

In all cases, 12-μm thick horizontal (non-MCAO mice) and coronal tissue sections

165

(MCAO mice) were prepared at -20 °C using a cryostat, and thaw-mounted onto ITO-

166

coated glass slides. For MALDI MS imaging of endogenous metabolite in non-MCAO

167

mouse brain, the horizontal sections were applied due to better display of internal

168

structures. For MALDI MS imaging of MCAO mouse brain, the coronal sections were

169

applied because the infarcts generated by MCAO are mainly located at the striatum

170

and the dorsolateral cortex. A laboratory-constructed electrospray was used for the

171

uniform application of matrix solution. The matrix application system and coating

172

procedure was similar to previously published work with some modifications.33 Briefly,

173

for MALDI MS imaging experiments, all candidate matrices were dissolved in methanol

174

containing 1.2% NH4OH at a concentration of 8.7 mg mL-1. Additionally, 30 mg mL-1

175

DHB (MeOH-H2O, 7:3, v/v), 10 mg mL-1 CHCA (ACN-H2O, 7:3, v/v), and 10 mg mL-1

176

9-AA (MeOH-H2O, 7:3, v/v) were prepared for the comparative analysis. For

177

homogenous deposition of matrix solution onto the brain tissues, a voltage of 5.9 kV

178

was applied to the spray nozzle with the ITO-slide held at ground. The emitter-to-tissue

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distance was approximately 13 cm. The flow rate was set to 1 mL h-1 and gas pressure

180

to 50 psi to deliver and nebulize matrix solution, respectively. 6 ACS Paragon Plus Environment

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MALDI FT-ICR MS Imaging Instrumentation.

182

All measurements were performed using a 9.4T solariX FT-ICR mass spectrometer

183

(Bruker Daltonics, USA) equipped with a dual ion source (ESI and MALDI) and a

184

Smartbeam II laser (1 kHz). An m/z range of 150-2000 was acquired in positive ion

185

mode and m/z 150-3000 acquired in negative ion mode. Single-scan spectra consisted

186

of 100 accumulated laser shots at 1 kHz with a laser focus set to “medium”. Laser

187

power was 40% for both positive and negative ion mode, respectively. MALDI images

188

were acquired at a 150 µm spatial resolution for normal brain tissues and 225 µm for

189

ischemic mouse brain tissues. Mass spectrometer calibration was performed

190

externally in dual polarities using DHB matrix peaks and a Peptide Calibration

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Standard Kit II (Bruker Daltonics, USA). Calibration of the m/z scale of the MALDI FT-

192

ICR MS in both positive and negative ion modes is an important step in obtaining

193

accurate mass. Data was analyzed using Data Analysis version 4.0 and flexImaging

194

version 4.1 software (Bruker Daltonics, USA).

195 196

RESULTS AND DISCUSSION

197

Evaluation of Phthalhydrazide and Its Homologues as Matrix for UV-

198

MALDI FT-ICR MS

199

In UV-MALDI MS, high optical absorption at the operational laser wavelength is

200

considered to be one of the crucial advantageous properties of UV-MALDI matrix.34-36

201

Currently, solid-state laser and gas laser operating at 355 nm and 337 nm respectively

202

are the predominant laser source equipped in most commercial MALDI MS

203

instruments. The UV absorption bands of many widely applied matrices such as DHB,

204

CHCA, and 9-AA match well with solid-state and N2 gas laser sources.37 Many studies

205

demonstrated significant differences between the solution and solid phase UV

206

absorbance spectra of matrix compounds.37 Usually, UV absorption profile of a target

207

compound in a solution is measured to evaluate its matrix potential. However, such

208

measurements acquired is potentially problematic due to solvent dependent effects

209

since UV-MALDI matrix is typically used in the solid state.35 Therefore, acquiring the

210

UV absorption profiles of the five candidate matrices in the solid-state was preferred

211

for the initial assessment. In Figure 1, phthalhydrazide (purple line) and 3-NPH (green

212

line) have three absorption peaks at around 220 nm, 266 nm, 308 nm and 224 nm,

213

267 nm, 300 nm, respectively. 3-APH (red line) and its sodium salts (black line) exhibit

214

similar absorption profile in solid state with three distinct maxima at around 224 nm,

215

308 nm, and 355 nm, respectively. However, comparing the peak intensity at 355 nm 7 ACS Paragon Plus Environment


216

the absorption of 3-APH sodium salts is lower than 3-APH. Compared to 3-APH, no

217

absorption maximum at 355 nm was observed for 3-NPH and phthalhydrazide. The

218

auxochrome −NH2 directly conjugated with the pi-system of the phthalhydrazide

219

contributes to the variation of the absorption profile and intensity. Unlike other

220

candidate matrices, 4-APH (blue line) exhibits only two distinct maxima at 236 and 264

221

nm, respectively. Although 4-APH displayed the strongest absorption band among the

222

five candidate matrices, the change in position of the −NH2 on the phthalazine ring

223

caused a blue-shift of λmax to 264 nm. The maximum UV-absorption of 3-APH and its

224

sodium salts in the solid state matched well with the MALDI FT-ICR MS equipped with

225

a 355 nm Nd:YAG UV laser.

226 227 228 229

FIGURE 1. Chemical structures of five candidate matrix compounds and their solid-state UV absorption spectra measured on dry and crystalized matrices.

230

The maximum UV absorption of potential matrix compound matching to the

231

operational laser wavelength is one of many properties that have been considered as

232

a critical requirement for UV-MALDI MSI matrix. The ion yields in UV-MALDI FT-ICR

233

MS were next evaluated by spraying each candidate matrix onto three sections of

234

tissue surrogates and measured consistently for day-to-day repeatability (Figure S1).

235

The respective MALDI FT-ICR mass spectra were acquired in both positive and 8 ACS Paragon Plus Environment

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236

negative ion modes for the individual candidate matrices (Figure 2). In Figure 2,

237

intense MALDI signals mainly consisting of lipids were observed in the two polarities

238

for 3-APH and its sodium salt in comparison with 3-NPH, 4-APH and phthalhydrazide.

239

As shown, in dual-polarity detection, there were almost no endogenous compound

240

signals detected with phthalhydrazide, and very low signal intensity observed when 3-

241

NPH and 4-APH were used as the matrix. Additionally, in the negative ion mode, high

242

background signals within the range of m/z 400-600 were observed with 3-NPH as a

243

matrix (Figure 2 and Figure S2).

244 245 246 247

FIGURE 2. MALDI FT-ICR mass spectra acquired from sections of tissue surrogates coated with five candidate matrices in the positive (left) and negative (right) ion modes.

248

We therefore compared the performance of 3-APH with its sodium salt. As shown

249

in Figure 2, minor differences were observed in 3-APH and its sodium salt in positive

250

ion mode. However, less number of ions in the mass range of m/z 1000-3000 were

251

detected when using 3-APH sodium salt in negative ion mode. Further comparison of

252

3-APH with its sodium salt was made by considering the molecular coverage in MALDI

253

FT-ICR MS imaging. As demonstrated in Figure S3, the use of 3-APH and its sodium

254

led to on-tissue MALDI FT-ICR MS detection and imaging of 159 and 133 endogenous

255

compounds from a mouse brain section in the positive ion mode, respectively, and 207

256

and 149 compounds detected respectively in the negative ion mode. Therefore, MALDI

257

MS results suggested that 3-APH is superior to its sodium salts particularly for negative

258

ion MALDI FT-ICR MS with 355 nm laser.

259

Next, we compared the performance of 3-APH with three commonly used MALDI 9 ACS Paragon Plus Environment


260

matrix DHB, CHCA and 9-AA. Initially, optimization of laser power was performed

261

(Figure S4). As shown in Figure S4 and S5, although the observed single mass

262

spectral profiles from sections of tissue surrogates were similar across 3-APH and

263

DHB matrices in the positive ion mode, the total ion intensity obtained by using 3-APH

264

was higher than DHB, consistent with laser power optimization results. While the total

265

ion intensity obtained using CHCA was higher than 3-APH, the chemical entities

266

acquired from a single mass spectrum using CHCA (71) was less than 3-APH (79). As

267

shown in Figure S4, high laser power could result in the high ion intensity. However,

268

mass measurement errors caused by frequency perturbations in FT-ICR MS were

269

observed at 50% laser power. This could be attributed mainly to space charge effects

270

resulted from an excess of ions trapped in the ICR cell. In the negative ion mode,

271

intense MALDI signals and a wide range of chemical entities were observed using 3-

272

APH compared with 9-AA at 40 % laser power. Finally, laser power set at 40% was

273

adopted because it produced the highest ion intensities with acceptable mass

274

measurement errors in both positive and negative ion modes. Compared with most

275

often used matrices, 3-APH resulted in very rich metabolite signatures and as well

276

suitable for MALDI FT-ICR imaging in two polarities with good sensitivity.

277

Besides the general properties of the ideal MALDI matrix; maximum absorption at

278

the operational laser wavelength, ability to promote analyte desorption and ionization,

279

3-APH met more criteria for MALDI MS imaging measurements. Figure S6 shows the

280

morphology of the matrix crystal layer coated on mouse brain tissues obtained by

281

optical microscopy. 3-APH coating exhibits a homogeneous sample coverage with the

282

crystal sizes at µm scale which can mitigate the possible analyte delocalization,

283

improve spot-to-spot reproducibility and provide the potential for high spatial resolution

284

MALDI MS imaging. Moreover, other characteristics of 3-APH used as MALDI MS

285

imaging matrix such as its high vacuum stability (Figure S7), the low yield of matrix-

286

related ions (Figure S2) and low matrix concentrations (8.7 mg mL-1), were also

287

demonstrated.

288 289

Optimization of the Matrix Solvent

290

It has been demonstrated that the matrix solvent composition directly influences

291

matrix-analyte interaction, matrix crystal size, and ion yields. These must be optimized

292

to obtain high sensitivity detection of analytes.14 We examined multiple combinations

293

of matrix solvents to identify a set of optimized conditions for subsequent MALDI MSI

294

experiments. 3-APH is comparatively insoluble in water and less soluble in commonly

295

used organic solutions such as methanol but is soluble in base. To ascertain which

296

solvent combination will be suitable, three solvent systems was used with each spiked 10 ACS Paragon Plus Environment

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297

with NH4OH. The three solvent combinations include solution A (pure MeOH

298

containing 1.2% NH4OH), solution B (MeOH-H2O (1:1, v/v) containing 1.2% NH4OH)

299

and solution C (MeOH-H2O (4:1, v/v) containing 1.2% NH4OH). As shown in Figure

300

S8, the mean peak intensities of most selected lipids detected in positive ion mode

301

were improved with solution A. However, in the negative ion mode, the three solutions

302

did not appear to provide a universal advantage in promoting intensities of selected

303

lipids. For example, selected lipid ions at m/z 699.50, m/z 806.51, m/z 885.55, m/z

304

888.62, m/z 945.55 and m/z 973.58 have higher intensities with solution A, and ions at

305

m/z 856.51, m/z 883.53 were better detected with solution C compared with A.

306

However, for m/z 1857.95 and m/z 2215.07 no statistical significant difference was

307

observed using the three solvent system. This is likely related to the different degree

308

of analyte-matrix interactions for each solvent toward the different lipid species.

309

Solution A was consequently used in the subsequent dual-polarity MALDI FT-ICR MSI

310

experiments.

311 312

Characterization of Metabolites in Normal Mouse Brain with 3-APH MALDI

313

FT-ICR MS

314

After 3-APH has been ascertained as a suitable MALDI matrix for the detection of

315

endogenous metabolites particularly for lipid species in both positive and negative ion

316

modes, its application in MALDI FT-ICR MS imaging of endogenous metabolites was

317

illustrated using the brain tissue sections of the normal adult mouse. As shown in

318

Figure 3, Table S1 and S2, 159 and 207 endogenous entities were detected from

319

mouse brain tissue with 3-APH in positive and negative polarities, respectively. In

320

positive ion mode, intense peaks were putatively attributed to phosphatidylcholines

321

(PCs), sphingomyelin (SMs) and phosphatidylethanolamines (PEs) identified by

322

accurate mass and/or tandem mass spectra matching in LIPID MAPS database

323

(www.lipidmaps.org), and several published literatures (Table S1 and S2). Moreover,

324

other lipid species including glycerolipids (GLs), ceramides (CERs), glycosphingolipids

325

(GSLs) and sterols were also detected (Table S1). In negative ion mode, the major

326

metabolites detected corresponded to phosphatidic acids (PAs), phosphatidylinositol

327

(PIs), phosphatidylserine (PSs), cardiolipin (CLs), PEs, and GSLs. Other molecules

328

were also detected such as phosphatidylglycerol (PGs), cyclic phosphatidic acids

329

(cPAs), phosphtatidylinositol phosphates (PIPs), fatty acids (FAs) and nucleotides with

330

3-APH (Table S2). The identification workflow of ions detected in the negative ion

331

mode is similar to those used for positive ions. The different kinds of adduct ions were

332

only counted as one compound.



333 334 335 336 337 338

FIGURE 3. Venn diagram showing the species and numbers of putatively identified endogenous metabolites detected using MALDI FT-ICR MS imaging with 3-APH matrix in positive and negative ion modes.

Furthermore, in situ tandem MS on the intense lipid signals were conducted in

339

order to evaluate the fragmentation patterns using 3-APH as a matrix. For example, in

340

positive ion mode (Figure S9), MS/MS of m/z 756.5513 produced a characteristic

341

tandem mass spectrum corresponding to the PC(32:0) with characteristic loss of 59.07

342

(N(CH3)3) and 183.06 (phosphocholine). MS/MS of m/z 830.5079 produced a

343

characteristic tandem mass spectrum corresponding to the PE(40:6) with

344

characteristic loss of 43.04 (C2H5N) and 141.02 (PE head group). In negative ion mode,

345

as demonstrated in Figure S10, m/z 1544.8694 and 1572.9007 correspondent to

346

monoisotopic masses of GM1(d36:1) and GM1(d38:1) were subjected to tandem MS

347

analysis, respectively. The fragmentation patterns of these two gangliosides are

348

consistent with previous research.38 For example, the tandem mass spectrum of

349

GM1(d36:1) ([M-H]-, m/z 1544.8694) exhibits product ions at 888.64 and 726.59, which

350

are responsible for [M-H-NeuAc-Hex-HexNAc]- and [M-H-NeuAc-Hex-HexNAc-Hex]-,

351

respectively (Figure S10). Another characteristic fragment ion at m/z 290.08 was

352

observed, confirming the existence of a sialic acid moiety.39

353 354

MALDI FT-ICR MSI of Spatial Distribution of Metabolites in Mouse Brain

355

The negative and positive ion MALDI FT-ICR MS imaging was serially performed on

356

one brain tissue section using 3-APH with a spatial resolution of 150 μm. In this case,

357

the negative grid array was aligned with an offset of 100 μm in both x and y dimensions

358

with respect to the array defined for positive data acquisition. In Figure 4, the Nissl-

359

stained brain section shows histologically distinguishable structures, including

360

cerebellum white matter and granular layers, inferior colliculus (IC), superior colliculus

361

(SC), hippocampus (HIP), thalamic nucleus (TN), fimbria, lateral ventricle (LV),

362

striatum, corpus callosum (CC) and cerebral cortex (CTX). As shown in Figure 4, 312 ACS Paragon Plus Environment

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363

APH yields a significant amount of spatial information on endogenous chemical entities

364

by serially imaging the same tissue section with the dual polarities. Different species

365

of metabolites exhibit tissue-specific distribution across the mouse horizontal brain

366

tissue section. Compared to commonly used 9-AA, 3-APH matrix exhibits a distinct

367

advantage over 9-AA in the detection sensitivity and species coverage of lipids in

368

negative ion mode. For example, using 3-APH, [PE(38:5)-H]- was detected but not with

369

9-AA, and the S/N ratios of some lipids (e.g. [PE(38:4)-H]-, [PI(36:6)-H]- and

370

[ST(d40:1)-H]-) were significantly increased using 3-APH as matrix as compared with

371

9-AA, which are consistent with previous studies.18,40 One of the major advantages of

372

3-APH matrix lies in its superior detection of many species of GSLs a subclass of

373

sphingolipids in negative ion mode, including neutral-, acidic- and amphoteric-GSLs

374

(Table S2). GSLs present rich and various chemical structures with biological functions

375

and have attracted intense interest to study this type of molecules. By matching

376

accurate mass and/or tandem MS obtained by FT-ICR MS with online databases and

377

literatures, the identity of GSLs were putatively assigned and listed in Table S2. In

378

Figure 4, tissue-specific spatial distributions of gangliosides containing one or more

379

sialic acids, such as GMs, GDs and GTs, are clearly revealed with 3-APH matrix, which

380

are consistent with previous data.41,42 For example, [GM1(d36:1)-H]- (m/z 1544.8694)

381

and [GM1(d38:1)-H]- (m/z 1572.9007) present similar and different tissue-specific

382

distribution patterns. These two gangliosides are mainly distributed in HIP, CTX and

383

striatum, and less expressed in cerebellum region. However, in HIP region GM1(d36:1)

384

is widely distributed, but GM1(d38:1) is confined in the dentate gyrus molecular layer

385

and the stratum lacunosum moleculare. This observation is consistent with previous

386

high resolution MALDI MS imaging results.43 Furthermore, the spatial distribution

387

patterns of some small molecules such as fatty acids and nucleosides were also

388

visualized (Figure 4) and consistent with previous data.33,40 For example, FA (22:6)

389

were mainly found in the cerebellum granular layers, HIP, TN, striatum and CTX.40

390

In positive ion MALDI MS imaging, 3-APH also enabled the revelation of spatial

391

distribution of various lipid species. For example, some lipids from the same species

392

exhibited heterogenous distribution patterns. [SM(d36:1)+K]+ is mainly observed in the

393

cerebellum granular layers, HIP, lateral septal nucleus (LSN) and TN, while

394

[SM(d42:2)+K]+ is observed in the complementary regions including cerebellum white

395

layers, IC, SC, fimbria and striatum. In addition, high abundancies of [DG(40:8)+Na]+,

396

[Cer(42:2)+K]+ and [SM(d38:1)+K]+ were found in LV and ventral hippocampal

397

commissure (VHC) areas. Besides being the building blocks of the outer and inner cell

398

membrane, lipids are critically important for brain function and regulate plenty of

399

physiological and pathological processes.6,44 Tissue-specific accumulation of lipids can 13 ACS Paragon Plus Environment


400

improve our understanding of the distinct function of individual lipids or their classes,

401

aiding to explain their roles and functions in various diseases. For example,

402

[Cer(42:2)+K]+ is highly confined in the LV, the largest cavities of the ventricular system

403

containing the cerebrospinal fluid (CSF), its presence may be involved in brain protect

404

or may cause neurological diseases if the ceramide homeostasis is interrupted.45,46

405 406 407 408 409 410

FIGURE 4. MALDI FT-ICR MS images of selected metabolites acquired in the positive and negative ion modes from a mouse horizonal brain section coated with 3-APH. Ion images are correlated to the same brain section stained with cresyl violet after MALDI MS imaging and removal of 3-APH. Ion images were recorded with a step size of 150 μm with a 100 μm offset between the positive and negative grid arrays.


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411

Visualization of Abnormal Metabolism in Mouse Brain Subjected to MCAO

412

To investigate its versatility as a matrix, 3-APH was also employed for MALDI FT-ICR

413

MSI of pathological specimens. Tests were performed using ischemic stroke (IS)

414

model i.e. mice subjected to MCAO. Ischemic strokes occur due to the sudden loss of

415

fresh blood circulation to a region of the brain, resulting in a corresponding neurologic

416

dysfunction.47 Abnormal metabolism of endogenous chemicals has been implicated in

417

the pathogenesis of IS.23,48-50 The use of 3-APH for endogenous metabolite MALDI MS

418

imaging may provide additional information for the understanding of complex

419

mechanistic insights associated with IS or for the discovery of novel biomarkers. As

420

shown in Figure S11 and S12, repeatability measurements and statistical analysis of

421

abnormal metabolism in mouse brain subjected to MCAO were conducted with three

422

replicates. Ratios of ion intensity of selected metabolites in left/contralateral

423

hemisphere to ion intensity of the same metabolites in right/ischemic hemisphere were

424

calculated. A bar chart plotted display the fold change in selected ions (Figure S12).

425

As shown in Figure 5, Figure S11 and Table S3, many molecular species exhibit

426

significant changes in coronal sections of the infarcted mouse brain. Interestingly,

427

three different adduct ions of LPC(16:0), i.e. [LPC(16:0)+H]+, [LPC(16:0)+Na]+ and

428

[LPC(16:0)+K]+, presented an inconsistent change in ischemic mouse brain. In the

429

ischemic region, IS elevated [LPC(16:0)+Na]+ signals, and lowered [LPC(16:0)+K]+

430

signals, but [LPC(16:0)+H]+ presents relative homogeneous distribution across

431

ipsilateral and contralateral hemispheres. This observation is consistent with previous

432

studies that the Na+/K+ homeostasis in the brain after IS was altered, leading to the

433

alteration of the cationization profile of the brain lipids.49,50 However, [PC(32:0)+H]+ and

434

[PC(32:0)+K]+presented a consistent change in ischemic mouse brain, both were

435

lowed in ischemic hemisphere. In negative ion mode, several small molecules such as

436

AMP, ADP, GMP, GSH and FAs exhibited a noticeable decrease in signals within the

437

ischemic region. This observation is in agreement with previous research.23,48 Here,

438

ATP signal was undetectable, mainly due to its very quick post-mortem degradation.48

439

IS also changed ganglioside signals in the ischemic region in contrast to contralateral

440

hemisphere. Gangliosides are considered to be intimately involved in the development

441

of various brain diseases.42,51,52 MALDI MSI results showed that [GM1(d38:1)-H]-

442

decreased but [GM2(d36:1)-H]- increased within the MCAO-induced infarcted

443

hemisphere, and no large difference in [GM3(d36:1)-H]- signal was observed in brain

444

section (Figure S12). In a previous study, the highest level of GM1, GM2, GM3 change

445

was observed in the MCAO mouse model at the 3-day reperfusion.51



446 447 448 449 450 451 452 453 454

FIGURE 5. MALDI FT-ICR MS images of selected metabolites acquired in the positive and negative ion modes from a non-MCAO (control) and MCAO mice coronal brain section coated with 3-APH. Ion images are correlated to the same brain sections stained with cresyl violet after MALDI MS imaging and removal of 3-APH. According to the cresyl violet stained images, the left side of the MCAO mouse brain is contralateral hemisphere, and the right side shows ischemic damage. Ion images were recorded with a step size of 225 μm with a 150 μm offset between the positive and negative grid arrays.

455

CONCLUSIONS

456

UV-MALDI MS is a complex multiple-step process where matrix factor plays a

457

significant role in the determination of the final imaging quality. Visualization of the

458

spatial distribution of endogenous metabolites in dual polarities with one matrix

459

significantly broadens the molecular coverage. In this work, we comprehensively

460

evaluated commercially available 3-APH and related analogs for their application as

461

UV-MALDI matrix in terms of optical absorption, ion yields and tissue imaging. In

462

general, MALDI MS results were consistent with the UV absorption properties of

463

individual matrix compounds in the solid state. Among the five candidate matrices, 3-

464

APH and its sodium salts provided more endogenous chemical entities in both positive

465

and negative ion detection, and were associated with low background interferences, 16 ACS Paragon Plus Environment

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466

high molecular coverage and high-vacuum stability. The best matrix performance was

467

observed with the use of 3-APH, particularly in negative polarity. In mouse brain, 159

468

and 207 endogenous entities were detected with 3-APH in positive and negative

469

polarities, respectively (Table S1 and S2).

470

To further demonstrate the application of 3-APH as a new MALDI matrix, mouse

471

brain sections subjected MCAO was analyzed with 3-APH MALDI FT-ICR MS imaging.

472

The complex metabolite alterations such as nucleotides, fatty acids, phospholipids,

473

and sphingolipids were visualized in the ipsilateral and contralateral hemispheres. The

474

alteration in Na+ and K+ homeostasis induced by ischemic brain injury were also

475

visualized by the alkali metal adduct ions for lipids. In total, 57 and 48 endogenous

476

metabolites exhibited large change in the ipsilateral hemispheres when detected in

477

positive and negative ion modes, respectively (Table S3). This demonstrates the

478

paramount importance for a comprehensive illustration of molecular mechanisms at

479

sites of tissue injury induced by different diseases. The MSI results generally agreed

480

with previous findings concerning the role of endogenous metabolite species in

481

ischemic injury. However, more work is further required to validate observed metabolite

482

alterations in brain by MALDI MSI.

483

ASSOCIATED CONTENT

484

Supplementary data of an additional Figure S1−S8, and Table S1- S3 (PDF).

485

AUTHOR INFORMATION

486

*Corresponding Authors:

487

E-mail: [email protected] (B.L.,

https://orcid.org/0000-0002-7713-159X)

488

[email protected] (P.L.) and [email protected] (H.Y.)

489

†These

490

ACKNOWLEDGMENTS

491

This work was supported by the National Natural Science Foundation of China (No.

492

81730104, No. 81773873, and No. 81722048), the National Standardization Program

493

for Chinese Medicine (ZYBZH-C-JS-35), the National Science and Technology Major

494

Projects

495

2017ZX09301012003), the 111 Project (No. B16046), and the "Double First-Class"

496

University Project (CPU2018GY09). The content is solely the responsibility of the

497

authors and does not necessarily represent the official views of the funding agencies.

authors contributed equally to this work.

for

“Major

New

Drugs

Innovation


and

Development

(No.


498

CONFLICT OF INTEREST

499

The authors declare that they have no conflict of interest.

500

REFERENCES

501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553

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3-Aminophthalhydrazide (Luminol) as A Novel Matrix for Dual-Polarity

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