Hydroxyflavones as a New Family of Matrices for MALDI Tissue. Imaging

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1 Supporting Information: Hydroxyflavones as a New Family of Matrices for MALDI Tissue Imaging Xiaodong Wang, Jun Han, Albert Chou, Juncong Yang, Jingxi Pan, and Christoph H. Borchers *,, University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, # Markham St., Victoria, BC V8Z 7X8, Canada Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada *Corresponding author: Christoph H. Borchers, Ph.D. christoph@proteincentre.com Tel.: (250) ; Fax: (250) S-1

2 Supporting Information - Methods Lipid Extraction and LC-MS/MS Analysis. Total lipids from the same rat liver and brain, which have been subjected to MALDI profiling or imaging, were extracted according to a previously described protocol, 1 with slight modifications. Briefly, the tissue aliquots in an amount of ca. 20 mg were homogenized in 200 µl of water inside 1.5 ml Eppendorf tubes with a Retsch MM400 mixer mill (Haan, Germany) with the aid of two 5-mm stainless steel metal balls for 30 s x 2 at a vibration frequency of 30 Hz. Next, 800 µl of a mixed chloroform-methanol (1:3, v/v) solvent was added to each tube, followed by another 30-s homogenization step using the same mixer mill settings. The tubes were then centrifuged at 4,000 x.g. and 4 o C for 20 minutes in a Beckman Coulter Allegra X-22R centrifuge (Brea, CA). The supernatants were collected and mixed with 250 µl of chloroform and 100 µl of water. After vortex mixing for 15 s and centrifugation at 10,600 x.g. for 5 min in a microcentrifuge, the lower organic phase in each tube was carefully removed using a 200-µL gel loading pipette tip, and then dried in a Savant SPD1010 speed-vacuum concentrator (Thermo Electron Corporation, Waltham, MA). The residues were stored at -80 o C until used. LC-MS/MS. Because of the low-mass cut-off (ca. m/z 130) and the relatively low sensitivity of the 12-Tesla FTICR MS instrument for MALDI-MS/MS of many of the lipids, hydrophilic interaction liquid chromatography (HILIC)-MS/MS was performed for verification of the lipid identities. Briefly, a Waters ACQUITY UPLC system coupled to a Waters Synapt HDMS quadrupole-time-of-flight (Q-TOF) mass spectrometer (Beverly, MA) was employed. The dried lipid extract residues were reconstituted in 100 µl of chloroform, and 10-µL aliquots were injected onto a Waters Atlantis HILIC silica column (3 µm particle size, 4.6 mm i.d. x150 mm; Beverly, MA) for separation of different lipid species based on their head groups. 2-4 The mobile phase for S-2

3 binary gradient elution was composed of methanol-water (75:5, v/v) containing 0.2% ammonium formate (ph adjusted to 5.9 with formic acid) as solvent A and acetonitrile-methanol-water (95:2:3, v/v/v) containing 0.2% ammonium formate (ph 5.9) as solvent B. The elution gradient was as follows: 100% B, 0 to 5 min; 100% to 90% B, 5 to 6 min; 90% to 35% B, 6 to 30 min; 35% to 10% B, 30 to 32 min, and 10% solvent B, 32 to 34 min. The column was then re-equilibrated at 100% B for 6 min between injections. The flow rate was 0.6 ml/min and the HILIC column was maintained at 30 o C. LC/MS data were collected in both positive and negative ESI modes, with respective injections. The detection mass range was m/z 80 to 1,200, with a scan rate of 0.25 s. MS/MS experiments were conducted using collision-induced dissociation (CID) at three collision energies (10, 20, and 30 V), which were applied to the trapping collision cell of the Q-TOF instrument. UPLC-full-scan MS data were processed with the MarkerLynx module embedded in the Waters MassLynx software (version 4.1) suite. Lipid identities were assigned by combining mass-matched metabolome database searching against the METLIN database 5 with MS/MS spectral searching against the standard MS/MS libraries in the METLIN, HMDB 6, 7 or LIPID MAPS databases, 8, 9 or using manual mass spectral interpretation. The parameters for peak extraction and alignment within the MarkerLynx module were as follows: allowable mass window, 50 ppm; retention time shift tolerance, ±0.1 min; ion intensity threshold, 50; and LC retention time range for data extraction, 1 to 27 min. S-3

4 Supporting Information Figure S1. Orthogonal array testing for optimization of quercetin matrix solution compositions. (A) Three variables (% of methanol in water, quercetin concentration, and % of NH 4 OH) x three levels for each factor tested. (B) Range analysis ( N max) for nine combinations of three factors x three levels for each factor. The % of NH 4 OH was shown as a dominant factor, while the % of methanol in water was a factor with the weakest impact. (C) Coated quercetin matrix appearances on rat liver tissue sections from spray of the nine matrix solutions. Combinations 2 and 5 showed good matrix application; combinations 3, 6, 8, and 9 led to matrix over-coating; and combinations 1, 4, and 7 led to deficient matrix coating. (D-F) Mass spectra of on-tissue lipid detection with quercetin as the matrix, using three different solution compositions. The solution composing 2.6 mg/ml quercetin in 80% methanol with 0.1% NH 4 OH was the best. (G) Overlay of mass spectra of the quercetin adduct ion [2M+K] +, before and after matrix solution optimization, showing the reduction of [2M+K] + ion after optimization. Two ESIgenerated ions from the ESI tuning mix solution, i.e., m/z and m/z , were used as the controls. S-4

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6 Supporting Information Figure S2. (A) Comparison of the matrix morphology of quercetin and 2-MBT on rat liver tissue sections, examined by scanning electron microscopy after matrix coating. (B) Comparison of MALDI-FTICR mass spectra of lipid profiles detected on two serial rat liver tissue sections with 2-MBT (upper) and quercetin (lower) as the MALDI matrices, respectively. The matrix-related signals have been labeled with triangles. (C) collision-induced dissociation mass spectra of m/z [M-H] - and m/z [M-H] - by LC-(-)ESI MS/MS. These two phosphotidic acids (PAs) were identified as PA(20:4/0:0) and PA(18:0/22:5), respectively. These two PAs were detected as their [M+K] + ions, on the same rat liver tissue sections, with positive-ion MALDI-FTICR MS with quercetin as the matrix. S-6

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9 Supporting Information Figure S3. Venn diagram comparison of lipid detection on rat liver sections by MALDI-FTICR MS in the positive-ion mode using quercetin or 2-MBT as the matrices. S-9

10 Supporting Information Figure S4. Evaluation of the stability of quercetin as a MALDI matrix. Rat liver sections were coated with quercetin and loaded onto the FTICR ion sources operated at a ~10-7 mbar vacuum for different time periods. Mass spectra were acquired at 0 hour (A), 6 hours (B), 12 hours (C), and 24 hours (D); and re-measured by MALDI-FTICR MS after being kept at room temperature for 13 days (E) and 28 days (F). A total of 161, 159, 159, 158, 157, 158 lipid entities were detected and identified, and no change in lipid detection was found, demonstrating the low volatility of quercetin under both the high vacuum and atmospheric pressure conditions. S-10

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12 Supporting Information Figure S5. Optical images of a H&E-stained rat brain section before the matrix coating and after removal of the coated quercetin matrix. S-12

13 Supporting Information Figure S6. MALDI-FTCIR mass spectrum acquired from a 12-µm thick rat brain tissue section with quercetin as the matrix. A total of 212 lipids were identified. S-13

14 Supporting Information Table S1. Optimized matrix solution compositions for the ten flavonoid compounds. Compounds Final concentration (mg/ml) Methanol (%) NH 4 OH (%) Flavone ,3,4 - trihydroxyflavone hydroxyflavone Saturated (<2.5) hydroxyflavone Saturated (<2.5) ,7-dihydroxyflavone Chrysin Fisetin Luteolin Quercetin Morin S-

15 Supporting Information Table S2. Comparison of lipid detection on rat liver sections by MALDI-FTICR MS in the positive-ion mode with ten flavone-type compounds as the MALDI matrices. Classification Glycerophospholipids Phosphatidylcholines (PCs) Flavone 3-HF 5-HF 3,7-DHF Chrysin Peak lists of lipid candidates 7, 3, 4 - THF Fisetin Luteolin Quercetin Morin Compounds PC(16:0/0:0) PC(18:3) LysoPC(18:3) PC(18:2) LysoPC(18:2) PC(O-18:1) PC(18:1) PC(18:0/0:0) PC(16:0/0:0) PC(18:2) LysoPC(18:2) PC(20:4/0:0) PC(18:0/0:0) PC(18:2) LysoPC(18:2) PC(18:1/0:0) PC(18:0/0:0) PC(20:4/0:0) PC(22:6) LysoPC(22:6) PC(20:4/0:0) Structur ally specific CID ions (m/z) a) 104, 184, 478, , 184, 506, , 184, 478, , 184, 526, , 184, 506, , 184, 504, , 184, 506, , 184, 526, , 184, 526, 544 S-15

16 Classification Flavone 3-HF 5-HF 3,7-DHF Chrysin Peak lists of lipid candidates 7, 3, 4 - THF Fisetin Luteolin Quercetin Morin Compounds PC(22:6) LysoPC(22:6) LysoPC(22:5) PC(16:0/16:0) PC(16:0/16:0) Structur ally specific CID ions (m/z) a) 104, 184, 478, , 184, 478, PC(34:2) 184, PC(16:0/18:1) PC(16:0/16:0) PC(34:3) PC(34:2) 86, 184, , 184, 478, PC(36:4) 184, PC(36:3) 184, PC(36:2) 184, PC(36:1) 184, hexadecanyl-2-(8-[3]- ladderane-octanyl)-snglycerophosphocholine PC(34:3) PC(34:2) 184, PC(16:0/18:1) PC(34:0) PC(36:4) 184, PC(36:2) 184, PC(18:0/20:4) 184, , 184, , 627, 752, PC(36:5) 184, PC(36:4) 184, PC(36:3) 184, 785 S-16

17 Classification Flavone 3-HF 5-HF 3,7-DHF Chrysin Peak lists of lipid candidates 7, 3, 4 - THF Fisetin Luteolin Quercetin Morin Compounds Structur ally specific CID ions (m/z) a) PC(36:2) 184, PC(36:1) 184, PC(38:6) 184, PC(18:0/20:4) PC(38:2) 184, 627, 752, PC(38:6) 184, PC(38:5) 184, PC(18:0/20:4) PC(18:0/22:6) PC(40:7) PC(18:0/22:6) 184, 627, 752, , 184, 776, , 184, 776, PC(40:5) 184, PC(42:4) Phosphatidylethanolamines (PEs) PE(16:0/0:0) PE(20:0) LysoPE(20:0) PE(18:2) LysoPE(18:2) PE(18:1) LysoPC(18:1) PE(18:0/0:0) PE(22:2) 153, 196, 2, 255, 391, 409, 452 0, 153, 196, 2, 283, 419, 437, 480 S-17

18 Classification Flavone 3-HF 5-HF 3,7-DHF Chrysin Peak lists of lipid candidates 7, 3, 4 - THF Fisetin Luteolin Quercetin Morin Compounds LysoPE(22:2) PE(22:0) LysoPE(22:0) PE(20:4/0:0) PE(20:2) LysoPE(20:2) PE(20:0) LysoPE(20:0) PE(22:6) LysoPE(22:6) PE(22:4) LysoPE(22:4) PE(26:1) Structur ally specific CID ions (m/z) a) 153, 195, 259, 303, PE(34:2) 255, PE(38:2) 283, PE(16:0/20:4) PE(18:1/18:1) PE(P-40:7) or 1-(8-[3]-ladderane- octanoyl)-2-(8-[3]- ladderane-octanyl)snglycerophosphoethanola mine PE(P-18:0/22:6) PE(P-40:5) PE(40:2) 195, 255, 259, 303, 434, 452, 482, 500, , 281, 460, 478, , 327, 446, 464, 774 S-18

19 Classification Flavone 3-HF 5-HF 3,7-DHF Chrysin Peak lists of lipid candidates 7, 3, 4 - THF Fisetin Luteolin Quercetin Morin Compounds Structur ally specific CID ions (m/z) a) PE(38:6) 279, PE(38:5) PE(38:4) PE(42:4) PE(P-42:6) PE(40:6) PE(40:4) PE(44:9) PE(44:9) 259, 283, 303, 462, 480, 482, 500, , 327, 462, 480, 506, 524, 791 Phosphatidic acids (PAs) PA(18:1/0:0) PA(18:0/0:0) LPA(18:0/0:0) PA(20:1/0:0) PA(20:4/0:0) PA(20:3/0:0) PA(20:2/0:0) PA(20:1/0:0) PA(P-32:1) PA(O-32:1) or PA(P- 32:0) PA(O-18:0/15:0) PA(P-34:3) or PA(O- 34:4) 79, 153, 171, 283, , 171, 259, 303, 457 S-19

20 Classification Flavone 3-HF 5-HF 3,7-DHF Chrysin Peak lists of lipid candidates 7, 3, 4 - THF Fisetin Luteolin Quercetin Morin Compounds PA(P-34:1) or PA(O- 34:2) PA(P-16:0/20:5) PA(P-36:3) or PA(O- 36:4) PA(16:0/18:2) PA(16:0/18:1) PA(36:4) PA(18:1/18:2) PA(18:0/18:2) PA(38:3) PA(16:0/22:6) PA(38:5) PA(38:4) PA(38:3) PA(38:2) PA(40:5) PA(42:8) PA(40:7) PA(18:0/22:6) Structur ally specific CID ions (m/z) a) 79, 153, 255, 279, 391, 409, , 255, 281, , 417, 435, , 281, 415, 417, 433, , 153, 279, 283, 415, 419, 433, 437, , 255, 283, 391, 409, 463, 481, , 283, 327, 419, 437, 463, S-20

21 Classification Flavone 3-HF 5-HF 3,7-DHF Chrysin Peak lists of lipid candidates 7, 3, 4 - THF Fisetin Luteolin Quercetin Morin Compounds PA(18:0/22:5) PA(42:7) Structur ally specific CID ions (m/z) a) 481, , 283, 329, 419, 437, 465, 483, 749 Phosphoglycerols (PGs) PG(P-16:0) PG(18:0/20:4) 152, 259, 283, 303, 419, 437, 438, 457, 593, 511, 513, 531, 798 Phosphatidylserine (PS) PS(O-40:4) or PS(P- 40:3) phosphatidylinositols (PIs) PI(34:6) PI(18:0/20:4) PI(40:6) 240, 259, 283, 303, 419, 437, 439, 457, 581, 599, 601, 619, Glycerophosphoglycerophosphoglycerols CL(1\'- [18:2(9Z,12Z)/0:0],3\'- [18:2(9Z,12Z)/0:0]) S-21

22 Classification Flavone 3-HF 5-HF 3,7-DHF Chrysin Peak lists of lipid candidates 7, 3, 4 - THF Fisetin Luteolin Quercetin Morin Compounds Structur ally specific CID ions (m/z) a) Cyclic phosphatidic acids (cpas) CPA(16:0) CPA(18:2) CPA(18:1) CPA(18:0) Sphingolipids Ceramides (Cers) CerP(d18:1/8:0) Cer(d18:1/24:0) CerP(d18:1/24:1) 264, 470, 488, , 671, , 749, 767 Sphingomyelins (SMs) SM(d18:1/16:0) 644, SM(d18:1/16:0) SM(d18:1/16:0) SM(d18:1/18:0) SM(d18:1/20:0) SM(d18:1/24:1) SM(d18:1/24:0) SM(d18:1/22:0) 7, 666, , 163, , 703, SM(d18:1/24:0) 778, SM(d18:1/24:1) SM(d18:1/24:0) S-22

23 Classification Flavone 3-HF 5-HF 3,7-DHF Chrysin Peak lists of lipid candidates 7, 3, 4 - THF Fisetin Luteolin Quercetin Morin Compounds Structur ally specific CID ions (m/z) a) Neutral Lipids Glycerolipids Monoacyllglycerol (MAG) MG (16:0) MG (18:0) MG (20:4) 239, 257, 313, 331, 369 Diacylglycerols (DAGs) DG(P-:0/18:1(9Z)) DG(34:2) DG(34:1) DG(16:0/19:0/0:0)[iso2] (-methyl- pentadecanoyl)-2-(8-[3]- ladderane-octanyl)-snglycerol 1-(6-[5]-ladderane- hexanoyl)-2-(8-[3]- ladderane-octanyl)-snglycerol 1-(8-[3]-ladderane- octanoyl-2-(8-[3]- ladderane-octanyl)-snglycerol DG(36:4) DG(36:2) DG(38:6) DG(40:8) DG(38:4) Sterol Lipids S-23

24 Classification Flavone 3-HF 5-HF 3,7-DHF Chrysin Peak lists of lipid candidates 7, 3, 4 - THF Fisetin Luteolin Quercetin Morin Compounds Conicasterol B :2 Cholesteryl ester or zymosteryl oleate 3-Oxo-5β-cholan-24-oic Acid Structur ally specific CID ions (m/z) a) Fatty acyls Fatty acids (FAs) FA(18:2) FA(18:1) FA(18:2) FA(20:4) FA(18:2) FA(22:6) FA(26:2) FA(26:2) FA(26:1) 195, 277, , 167, 195, 249, , 229, 249, 283, 299, 327 Lipids numbers a), Structurally specific CID ions of extracted lipids were detected by LC-MS/MS using CID. Red fragment ions were detected in the positive ion mode, and blue fragment ions were detected in the negative ion mode. S-24

25 Supporting Information Table S3A. Comparison of lipid detection on rat liver sections by MALDI-FTICR MS in the positive-ion mode using quercetin and 2-MBT as the matrices, respectively. Classification Glycerophospholipids Phosphatidylcholines (PCs) No. Measured m/z Error (ppm) Assignment Quercetin 2-MBT Calculated m/z Quercetin 2-MBT Ion form Compound Molecular formula [M+H] + PC(16:0/0:0) C 24 H 50 NO 7 P [M+H] + PC(18:3) LysoPC(18:3) [M+H] + PC(18:2) LysoPC(18:2) [M+H] + PC(O-18:1) PC(18:1) C 26 H 48 NO 7 P Ref. C 26 H 50 NO 7 P 10, 13 C 26 H 52 NO 7 P [M+H] + PC(18:0/0:0) C 26 H 54 NO 7 P [M+K] + PC(16:0/0:0) C 24 H 50 NO 7 P [M+Na] + PC(18:2) LysoPC(18:2) [M+H] + PC(20:5) C 28 H 48 NO 7 P C 26 H 50 NO 7 P [M+H] + PC(20:4/0:0) C 28 H 50 NO 7 P [M+Na] + PC(18:0/0:0) C 26 H 54 NO 7 P [M+H] + PC(20:3) LysoPC(20:3) [M+K] + PC(18:2) LysoPC(18:2) C 28 H 52 NO 7 P C 26 H 50 NO 7 P [M+K] + PC(18:1/0:0) C 26 H 52 NO 7 P [M+K] + PC(18:0/0:0) C 26 H 54 NO 7 P [M+Na] + PC(20:4/0:0) C 28 H 50 NO 7 P [M+H] + PC(22:6) LysoPC(22:6) C 30 H 50 NO 7 P [M+K] + PC(20:4/0:0) C 28 H 50 NO 7 P [M+K] + PC(22:6) LysoPC(22:6) C 30 H 50 NO 7 P S-25

26 Classification No. Measured m/z Error (ppm) Assignment Quercetin 2-MBT Calculated m/z Quercetin 2-MBT Ion form Compound Molecular formula [M+K] + LysoPC(22:5) C 30 H 52 NO 7 P Ref [M+H] + PC(32:1) C 40 H 78 NO 8 P [M+H] + PC(32:0) C 40 H 80 NO 8 P [M+Na] + PC(16:0/16:0) C 40 H 80 NO 8 P 16, [M+H] + PC(34:2) C 42 H 80 NO 8 P 13, [M+H] + PC(34:1) C 42 H 82 NO 8 P 10, 11, 13-16, 22, [M+H] + PC(34:0) C 42 H 84 NO 8 P [M+K] + PC(16:0/16:0) C 40 H 80 NO 8 P, 16, 18, 24, [M+Na] + PC(34:3) C 42 H 78 NO 8 P [M+Na] + PC(34:2) C 42 H 80 NO 8 P 13, 21, [M+H] + PC(36:5) C 44 H 78 NO 8 P [M+H] + PC(36:4) C 44 H 80 NO 8 P 10, 13, 15, [M+Na] + PC(34:0) C 42 H 84 NO 8 P [M+H] + PC(36:3) C 44 H 82 NO 8 P 10, [M+H] + PC(36:2) C 44 H 84 NO 8 P 10, 13, [M+H] + PC(36:1) C 44 H 86 NO 8 P 10,, 16, [M+K] + ladderane-octanyl)-snglycerophosphocholine 1-hexadecanyl-2-(8-[3] [M+K] + PC(34:3) C 42 H 78 NO 8 P C 44 H 84 NO 6 P [M+K] + PC(34:2) C 42 H 80 NO 8 P 13,, [M+K] + PC(16:0/18:1) C 42 H 82 NO 8 P 13-16, 18, 20, 22, [M+K] + PC(34:0) C 42 H 84 NO 8 P [M+Na] + PC(36:4) C 44 H 80 NO 8 P 13,, 27, [M+Na] + PC(36:2) C 44 H 84 NO 8 P 13, [M+H] + PC(38:5) C 46 H 82 NO 8 P [M+H] + PC(18:0/20:4) C 46 H 84 NO 8 P 10, [M+K] + PC(36:5) C 44 H 78 NO 8 P [M+K] + PC(36:4) C 44 H 80 NO 8 P 10,, 20, [M+K] + PC(36:3) C 44 H 82 NO 8 P 13, 26 S-26

27 Classification No. Measured m/z Error (ppm) Assignment Quercetin 2-MBT Calculated m/z Quercetin 2-MBT Ion form Compound Molecular formula [M+K] + PC(36:2) C 44 H 84 NO 8 P 13, [M+K] + PC(36:1) C 44 H 86 NO 8 P, 15, 23, 25, 29, [M+Na] + PC(38:6) C 46 H 80 NO 8 P 10,, 18, [M+H] + PC(40:9) C 48 H 78 NO 8 P [M+Na] + PC(38:4) C 46 H 84 NO 8 P 13,, [M+H] + PC(40:7) C 48 H 82 NO 8 P [M+H] + PC(40:6) C 48 H 84 NO 8 P 10,, [M+Na] + PC(38:2) C 46 H 88 NO 8 P [M+H] + PC(40:5) C 48 H 86 NO 8 P [M+K] + PC(38:7) C 46 H 78 NO 8 P [M+K] + PC(38:6) C 46 H 80 NO 8 P 10,, 18, 24, 25, [M+K] + PC(38:5) C 46 H 82 NO 8 P, [M+K] + PC(18:0/20:4) C 46 H 84 NO 8 P 13, 20, 24, [M+Na] + PC(18:0/22:6) C 48 H 84 NO 8 P 10,, [M+K] + PC(40:7) C 48 H 82 NO 8 P [M+K] + PC(18:0/22:6) C 48 H 84 NO 8 P 18, [M+K] + PC(40:5) C 48 H 86 NO 8 P [M+NH 4] + PC(42:4) C 50 H 92 NO 8 P Ref. Phosphatidylethanolamines (PEs) [M+K] + PE(16:0) C 21 H 44 NO 7 P [M+H] + PE(20:0) LysoPE(20:0) [M+K] + PE(18:2) LysoPE(18:2) [M+K] + PE(18:1) LysoPC(18:1) C 25 H 52 NO 7 P C 23 H 44 NO 7 P C 23 H 46 NO 7 P [M+K] + PE(18:0/0:0) C 23 H 48 NO 7 P [M+H] + PE(22:2) LysoPE(22:2) [M+H] + PE(22:0) LysoPE(22:0) C 27 H 52 NO 7 P C 27 H 56 NO 7 P S-27

28 Classification No. Measured m/z Error (ppm) Assignment Quercetin 2-MBT Calculated m/z Quercetin 2-MBT Ion form Compound Molecular formula [M+K] + PE(20:4/0:0) C 25 H 44 NO 7 P [M+K] + PE(20:2) LysoPE(20:2) [M+K] + PE(20:0) LysoPE(20:0) [M+K] + PE(22:6) LysoPE(22:6) [M+K] + PE(22:4) LysoPE(22:4) C 25 H 48 NO 7 P C 25 H 52 NO 7 P C 27 H 44 NO 7 P C 27 H 48 NO 7 P [M+NH 4] + PE(26:1) C 31 H 60 NO 8 P [M+K] + PE(34:2) C 39 H 74 NO 8 P [M+H] + PE(38:2) C 43 H 82 NO 8 P 13, [M+K] + PE(16:0/20:4) C 41 H 74 NO 8 P [M+K] + PE(18:1/18:1) C 41 H 78 NO 8 P [M+Na] + PE(P-40:7) C 45 H 76 NO 7 P [M+Na] + PE(P-18:0/22:6) C 45 H 78 NO 7 P [M+Na] + PE(P-40:5) C 45 H 80 NO 7 P [M+H] + PE(40:2) C 45 H 86 NO 8 P [M+K] + PE(38:6) C 43 H 74 NO 8 P [M+K] + PE(38:5) C 43 H 76 NO 8 P [M+K] + PE(38:4) C 43 H 78 NO 8 P [M+H] + PE(42:4) C 47 H 86 NO 8 P [M+Na] + PE(P-42:6) C 47 H 82 NO 7 P [M+K] + PE(40:6) C 45 H 78 NO 8 P [M+K] + PE(40:4) C 45 H 82 NO 8 P [M+H] + PE(44:9) C 49 H 80 NO 8 P [M+Na] + PE(44:9) C 49 H 80 NO 8 P [M+K] + PE(44:9) C 49 H 80 NO 8 P Ref. Phosphatidic acids (PAs) [M+K] + PA(18:1/0:0) C 21 H 41 O 7 P S-28

29 Classification No. Measured m/z Error (ppm) Assignment Quercetin 2-MBT Calculated m/z Quercetin 2-MBT Ion form Compound [M+K] + PA(18:0) LPA(18:0) Molecular formula C 21 H 43 O 7 P [M+Na] + PA(20:1) C 23 H 45 O 7 P [M+K] + PA(20:4/0:0) C 23 H 39 O 7 P [M+K] + PA(20:3) C 23 H 41 O 7 P [M+K] + PA(20:2) C 23 H 43 O 7 P [M+K] + PA(20:1) C 23 H 45 O 7 P [M+H] + PA(P-32:1) C 35 H 67 O 7 P [M+H] + PA(O-32:1) or PA(P-32:0) C 35 H 69 O 7 P [M+H] + PA(O-18:0/15:0) C 36 H 73 O 7 P [M+H] + PA(P-34:3) C 37 H 67 O 7 P [M+H] + PA(P-34:1) or PA(O-34:2) C 37 H 71 O 7 P [M+H] + PA(P-16:0/20:5) C 39 H 67 O 7 P [M+H] + PA(P-36:3) or PA(O-36:4) C 39 H 71 O 7 P Ref [M+K] + PA(16:0/18:2) C 37 H 69 O 8 P [M+K] + PA(16:0/18:1) C 37 H 71 O 8 P [M+K] + PA(36:4) C 39 H 69 O 8 P [M+K] + PA(18:1/18:2) C 39 H 71 O 8 P [M+K] + PA(18:0/18:2) C 39 H 73 O 8 P [M+Na] + PA(38:3) C 41 H 75 O 8 P [M+K] + PA(16:0/22:6) C 41 H 69 O 8 P [M+K] + PA(38:5) C 41 H 71 O 8 P [M+K] + PA(38:4) C 41 H 73 O 8 P [M+K] + PA(38:3) C 41 H 75 O 8 P S-29

30 Classification No. Measured m/z Error (ppm) Assignment Quercetin 2-MBT Calculated m/z Quercetin 2-MBT Ion form Compound Molecular formula [M+K] + PA(38:2) C 41 H 77 O 8 P [M+Na] + PA(40:5) C 43 H 75 O 8 P [M+H] + PA(42:8) C 45 H 73 O 8 P [M+K] + PA(40:7) C 43 H 71 O 8 P [M+K] + PA(18:0/22:6) C 43 H 73 O 8 P [M+K] + PA(18:0/22:5) C 43 H 75 O 8 P [M+K] + PA(42:7) C 45 H 75 O 8 P Ref. Phosphoglycerols (PGs) [M+K] + PG(P-16:0) C 22 H 45 O 8 P [M+K] + PG(18:0/20:4) C 44 H 79 O 10 P Phosphatidylserine (PS) [M+K] + PS(O-40:4) or PS(P-40:3) C 46 H 84 NO 9 P Phosphatidylinositols (PIs) [M+Na] + PI(34:6) C 43 H 71 O 13 P [M+K] + PI(18:0/20:4) C 47 H 83 O 13 P [M+K] + PI(40:6) C 49 H 83 O 13 P Glycerophosphoglycerophosphoglycerols [M+K] + CL(1\'-[18:2(9Z,12Z)/0:0],3\'- [18:2(9Z,12Z)/0:0]) C 45 H 82 O 15 P 2 Cyclic phosphatidic acids (cpas) [M+K] + CPA(16:0) C 19 H 37 O 6 P [M+K] + CPA(18:2) C 21 H 37 O 6 P [M+K] + CPA(18:1) C 21 H 39 O 6 P [M+K] + CPA(18:0) C 21 H 41 O 6 P S-30

31 Classification No. Measured m/z Error (ppm) Assignment Quercetin 2-MBT Calculated m/z Quercetin 2-MBT Ion form Compound Molecular formula Ref. Sphingolipids Ceramides (Cers) [M+H] + CerP(d18:1/8:0) C 26 H 52 NO 6 P [M+K] + Cer(d18:1/24:0) C 42 H 83 NO [M+K] + CerP(d18:1/24:1) C 42 H 82 NO 6 P Sphingomyelins (SMs) [M+H] + SM(d18:1/16:0) C 39 H 79 N 2 O 6 P 10, [M+Na] + SM(d18:1/16:0) C 39 H 79 N 2 O 6 P, 15, 20, [M+K] + SM(d18:1/16:0) C 39 H 79 N 2 O 6 P [M+K] + SM(d18:1/18:0) C 41 H 83 N 2 O 6 P, [M+H] + SM(d18:1/22:0) C 45 H 91 N 2 O 6 P [M+K] + SM(d18:1/20:0) C 43 H 87 N 2 O 6 P, [M+H] + SM(d18:1/24:1) C 47 H 93 N 2 O 6 P [M+H] + SM(d18:1/24:0) C 47 H 95 N 2 O 6 P [M+K] + SM(d18:1/22:0) C 45 H 91 N 2 O 6 P [M+Na] + SM(d18:1/24:0) C 47 H 95 N 2 O 6 P, [M+K] + SM(d18:1/24:1) C 47 H 93 N 2 O 6 P [M+K] + SM(d18:1/24:0) C 47 H 95 N 2 O 6 P Neutral Lipids Glycerolipids Monoacylglycerols (MAGs) [M+K] + MG (16:0) C 19 H 38 O [M+K] + MG (18:0) C 21 H 42 O [M+K] + MG (20:4) C 23 H 38 O 4 Diacylglycerols (DAGs) [M+H] + DG(P-:0/18:1(9Z)) C 35 H 66 O 4 S-31

32 Classification No. Measured m/z Error (ppm) Assignment Quercetin 2-MBT Calculated m/z Quercetin 2-MBT Ion form Compound [M+H] + 2-(8-[3]- 1-(-methyl-pentadecanoyl)- ladderane-octanyl)-sn-glycerol [M+H] + 2-(8-[3]-ladderane-octanyl)- 1-(6-[5]-ladderane-hexanoyl)- sn-glycerol Molecular formula C 39 H 70 O [M+K] + DG(34:2) C 37 H 68 O [M+K] + DG(34:1) C 37 H 70 O [M+K] + DG(16:0/19:0/0:0)[iso2] C 38 H 74 O [M+H] + (8-[3]-ladderane-octanyl)-snglycerol 1-(8-[3]-ladderane-octanoyl-2- Ref. C 34 H 69 O 7 P C 43 H 70 O [M+K] + DG(36:4) C 39 H 68 O [M+K] + DG(36:2) C 39 H 72 O [M+K] + DG(38:6) C 41 H 68 O [M+Na] + DG(40:8) C 43 H 63 D 5 O [M+K] + DG(38:4) C 41 H 72 O 5 Sterol Lipids [M+Na] + Conicasterol B C 29 H 44 O [M+K] + 18:2 Cholesteryl ester or zymosteryl oleate C 45 H 76 O [M+Na] + 3-Oxo-5β-cholan-24-oic Acid C 24 H 38 O 3 Fatty acyls Fatty acids (FAs) [M+K] + FA(18:2) C 18 H 32 O [M+K] + FA(18:1) C 18 H 34 O [M+K] + FA(18:2) C 18 H 32 O [M+K] + FA(20:4) C 20 H 32 O [M+K] + FA(18:2) C 18 H 32 O [M+K] + FA(22:6) C 22 H 32 O 2 S-32

33 Classification No. Measured m/z Error (ppm) Assignment Quercetin 2-MBT Calculated m/z Quercetin 2-MBT Ion form Compound Molecular formula Ref [M+H] + FA(22:0) C 22 H 42 O [M+Na] + FA(22:0) C 22 H 42 O [M+Na] + FA(26:2) C 26 H 48 O [M+K] + FA(26:2) C 26 H 48 O [M+K] + FA(26:1) C 26 H 50 O 2 S-33

34 Supporting Information Table S3B. Classification of lipid entities shown in Supporting Information Table S3A. Lipid classification Number of Lipidsquercetin (LN Q ) Number of Lipids-Main Class (LN MC ) LN MC /LN Total Number of Lipids-2-MBT (LN 2-MBT ) Number of Lipids-Main Class (LN MC) LN MC /LN Total LN Q /LN 2- MBT Glycerophospholipids LN MC- LN MC- Glycerophospholipids Glycerophospholipids Phosphatidylcholines (PCs) % % 0.9 Phosphatidylethanolamines (PEs) % 15.7% 2.1 Phosphatidic acids (PAs) % 0 0 Phosphoglycerols (PGs) 2 1.2% 1 1.1% 2.0 Phosphatidylserine (PS) % 75.8% % 77.5% 1.0 phosphatidylinositols (PIs) 3 1.9% 0 0 Glycerophosphoglycerophosphoglycerols 1 0.6% 0 0 Cyclic phosphatidic acids (cpas) 4 2.5% 0 0 Sphingolipids LN MC-Sphingolipids LN MC-Sphingolipids Ceramides (Cers) 3 1.9% 1 1.1% 3.0 Sphingomyelins (SMs) % 7.5% % 13.5% 0.8 Neutral Lipids LN MC-NL LN MC-NL Glycerolipids LN MC-NL: Glycerolipids LN MC-NL: Glycerolipids Monoacylglycerols (MAGs) 3 1.9% 1 1.1% % 5 5.6% Diacylglycerols (DAGs) % 4 4.5% 3.0 Sterol Lipids LN MC-NL: Sterol lipids LN MC-NL: Sterol lipids % 1.9% Fatty acyls LN MC-Fatty acyls LN MC-Fatty acyls Fatty acids (FAs) % 5.6% % 3.4% 3.0 S-34

35 Lipid classification Number of Lipidsquercetin (LN Q ) Number of Lipids-Main Class (LN MC ) LN MC /LN Total Number of Lipids-2-MBT (LN 2-MBT ) Number of Lipids-Main Class (LN MC) LN MC /LN Total LN Q /LN 2- MBT LN Total S-35

36 Supporting Information Table S4A. Imaged and identified lipids detected on transversally-cut rat brain tissue sections by MALDI- FTICR MS in the positive-ion mode using quercetin as a matrix. Classification No. Measured m/z Calculated m/z Error (ppm) Glycerophospholipids Phosphatidylcholines (PCs) Ion form Assignment Compound Molecular formula [M+H] + PC(O-16:2) C 24 H 48 NO 6 P [M+H] + PC(P-18:1) C 26 H 52 NO 6 P [M+K] + PC(O-16:2) C 24 H 48 NO 6 P [M+K] + PC(O-16:1) or LysoPC(P-16:0) LysoPC(18:0) [M+H] + PC(18:0) PC(O-18:0) C 24 H 50 NO 6 P [M+Na] + PC(O-18:2) or PC(P-18:1) C 26 H 52 NO 6 P [M+K] + LysoPC(16:0) PC(16:0) PC(O-16:0) [M+K] + PC(O-18:2) or PC(P-18:1) C 26 H 52 NO 6 P [M+K] + PC(18:1) PC(O-18:1) LysoPC(18:1) [M+K] + LysoPC(18:0) PC(18:0) PC(O-18:0) [M+K] + PC(20:1) C 28 H 54 NO 8 P [M+K] + PC(22:6) LysoPC(22:6) Ref. C 26 H 54 NO 7 P 13 C 24 H 50 NO 7 P C 26 H 52 NO 7 P C 26 H 54 NO 7 P C 30 H 50 NO 7 P [M+H] + PC(32:0) C 40 H 80 NO 8 P [M+Na] + PC(32:0) C 40 H 80 NO 8 P 16, [M+H] + PC(34:1) C 42 H 82 NO 8 P 10, 11, 13-16, 22, [M+K] + PC(32:1) C 40 H 78 NO 8 P S-36

37 Assignment Classification No. Measured m/z Calculated m/z Error (ppm) Ion form Compound Molecular formula Ref [M+K] + PC(32:0) C 40 H 80 NO 8 P, 16, 18, 24, [M+Na] + PC(34:1) C 42 H 82 NO 8 P [M+H] + (8-[3]-ladderane-octanyl)-snglycerophosphocholine 1-(6-[5]-ladderane-hexanoyl) [M+K] + ladderane-octanyl)-snglycerophosphocholine 1-hexadecanyl-2-(8-[3]- C 46 H 76 NO 7 P C 44 H 84 NO 6 P [M+K] + PC(34:1) C 42 H 82 NO 8 P 13-16, 18, 20, 22, [M+Na] + PC(36:2) C 44 H 84 NO 8 P 13, [M+Na] + (8-[3]-ladderane-octanyl)-snglycerophosphocholine 1-(6-[3]-ladderane-hexanoyl)-2- C 46 H 78 NO 7 P [M+H] + PC(38:4) C 46 H 84 NO 8 P 10, [M+Na] + PC(P-38:6) C 46 H 80 NO 7 P [M+K] + PC(36:4) C 44 H 80 NO 8 P 10,, 20, [M+K] + PC(36:2) C 44 H 84 NO 8 P 13, [M+K] + PC(36:1) C 44 H 86 NO 8 P, 15, 23, 25, 29, [M+Na] + PC(38:4) C 46 H 84 NO 8 P 13,, [M+Na] + PC(38:2) C 46 H 88 NO 8 P [M+K] + PC(38:6) C 46 H 80 NO 8 P 10,, 18, 24, 25, [M+K] + PC(38:5) C 46 H 82 NO 8 P, [M+K] + PC(38:4) C 46 H 84 NO 8 P 13, 20, 24, [M+K] + PC(38:1) C 46 H 90 NO 8 P [M+Na] + PC(40:6) C 48 H 84 NO 8 P 10,, [M+K] + PC(40:6) C 48 H 84 NO 8 P 18, [M+K] + PC(40:5) C 48 H 86 NO 8 P [M+K] + PC(40:4) C 48 H 88 NO 8 P [M+K] + PC(40:1) C 48 H 94 NO 8 P [M+K] + PC(42:10) C 50 H 80 NO 8 P [M+K] + PC(42:2) C 50 H 96 NO 8 P [M+K] + PC(42:1) C 50 H 98 NO 8 P S-37

38 Classification No. Measured m/z Calculated m/z Error (ppm) Phosphatidylethanolamines (PEs) Ion form Assignment Compound Molecular formula [M+K] + PE(P-16:0) C 21 H 44 NO 6 P [M+K] + PE(16:0) C 21 H 44 NO 7 P [M+K] + PE(O-18:1) or PE(P-18:0) C 23 H 48 NO 6 P [M+K] + PE(18:2) LysoPE(18:2) [M+K] + PE(18:1) LysoPE(18:1) C 23 H 44 NO 7 P C 23 H 46 NO 7 P [M+K] + PE(18:0) or LysoPE(18:0) C 23 H 48 NO 7 P + PE(20:1) or [M+K] LysoPE(20:1) PE(22:6) [M+K] + LysoPE(22:6) [M+K] + PE(22:4) LysoPE(22:4) C 25 H 50 NO 7 P C 27 H 44 NO 7 P C 27 H 48 NO 7 P [M+H] + PE(36:0) or PE-NMe2(17:0/17:0) C 41 H 82 NO 8 P [M+K] + PE(34:1) C 39 H 76 NO 8 P [M+K] + PE(34:0) or PE-NMe2(16:0/16:0) C 39 H 78 NO 8 P [M+K] + PE(P-36:1) or PE(O-36:2) C 41 H 80 NO 7 P [M+H] + PE(38:2) C 43 H 82 NO 8 P 13, [M+H] + PE(38:1) C 43 H 84 NO 8 P [M+K] + PE(36:1) or PE-NMe2(34:1) C 41 H 80 NO 8 P [M+K] + PE(P-38:6) C 43 H 74 NO 7 P [M+K] + PE(P-38:5) or PE(O-38:6) C 43 H 76 NO 7 P [M+K] + PE(P-38:4) or PE(O-38:5) C 43 H 78 NO 7 P [M+H] + PE(40:2) C 45 H 86 NO 8 P [M+K] + PE(38:6) or PE(P-38:6) C 43 H 74 NO 8 P [M+H] + PE(40:1) C 45 H 88 NO 8 P [M+K] + PE(38:5) C 43 H 76 NO 8 P [M+K] + PE(38:4) or PE- C 43 H 78 NO 8 P Ref. S-38

39 Classification No. Measured m/z Calculated m/z Error (ppm) Ion form Assignment Compound NMe2(16:0/20:4(5Z,8Z,11Z,Z) )[S] or PE(P-38:4) Molecular formula [M+K] + PE(P-40:7) C 45 H 76 NO 7 P [M+K] + PE(P-40:6) C 45 H 78 NO 7 P [M+K] + PE(P-40:5) or PE(O-40:6) C 45 H 80 NO 7 P [M+K] + PE(P-40:4) or PE(O-40:5) C 45 H 82 NO 7 P [M+NH 4] + PE(40:1) C 45 H 88 NO 8 P [M+H] + PE(42:5) C 47 H 84 NO 8 P [M+K] + PE(40:6) C 45 H 78 NO 8 P [M+K] + PE(40:4) C 45 H 82 NO 8 P [M+NH 4] + PE(42:2) C 47 H 90 NO 8 P [M+K] + PE(P-42:2) C 47 H 90 NO 7 P [M+K] + PE(42:9) C 47 H 76 NO 8 P [M+NH 4] + PE(44:2) C 49 H 94 NO 8 P Ref. Phosphatidic acids (PAs) [M+H] + PA(P-16:0/0:0) C 19 H 39 O 6 P [M+K] + PA(18:1) C 21 H 41 O 7 P [M+K] + PA(18:0) LPA(18:0) C 21 H 43 O 7 P [M+Na] + PA(20:1) C 23 H 45 O 7 P [M+K] + PA(20:4/0:0) C 23 H 39 O 7 P [M+K] + PA(20:3) C 23 H 41 O 7 P [M+K] + PA(20:2) C 23 H 43 O 7 P [M+K] + PA(20:1) C 23 H 45 O 7 P [M+H] + PA(P-32:1) C 35 H 67 O 7 P [M+H] + PA(P-34:1) or PA(O-34:2) C 37 H 71 O 7 P [M+H] + PA(P-36:5) C 39 H 67 O 7 P [M+H] + PA(O-16:0/20:5) or PA(P-36:4) C 39 H 69 O 7 P [M+H] + PA(P-36:3) C 39 H 71 O 7 P S-39

40 Classification No. Measured m/z Calculated m/z Error (ppm) Ion form Assignment Compound Molecular formula [M+K] + PA(32:1) C 35 H 67 O 8 P [M+K] + PA(32:0) C 35 H 69 O 8 P [M+H] + PA(P-36:1) or PA(O-36:2) C 39 H 75 O 7 P [M+H] + PA(P-36:0) or PA(O-36:1) C 39 H 77 O 7 P [M+Na] + PA(34:1) C 37 H 71 O 8 P [M+Na] + PA(P-36:5) C 39 H 67 O 7 P [M+H] + PA(P-38:5) or PA(O-38:6) C 41 H 71 O 7 P [M+K] + PA(34:2) C 37 H 69 O 8 P [M+K] + PA(34:1) C 37 H 71 O 8 P [M+Na] + PA(36:2) C 39 H 73 O 8 P [M+H] + PA(38:4) C 41 H 73 O 8 P [M+Na] + PA(P-38:6) C 41 H 69 O 7 P [M+K] + PA(36:3) C 39 H 71 O 8 P [M+K] + PA(36:2) C 39 H 73 O 8 P [M+NH 4] + PA(38:1) C 41 H 79 O 8 P [M+Na] + PA(38:2) C 41 H 77 O 8 P [M+K] + PA(38:5) C 41 H 71 O 8 P [M+K] + PA(38:4) C 41 H 73 O 8 P [M+K] + PA(38:3) C 41 H 75 O 8 P [M+K] + PA(38:2) C 41 H 77 O 8 P [M+NH 4] + PA(40:2) C 43 H 81 O 8 P [M+K] + PA(40:7) C 43 H 71 O 8 P [M+K] + PA(40:5) C 43 H 75 O 8 P [M+NH 4] + PA(42:2) C 45 H 85 O 8 P Ref. Phosphoglycerols (PGs) [M+K] + PG(O-34:1) or PG(P-34:0) C 40 H 79 O 9 P [M+Na] + PG(36:4) C 42 H 75 O 10 P [M+K] + PG(18:0/20:4) C 44 H 79 O 10 P S-40