Supporting Information

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1 Supporting Information Development of a High Coverage Pseudotargeted Lipidomics Method Based on Ultra-High Performance Liquid Chromatography-Mass Spectrometry Qiuhui Xuan 1,2#, Chunxiu Hu 1#, Di Yu 1,2, Lichao Wang 1,2, Yang Zhou 1,2, Xinjie Zhao 1, Qi Li 1, Xiaoli Hou 1, Guowang Xu 1* 1 CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning, , China 2 University of Chinese Academy of Sciences, Beijing , China. # These authors contribute equally to this manuscript. * Address correspondence to: Prof. Dr. Guowang Xu, CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian , China. Tel. / Fax: xugw@dicp.ac.cn. S-1

2 Contents Experimental details...s-3 Figure S1. Linear relationship between t R and No. of acyl carbon or No. of acyl double bond..s-7 Figure S2. Typical chromatograms of QC based on pseudotargeted lipidomics method...s-8 Figure S3. Reproducibility of pseudotargeted lipidomics method....s-9 Table S1. Clinical parameters of 30 patients with diabetes and 30 healthy controls..s-10 Table S2. Information of the detected and predicted lipids covering 3377 lipid ion pairs (Table S2 is provided by separated excel sheets)..s-10 Table S3. Each of lipid ion pair represents one or more lipid molecular structures due to different acyl chain combinations caused by different length of acyl chain and different No. of the acyl chain double bond. S-11 Table S4. Concentration range of 7 lipid internal standards....s-13 Table S5. The most significantly changed 163 lipids contributing to the discrimination between patients with diabetes and the healthy controls (Table S5 is provided with a separated excel sheet)..s-13 S-2

3 Sample Preparation Cell lipid extraction. One milliliter of cold MeOH was added to the cell culture dish followed by transferring into a 5 ml tube, and 2.5 ml of MTBE was added, and then the mixture was vortexed for 10 s both before and after adding MTBE. After that, the mixture was vibrated for 15 min. Subsequently, 700 μl of H2O was added and vortexed for 10 s to form a two-phase system. After equilibration for 10 min at 4 C, the mixture was centrifuged at 10,000 g for 10 min at 4 C. Seven hundred microliters of supernatant was lyophilized and stored at -80 C prior to LC-MS analysis. Tissue lipid extraction. Three hundred microliters of cold MeOH was added to accurately weight mg brain or liver tissues followed by the homogenization at 20 Hz for 2 min, and then 1 ml MTBE was added. The mixture was vortexed for 10 s both before and after adding MTBE. After that, the mixture was vibrated for 15 min. Subsequently, 300 μl of H2O was added and vortexed for 10 s to form a two-phase system. After equilibration for 10 min at 4 C, the mixture was centrifuged at 10,000 g for 10 min at 4 C. Four hundred microliters of supernatant was lyophilized and stored at - 80 C before LC-MS analysis. Plasma lipid extraction. An equal aliquot of plasma from 20 healthy subjects was mixed to form pooled quality control (QC) samples for method development. Three hundred microliters of cold MeOH was added to each QC sample followed by the addition of 1 ml of MTBE. The mixture was vortexed for 10 s both before and after adding MTBE. After that, the mixture was vibrated for 15 min. Subsequently, 300 μl H2O was added and vortexed for 10 s to form a two-phase system. After equilibration for 10 min at 4 C, the mixture was centrifuged at 10,000 g for 10 min at 4 C. Four hundred microliters of supernatant was lyophilized and stored at -80 C prior to LC-MS analysis. Specifically, serum samples from 30 diabetic and 30 age-matched healthy control subjects used for method application were prepared as the same manner as that of the plasma lipid extraction described above. Lipidomics Analysis S-3

4 Nontargeted lipidomics analysis using UHPLC/Q Exactive-HF MS A reversed-phase BEH C8 column (2.1 mm 100 mm, 1.7 µm, Waters, Milford, MA, U.S.A.) was used for the chromatographic separation of lipids. Mobile phases A and B were ACN/H2O (60:40, v/v) and IPA/ACN (90:10, v/v) respectively, both containing 10 mm AmAc. The flow rate was 0.26 ml/min. The column temperature was at 55 C. The elution gradient started with 32% B and was kept for 1.5 min, then linearly increased to 85% B at 15.5 min, and then to 97% B at 15.6 min, and held for 2.4 min. The gradient was back to 32% B at 18.1 min and kept for 1.9 min to equilibrate column. The temperature of the sample manager was set at 10 C. The mass spectrometer was operated with a capillary voltage of 3.5 kv in positive mode and 3.0 kv in negative mode. The capillary temperature was set as 300 C. Sheath gas flow rate and aux gas flow rate were set at 45 and 10 (in arbitrary units). Aux gas heater temperature was 350 C. The S- lens rf level was The resolutions of and were set for full scan MS and datadependent MS/MS (ddms 2 ) in both modes. The AGC target and maximum IT were 3x10 6 ions capacity and 100 ms in full scan MS settings while their values were 1x10 5 ions capacity and 50 ms in ddms 2 settings. The TopN (N, the number of top most abundant ions for fragmentation) was set to 15. The normalized collision energy (NCE) was set 25, 35, 45 ev, respectively. The scan range was set at m/z Freeze-dried samples were reconstituted in ACN/IPA/H2O (65:30:5, v/v/v/) containing 5mM AmAc, and 5 µl was injected into the LC-MS system. Pseudotargeted lipidomics analysis using UHPLC/QQQ MRM MS The LC conditions for pseudotargeted lipidomics analysis were exactly the same as those of nontargeted lipidomics analysis mentioned above. For MS detection, the QQQ MRM MS was operated with IonSpray Voltage 5500 V in positive mode; the temperature was set as 500 C; both Ion Source Gas 1 (GS1) and Ion Source Gas 2 (GS2) were set as 50. In negative mode, the QQQ MRM MS was operated with IonSpray Voltage V; the S-4

5 temperature was set as 550 C; both GS1 and GS2 were set as 40. The Collision Gas and Curtain Gas were set as high and 35 respectively in both modes. Lipidomics analysis based on UHPLC/LTQ-Orbitrap MS platform The LC conditions were the same as those used in the nontargeted lipidomics analysis. The LTQ- Orbitrap MS was operated with a capillary voltage of 4.5 kv in positive mode and 4.0 kv in negative mode. The capillary temperature was set as 325 C. Sheath gas flow rate and aux gas flow rate were set at 45 and 10 (in arbitrary units). The Tube Lens was 50 v. In full scan mode, the M/Z range was set in positive mode and in negative mode. The resolution of was set in both modes. Method validation of the developed UHPLC/QQQ MRM MS-based pseudotargeted lipidomics Validation of the developed method was performed in the presence of plasma matrix. Linearity, recovery, limit of detection (LOD), precision and repeatability were assessed referring to accepted guidelines 1. Linearity. Linearity was determined for the mixture of 7 different lipid internal standards (ISs) spiked to 40 µl of the pooled plasma sample at 10 different concentrations (Table S4) before lipid extraction. Considering the different concentration ranges of different lipid classes in biological samples, different lipid ISs with different concentration ranges were used for linearity. The mean peak area of six replicate measurements at each concentration level was calculated. And the calibration curve for each lipid IS was constructed based on the mean peak area versus its corresponding concentration. Precision. Precision was assessed for the 7 lipid ISs at the low, medium and high concentration levels by repeating pooled plasma sample preparation and analysis during three consecutive days (see Experimental Section). The intra-day precision was calculated as %RSD of the peak area of each lipid IS from 6 replicates (i.e., n = 3 2) in one day. The %RSDs of interday precision were calculated S-5

6 using the peak area of each lipid IS from 18 replicates in three consecutive days (Table S4). Recovery. Recoveries were carried out for the 7 lipid ISs at the low, medium and high concentration levels in the pooled plasma samples spiked before and after extraction (n = 3). The recoveries were calculated for each lipid IS as the ratio of the peak area of that lipid in the sample spiked prior to extraction and that in the sample spiked after sample extraction. Repeatability. Repeatability was evaluated by %RSD of peak area of the detected lipids obtained from 2 replicate injections of triplicate samples prepared in parallel (n = 3 2). The performance was carried out on UHPLC/QQQ MRM MS- and UHPLC/LTQ-Orbitrap MS-based lipidomics platforms. S-6

7 Figure S1. Linear relationship between tr and (A) No. of acyl carbon or (C) No. of acyl double bond of all identified PC lipids. (B) Black dot: tr prediction of PC (X:0) by equation y = x (DB=0, R 2 = 0.999); red triangle: tr of PC (X:0) lipids found by EIC operation. (D) Black dot: tr prediction of PC (38:X) by equation y = x (AC=38, R 2 =0.997); red triangle: tr of PC (38:X) lipids found by EIC operation. DB: Acyl chain double bond No., AC: Acyl chain carbon No. S-7

8 Figure S2. Representative chromatograms of serum QC based on the constructed high coverage of lipid ion pairs. MRM chromatograms of 1 st scan (A) and 2 nd scan (B) in positive mode. (C) MRM chromatogram in negative mode. S-8

9 Figure S3. Reproducibility of pseudotargeted lipidomics method for pooled QC with 6 replicates in positive (A) and negative (B) modes. S-9

10 Table S1. Clinical parameters of 30 patients with diabetes and 30 healthy controls. characteristics diabetes control No Sex (male/female) 15 / / 15 Age 52.1 ± ± 9.3 FBG (mmol/l) 11.9 ± ± 0.5 HbA1c (%) 11.2 ± FCP 1.9 ± 1.0 a - TC (mmol/l) 5.1 ± 1.3 b 4.6 ± 0.9 TG (mmol/l) 2.4 ± 2.2 c 1.5 ± 1.1 LDL(mmol/L) 3.2 ± 0.9 d 3.1 ± 0.9 HDL(mmol/L) 1.0 ± 0.3 e 1.3 ± 0.3 In 30 diabetic cases, a. 5 cases were lack of FCP information, b. one patient was lack of TC information, c. one case was lack of TG information, d. one case was lack of LDL information and e. 2 patients were lack of HDL information. Table S2. Information of the detected and predicted lipids covering 3377 lipid ion pairs (Table S2 is provided by separated excel sheets). Sheet 1 The detailed information of the detected and predicted lipids in positive mode. Sheet 2 The detailed information of the detected and predicted lipids in negative mode. Sheet 3 Information of all lipid ion pairs in positive mode. Sheet 4 The first acquisition method in positive mode. Sheet 5 The second acquisition method in positive mode. Sheet 6 Information of all lipid ion pairs in negative mode. Sheet 7 The acquisition method in negative mode. S-10

11 Table S3. Each of lipid ion pair represents one or more lipid molecular structures due to different acyl chain combinations caused by different length of acyl chain and different No. of the acyl chain double bond. Ion pairs Precursor ion Product ion Lipids Possible lipid composition structures PC (44:0) PC 18:0 _26:0, PC 19:0 _25:0, PC 20:0 _24:0, PC 21:0 _23:0, PC 22:0 _22: PC (44:1) PC 18:1 _26:0, PC 18:0 _26:1, PC 19:1 _25:0, PC 20:0 _24:1, PC 20:1 _24:0, PC 22:0 _22: PC (44:2) PC 26:1 _18:1, PC 18:2 _26:0, PC 20:1 _24:1, PC 20:2 _24:0, PC 22:0 _22:2, PC 22:1 _22: PC (44:3) PC 26:1 _18:2, PC 18:3 _26:0, PC 20:2 _24:1, PC 20:3 _24:0, PC 22:1 _22:2, PC 22:0 _22: PC (44:4) PC 26:1 _18:3, PC 18:4 _26:0, PC 20:3 _24:1, PC 20:4 _24:0, PC 22:0 _22:4, PC 22:1 _22:3, PC 22:2 _22: PC (44:5) PC 18:4 _26:1, PC 20:4 _24:1, PC 20:5 _24:0, PC 22:0 _22:5, PC 22:1 _22:4, PC 22:2 _22: PC (44:6) PC 20:5 _24:1, PC 22:0 _22:6, PC 22:1 _22:5, PC 22:2 _22:4,PC 22:3 _22: PC (44:7) PC 22:1 _22:6, PC 22:2 _22:5, PC 22:3 _22: PC (44:8) PC 22:2 _22:6, PC 22:3 _22:5, PC 22:4 _22: PC (44:9) PC 22:3 _22:6, PC 22:4 _22:5 S-11

12 PC (44:10) PC 22:4 _22:6, PC 22:5 _22: PC (44:11) PC 22:5 _22: PC (44:12) PC 22:6 _22:6 S-12

13 Table S4. Concentration range of 7 lipid internal standards for evaluating performance of pseudotargeted lipidomics method. Lipid IS Concentration (µg/ml) C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 FA 16:0-d LPC 19: PC 19:0/19: PE 17:0/17: SM d18:1/12: Cer d18:1/17: TG 15:0/15:0/15: Table S5. The most significantly changed 163 lipids contributing to the discrimination between patients with diabetes and the healthy controls (Table S5 is provided with a separated Excel sheet). REFERENCE (1) Health, U. D. O.; Human services, F.; Drug Administration, C. F. D. E.; Research, C. F. V. M. Federal Register 2001, 66, S-13