Supplementary Information

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1 Supplementary Information Nano-LC/NSI MS refines lipidomics by enhancing lipid coverage, measurement sensitivity, and linear dynamic range Niklas Danne-Rasche 1, Cristina Coman 1, Robert Ahrends 1 1) Leibniz-Institut für Analytische Wissenschaften-ISAS e.v. Otto- Hahn-Str. 6b, Dortmund, Germany S-1

2 Content of Supplementary Material List of lipid abbreviations used in this paper... 3 Supplementary Methods... 4 Table S1. Reagents and standards Text S1. Yeast extraction Text S2. Preparation of nano-bore columns... 6 Table S2. HPLC gradients Table S3. MS Parameters for QExactive Plus Table S4. Optimized collision energies for targeted PRM analyses Text S3. Semiautonomous lipid identification using LipidCreator and Skyline... 9 Figure S1. Strategy for lipid identification Figure S2. Hierarchy of lipid annotation Table S5. Solvent composition of the tested mixtures to resuspend lipids Supplementary Results Figure S3. Lipid resuspension in strong organic solvents interferes with chromatography Figure S4. Lipid solubility is dependent on solvent (comprehensive view) Figure S5. Lipid solubility is dependent on solvent (bar graphs) Figure S6. Phosphoric acid supplementation to eluents did not improve detection of lipids with terminal phosphates Figure S7. Phosphoric acid influences peak shape of lipids with terminal phosphate groups Figure S8. Repeatability of nlc-method over 25 repetitive injections Table S6. Repeatability of nlc-method over 25 repetitive injections (data) Table S7. Sensitivity benchmark Figure S9. Benchmark of the linear range (comprehensive view) Figure S10. Detection of PE (31:1) in the yeast lipid extract using the nlc method Figure S11. The yeast phospholipidome measured on the nlc Figure S12. Lipid class distribution in yeast Table S8. Intensity comparison of 3 lipids of different classes and retention times Table S9. Comparison of lipid identifications in the different chromatographic conditions Figure S13. Chromatograms and MS/MS spectra for PL 30: Figure S14. Chromatograms and MS/MS spectra of various yeast lipids Supplementary References Appendix S-2

3 List of lipid abbreviations used in this paper Cer Ceramide CerP Ceramide-1-Phosphate GlcCer Glucosyl-ceramide LacCer Lactosyl-ceramide SM Sphingomyelin LCBP Sphinganine-1-Phosphate / Sphingosine-1-Phosphate LCB Long-chain base (sphingoid bases) M(IP)2C Mannosyl-Diinositolphosphoceramide MIPC Mannosyl-Inositolphosphoceramide IPC Inositolphosphoceramide LPA Lyso-Phosphatidic acid LPC Lyso-Phosphatidylcholine LPG Lyso-Phosphatidylglycerol LPS Lyso-Phosphatidylserine PA Phosphatidic acid PC Phosphatidylcholine PE Phosphatidylethanolamine PG Phosphatidylglycerol PI Phosphatidylinositol PS Phosphatidylserine CL Cardilipin MAG Monoacylglycerol DAG Diacylglycerol TAG Triacylglycerol SE Steryl ester PL Phospholipid FA Fatty acyl chain S-3

4 Supplementary Methods Table S1. Reagents and standards. Solvents 1-Butanol (LiChrosolv ) 1-Pentanol (Emsure ) Isopropanol (IPA) (LiChrosolv ) Water (ULC/MS) Acetonitrile (ACN) (ULC/MS) Formic acid (FA) (99% ULC/MS) Chloroform (Chromasolv ) orthophosphoric acid (50%) Vendor Merck Millipore (Darmstadt, Germany) Merck Millipore (Darmstadt, Germany) Merck Millipore (Darmstadt, Germany) Biosolve (Valkenswaard, Netherlands) Biosolve (Valkenswaard, Netherlands) Biosolve (Valkenswaard, Netherlands) Sigma Aldrich (St. Louis, USA) Fluka (Sigma Aldrich) Lipid standards SE 27:1/17:0 (Cholesteryl ester) SE 27:1/22:0 (Cholesteryl ester) Ceramide STD Mix II CL 15:0-15:0-15:0-16:1 CL 15:0-15:0-15:0-16:1 DAG d5 17:0/17:0 LPA 17:1 LPC 13:0 LPG 17:1 LPS 17:1 MAG 16:0 PA 17:0/14:1 PC 17:0/14:1 PE 17:0/14:1 PE 21:0/22:6 PG 17:0/14:1 PI 17:0/14:1 PS 17:0/14:1 TAG 14:0/14:0/14:0 TAG 16:0/16:0/16:0 TAG d5 17:0-17:1-17:0 Vendor Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Avanti Polar Lipids (Birmingham, USA) Sigma Aldrich (St. Louis, USA) Sigma Aldrich (St. Louis, USA) Avanti Polar Lipids (Birmingham, USA) Reagents and Salts Ammonium bicarbonate (ABC) Sodium docecyl sulfate (SDS) Tris(hydroxymethyl)aminomethane (TRIS) Ammonium formate (AF) (LC/MS) Vendor Fluka (Sigma Aldrich) Roth (Karlsruhe, Germany) AppliChem GmbH (Darmstadt, Germany) Fluka (Sigma Aldrich) S-4

5 Text S1. Yeast extraction. Yeast was cultivated in defined yeast nitrogen base medium (YNB) (Sigma Aldrich) (13.4 g/l yeast nitrogen base supplemented with 4.0 g/l amino acid powder without uracil and 2 % Glucose (w/v)) for 15 hours at 30 C. The culture was diluted to OD in YNB containing Raffinose (13.4 g/l yeast nitrogen base supplemented with 4.0 g/l amino acid powder without uracil + 2 % Raffinose (w/v)). After an incubation period of 3 hours at 30 C (OD of O ) galactose was added (2% (w/v)) to the medium. The yeast cells were incubated for additional 6 hours at 30 C and subsequently were diluted with the galactose containing medium to an OD600 of 0.1. After a final incubation period of 20 hours at 30 C, the cells were harvested and stored at -80 C. For yeast extraction, the cells were thawed on ice and diluted in 150 mm ammonium bicarbonate buffer to 1.3x10 9 cells per µl. Acid washed 250 µl glass beads (Sigma Aldrich) were added to 500 µl of yeast suspension ( 6.7x10 8 cells). After the cells were counted with the help of a Thoma cell counting chamber, they were disrupted in the TissueLyser (Qiagen) for 30 min at a frequency of 20 Hz using precooled (-20 C) reaction tube racks. After disruption 400 µl of the yeast suspension were pipetted to a separate 5 ml polypropylene tube and 1980 µl of CHCL 3:MeOH 17:1 was added lipids were extracted for 2 hours at 4 C and 750 rpm on a Thermomixer comfort (Eppendorf, Germany). In order to induce phase separation, samples were centrifuged for 10 min at 4 C at 8000 RCF. The lower chloroform phase (containing the lipids) was pipetted into a separate vial. The upper (metabolite) phase and interphase (cell debris and protein phase) were re-extracted with 990 µl CHCL 3:MeOH 2:1 for 2 hours at 750 rpm and 4 c on a Thermomixer comfort and were subsequently centrifuged at 8000 RCF and 4 C for 10 min. The organic chloroform phase was combined with the first lipid phase, while upper phase and interphase were used for protein precipitation adding a fourfold volume of MeOH. Proteins were precipitated for 2 hours at -80 C. Samples were centrifuged for 30 min at 4 C and RCF. The supernatant was removed and the pellet was resuspended in 500 µl 1% SDS buffer (1% SDS, 150 mm NaCl, 50 mm Tris, ph 7.8 (HCL)). Samples were centrifuged for 30 min at RCF at room temperature. The supernatant was carefully removed to a 1.5 ml low binding tube (Eppendorf) followed by measurement of the protein concentration by a BCA assay (Pierce TM BCA Protein Assay Kit, Thermo Scientific). The yeast extract was used as matrix for the reproducibility experiment (spiked with standards to yield a final concentration of 0.4 µm) and determination of linear measurement range (spiked with standards to yield final standard concentrations of 0.64 nm 10 µm). For these experiments, 4 parallel extractions were dissolved in 250 µl CHCL 3: MeOH:H 2O 60:30:4.5, pooled evaporated and redissolved in 8000 µl 8Bu+ (1BuOH:IPA:H 2O 8:23: mm H 3PO 4) (lipid amount equals 8432 µg protein). For the lipidome coverage experiment three extraction replicates were dissolved in CHCL 3:MeOH:H 2O 60:30:4.5 and were pooled, evaporated and redissolved in 200 µl 8Bu+ (lipids equal 3174 µg protein). S-5

6 Text S2. Preparation of nano-bore columns Fused silica capillaries (100 µm inner diameter (ID), 360 µm outer diameter (OD)) were cut to a length of 35 cm and sealed with a frit of GF/F Whatman Filter (Maidstone, UK) that was wetted with a Kasil 1 Potassium Silicate (PQ Corporation, USA) - Formamid (Merck Millipore - Calbiochem, USA) solution. After an overnight polymerization at 85 C, the column was slurry packed at 100 bar with a methanolic suspension of Ascentis Express C18 core shell material (2.7 µm; 90 Å; Supelco). Subsequent to controlling of packing uniformity and polishing of capillary ends, columns with a length of 30 cm were selected for separation purposes. The nano-bore column was connected to the injection valve by a capillary with an ID of 20 µm thus enabling a direct injection of samples onto the column without using a trapping-column. Table S2. HPLC gradients. Nano-LC (600 nl/min) (Column: 30 cm x 100 µm) Narrow-bore HPLC (500 µl/min) Column (15 cm x 2.1 mm) Min [%B] Min [%B] S-6

7 Table S3. MS Parameters for QExactive Plus. Top 10 PRM Polarity positive negative positive negative Full scan Full scan Full scan Full scan Resolution AGC 1.E+06 1.E+06 1.E+06 1.E+06 Maximum IT [ms] Scan range [m/z] Top10 (ddms2) Top10 (ddms2) targeted MS2 targeted MS2 Resolution AGC 1.E+05 1.E+05 1.E+06 1.E+06 Maximum IT [ms] Isolation window [m/z] NCE 25,30 20,30,40 Class-specific optimized Class-specific optimized Dynamic exclusion [s] Minimum AGC 2.00E E AGC: Automatic gain control; IT: injection time; NCE: Normalized collision energy S-7

8 Table S4. Optimized collision energies for targeted PRM analyses. Lipidclass normalized CE (pos. mode) Adduct normalized CE (neg. mode) Adduct PC 26 [M+H] + 21 [M+HCOO] - LPC 26 [M+H] + 24 [M+HCOO] - PE 24 [M+H] + 21 [M-H] - PS 26 [M+H] + 25 [M-H] - LPS 26 [M+H] + 24 [M-H] - PG 21 [M+NH4] + 27 [M-H] - LPG 20 [M+NH4] + 27 [M-H] - PA 21 [M+NH4] + 26 [M-H] - LPA 21 [M+NH4] + 26 [M-H] - PI 29 [M+NH4] + 25 [M-H] - CL 20 [M+NH4] + 21 [M-H] - MAG 16 [M+NH4] + n.d. n.d. DAG 25 [M+NH4] + 24 [M+HCOO] - TAG 23 [M+NH4] + 24 [M+HCOO] - SE 14 [M+NH4] + 25 [M+HCOO] - /[M- H] - LCB 17:1 30 [M+H] + 35 [M+HCOO] - LCB 17:0 29 [M+H] + 35 [M+HCOO] - LCBP 17:1 16 [M+H] + 32 [M-H] - LCBP 17:0 23 [M+H] + 35 [M-H] - Cer 24 [M+H] + 30 [M+HCOO] - GlcCer 20 [M+H] + 26 [M+HCOO] - LacCer 22 [M+H] + 25 [M+HCOO] - Cer-1- Phosphate 28 [M+H] + 29 [M-H] - SM 16 [M+H] + 34 [M+HCOO] - M(IP)2C* 24 [M+H] + 27 [M-H] - MIPC* 24 [M+NH4] + /[M+H] + 27 [M-2H] 2- /[M-H] - IPC* 24 [M+H]+ 27 [M-H] - The collision energies were optimized using standards by direct infusion experiments (data not shown), or estimated when no standard was available (*). S-8

9 Text S3. Semiautonomous lipid identification using LipidCreator and Skyline As a starting point for the workflow, target transition list (of target lipid class) was created using lipid creator (Figure S1). The transition list was then imported to Skyline, which allowed to check whether qualifier fragments (e.g. NL of Da for PC) could be observed on MS2 level (< 3 ppm) that were generated by the corresponding precursor ion (< 3 ppm). Lipids elute in a gradient dependent pattern that can be approximated by polynomial functions (Figure 4). Here, lipids of the same class clearly separate on species level by cumulated carbon amount in the fatty acyl chains and degree of unsaturation (and further modifications such as oxidation). For this reason the retention time was used as an additional parameter to verify the lipid identifications, as already previously demonstrated 1-2. Accordingly, lipids could be characterized on species level even if no MS2 scan was triggered for a certain precursor when that precursor gave rise to a peak, which exactly fell into the class specific elution pattern. Those lipids were designated as putative IDs to discriminate them from the lipids that could be additionally verified by MS2 data. The identification of FA pairs in MS2 spectra further allowed lipid annotation molecular species level (e.g. PC 14:1-15:0). As a rule of thumb, FA intensities (of on FA pair) should not differ in intensity by more than factor of two. In most cases, lipids could be clearly separated on species level, while they were separated on molecular species level to a lesser extent. This gave rise to a coelution (pre-separation) of lipids that share the same species but have different FA combinations (Figure 4a; Figure S10). Hence, MS2 spectra were only rarely specific for one molecular species and more than one FA pairs were usually detectable in single MS2 scans. Peak integration was thus performed on MS1 level thereby summarizing multiple molecular species on the superordinate species level (e.g. PC 29:1) (Figure 4a). All peak integrations, performed for the lipidome evaluations on both chromatographic systems were carried out for negative mode analyses (n=3). S-9

10 Figure S1. Strategy for lipid identification. Left: Excerpt of a transition list generated with lipid creator. The precursor to precursor transitions allow the quantification of MS1 signals. Qualifier product ions or specific neutral losses (orange) allow to retrieve information that indicates the lipid species. Identification of FA fragments further designate the molecular lipid species level. The information, which can be retrieved by skyline, is shown in the chromatogram on the right. Figure S2. Hierarchy of lipid annotation. Figure adapted after Foster et al. (2013) 3 and Pauling et al. (2017) 4. Peak integration was performed on lipid species level; putative lipid identifications were assigned on this level as described in text S3. Lipids that were assigned by (i) lipid class-selective MS2 fragments, (ii) molecular lipid species-specific fragments (i.e. fatty acid related fragments), (iii) exact precursor mass and (iv) fitting retention time were annotated on molecular species level. Artificial standards that were used in this study were described on sub species level ( hydrocarbon chain position-defined molecular lipid species level). S-10

11 Table S5. Solvent composition of the tested mixtures to resuspend lipids. 0%B 25%B 50 %B 100 %B Sarafian Bird 8Bu 30Bu 1-BuOH IPA ACN H2O S-11

12 Supplementary Results Figure S3. Lipid resuspension in strong organic solvents interferes with chromatography. If moderate hydrophobic solvents like IPA (i.e. IPA:ACN 90:10 strong LC eluent) are used to dissolve lipid extracts, certain lipids are not retained on the column anymore, but are rather flushed through the column along with the injection volume. S-12

13 Figure S4. Lipid solubility is dependent on solvent (comprehensive view). Selected lipids of different hydrophobicity were dissolved in the indicated solvents (Table S5) and a 240 nm standard solution was directly injected onto the nano column at an injection volume of 1 µl ( injection amount of 240 fmol per lipid standard). The chromatograms demonstrate that peak shape, signal height and recovery are strongly influenced by the choice of the resuspension solvent. S-13

14 Figure S5. Lipid solubility is dependent on solvent (bar graphs). Selected lipids of different hydrophobicity were dissolved in the indicated solvents (Table S5) and a 240 nm standard solution was directly injected onto the nano column at an injection volume of 1 µl ( injection amount of 240 fmol per lipid standard). Peak area, normalized area, height, normalized height, retention time (RT) and half maximal peaks width (FWHM) were plotted for the indicated lipids (n=3). S-14

15 Figure S6. Phosphoric acid supplementation to eluents did not improve detection of lipids with terminal phosphates. Selected lipids of different lipids classes were dissolved in 8Bu (1-BuOH:IPA:H 2O 8:23:69) or 8Bu+ (1-BuOH:IPA:H 2O 8:23: mm H 3PO 4). Addition of phosphoric acid to the samples clearly improved peak shape and MS response of both lipids with terminal phosphates and cardiolipins (black), while other lipids like PG were not affected by supplementation of phosphoric acid to the samples (grey). The analysis was performed with eluents containing 5 µm phosphoric acid. S-15

16 Figure S7. Phosphoric acid influences peak shape of lipids with terminal phosphate groups. Selected lipids of different lipids classes were dissolved in 8Bu (1-BuOH:IPA:H2O 8:23:69) or 8Bu+ (1- BuOH:IPA:H2O 8:23: mm H 3PO 4). The analysis was performed under standard elution conditions. A) shows the fold change of the area between standard samples (in 8Bu) to samples with phosphoric acid (in 8Bu+), while B) depicts the fold change of the signal height. The half maximal peak width (FWHM) in the two sample types is depicted in absolute numbers in C) and relative fold change (+H 3PO 4 /-H 3PO 4) E). D) shows the calculation of retention time shifts from normal samples to samples with phosphoric. Here, the mean RT of the standard samples was subtracted in each case. S-16

17 S-17

18 Figure S8. Repeatability of nlc-method over 25 repetitive injections. The repeatability of the nlc-ms method was evaluated by 25 repetitive analyses of a yeast extract containing standards at a concentration of 0.4 fmol/µl (0.4 µm) normalization purposes. Retention times and peak areas were recorded for 66 selected lipids (endogenous & standards) using parallel reaction monitoring in positive mode focusing principal the MS/MS fragment (derived from the respective main adduct) for peak integration. A) RSD and maximal deviation from retention time from 25 repetitive analyses of 66 lipids are plotted against retention time. C) The retention time variances of 4 selected lipids were plotted more precisely. B) The plot illustrates the relative standard deviation (RSD) of the peak area plotted against retention time for all analyzed lipids. D) In order to reduce the effect of spray instabilities/varying MS response a normalization was carried out. Normalization of peak areas was either performed based on retention time (normalization to a close eluting standard) or with a standard of the same lipid class (see Table S6). The left plots depict the relative deviation from the average peak area in each of the repetitive analyses; the grey box marks acceptable deviations of ± 10 %. The boxes in the Box-Whisker plot indicate the 25th and 75th percentile while the whiskers represent the 90th and 10th percentile, respectively. S-18

19 Table S6. Repeatability of nlc-method over 25 repetitive injections (data). The repeatability of the nlc-ms method was evaluated by 25 repetitive analyses of yeast extract that contained standards of different classes at a concentration 0.4 µm for normalization purposes. The table lists standard deviations for retention time and peak area. The extract was analyzed in positive using mode parallel reaction monitoring focusing principal the MS/MS fragment (derived from the respective main adduct) for peak integration. (Peak area repeatability figures (compare Figure S8D) for the marked lipids are appended to this document) Molecule Retention time RSD Area [%] # Fig. Lipid Standard Class Mean [min] ± [min] ± [%] Δmax (s) Original RT based Class based RT norm. --> Class norm --> 1 yes LPC 13:0 STD LPC n.d LPG 17:1 LPC 13:0 2 yes LCBP 17:1;2 STD LCBP n.d LPG 17:1 LCBP 17:1;2 3 yes LCBP 17:0;2 STD LCBP LPG 17:1 LCBP 17:1;2 4 yes LCB 17:1;2 STD LCB n.d LPG 17:1 LCB 17:1;2 5 yes LPS 17:1 STD LPS n.d LPG 17:1 LPS 17:1 6 LCB 18:0;3 LCB LPG 17:1 LCB 17:1;2 7 yes LCB 17:0;2 STD LCB LPG 17:1 LCB 17:1;2 8 yes LPC 16:1 LPC LPG 17:1 LPC 13:0 9 yes LPG 17:1 STD LPG n.d n.d LPG 17:1 LPG 17:1 10 yes LPS 18:1 LPS LPG 17:1 LPS 17:1 11 yes LPA 17:1 STD LPA n.d LPG 17:1 LPA 17:1 12 LCB 18:0;2 LCB LPG 17:1 LCB 17:1;2 13 yes LPG 18:1 LPG LPG 17:1 LPG 17:1 14 yes LPA 18:1 LPA LPG 17:1 LPA 17:1 15 LCB 20:0;3 LCB LPG 17:1 LCB 17:1;2 16 LPC 18:1 LPC LPG 17:1 LPC 13:0 17 LCB 20:0;2 LCB LPG 17:1 LCB 17:1;2 18 yes PI 28:1 PI GlcCer 18:1;2/12:0;0 PI 31:1 19 PS 28:1 PS GlcCer 18:1;2/12:0;0 PS 31:1 20 yes CerP 18:1;2/12:0;0 STD CerP GlcCer 18:1;2/12:0;0 Cer 18:1;2/12:0;0 21 yes PG 28:1 PG GlcCer 18:1;2/12:0;0 PG 31:1 22 yes LacCer 18:1;2/12:0;0 STD LacCer GlcCer 18:1;2/12:0;0 Cer 18:1;2/12:0;0 23 yes SM 30:1;2 STD SM GlcCer 18:1;2/12:0;0 Cer 18:1;2/12:0;0 24 PC 28:1 PC GlcCer 18:1;2/12:0;0 PC 31:1 25 yes GlcCer 18:1;2/12:0;0 STD GlcCer n.d 5.07 GlcCer 18:1;2/12:0;0 Cer 18:1;2/12:0;0 26 yes PI 32:2 PI GlcCer 18:1;2/12:0;0 PI 31:1 27 PE 28:1 PE GlcCer 18:1;2/12:0;0 PE 31:1 28 yes PS 32:2 PS GlcCer 18:1;2/12:0;0 PS 31:1 29 yes PI 31:1 STD PI n.d GlcCer 18:1;2/12:0;0 PI 31:1 30 yes PS 31:1 STD PS n.d GlcCer 18:1;2/12:0;0 PS 31:1 31 yes PG 31:1 STD PG n.d GlcCer 18:1;2/12:0;0 PG 31:1 32 yes PA 32:2 PA GlcCer 18:1;2/12:0;0 PA 31:1 33 yes DAG 10:0-14:0 DG GlcCer 18:1;2/12:0;0 DAG 17:0/17:0 D5 34 yes Cer 18:1;2/12:0;0 STD Cer n.d GlcCer 18:1;2/12:0;0 Cer 18:1;2/12:0;0 35 yes PC 32:2 PC GlcCer 18:1;2/12:0;0 PC 31:1 36 PG 32:1 PG GlcCer 18:1;2/12:0;0 PG 31:1 37 yes PA 31:1 STD PA n.d GlcCer 18:1;2/12:0;0 PA 31:1 38 yes PE 32:2 PE GlcCer 18:1;2/12:0;0 PE 31:1 39 yes PC 31:1 STD PC n.d PE 31:1 PC 31:1 40 yes PE 31:1 STD PE n.d n.d PE 31:1 PE 31:1 41 PS 34:1 PS PE 31:1 PS 31:1 42 yes PG 34:1 PG PE 31:1 PG 31:1 43 PA 34:1 PA PE 31:1 PA 31:1 44 DG 32:2 DG PE 31:1 DG 17:0/17:0 D5 45 PE 36:2 PE PE 31:1 PE 31:1 46 PC 36:1 PC PE 31:1 PC 31:1 47 yes M(IP)2C 18:0,3/26:0,1 M(IP)2C PE 31:1 Cer 18:1;2/12:0;0 48 yes MIPC 18:0,3/26:0,1 MIPC DG 17:0/17:0 D5 Cer 18:1;2/12:0;0 49 yes PI 40:1 PI DG 17:0/17:0 D5 PI 31:1 50 yes IPC 18:0,3/26:0,1 IPC DG 17:0/17:0 D5 Cer 18:1;2/12:0;0 51 yes Cer 18:0;3/22:0;1 Cer DG 17:0/17:0 D5 Cer 18:1;2/12:0;0 52 CL 10:0-16:1-16:1-16:1 CL DG 17:0/17:0 D5 CL 15:0-15:0-15:0-16:1 53 yes DG 17:0/17:0 D5 STD DG n.d n.d DG 17:0/17:0 D5 DAG 17:0/17:0 D5 54 DG 18:0-18:1 DG DG 17:0/17:0 D5 DAG 17:0/17:0 D5 55 Cer 18:0,3/24:0,1 Cer DG 17:0/17:0 D5 Cer 18:1;2/12:0;0 56 Cer 18:0,3/26:0,1 Cer DG 17:0/17:0 D5 Cer 18:1;2/12:0;0 57 yes CL 15:0-15:0-15:0-16:1 STD CL n.d DG 17:0/17:0 D5 CL 15:0-15:0-15:0-16:1 58 yes TG 16:1-16:1-16:1 TG DG 17:0/17:0 D5 TAG 17:0-17:1-17:0 d5 59 yes CL 18:1-18:1-18:1-18:1 CL DG 17:0/17:0 D5 CL 15:0-15:0-15:0-16:1 60 SE 27:2/16:1 SE DG 17:0/17:0 D5 SE 27:1/22:0 61 yes SE 27:2/18:1 SE DG 17:0/17:0 D5 SE 27:1/22:0 62 SE 29:1/16:1 SE DG 17:0/17:0 D5 SE 27:1/22:0 63 yes TAG 17:0-17:1-17:0 d5 STD TG n.d DG 17:0/17:0 D5 TAG 17:0-17:1-17:0 d5 64 TAG 18:0-18:1-18:1 TG DG 17:0/17:0 D5 TAG 17:0-17:1-17:0 d5 65 SE 29:1/18:1 SE DG 17:0/17:0 D5 SE 27:1/22:0 66 yes SE 27:1/22:0 STD SE n.d DG 17:0/17:0 D5 SE 27:1/22:0 S-19

20 Table S7. Sensitivity benchmark. The sensitivity and the linear dynamic range of the nano-lc-ms (nlc) method was benchmarked against a corresponding narrow bore HPLC method (HPLC) in both positive and negative ionization mode (n=3) for different lipids. The experiment was designed to curtail the working ranges of the two methods comparatively. The quantification limits (LOQ) were approximated as the lowest detectable concentration within linear range in case no matrix response was observed, since high resolution orbitrap MS2 data do not give rise to a blank/background response when no target analyte is present. In cases where a matrix response was observed, the LOQ was LOQ was determined as the next highest concentration within linear range that could be reliably distinguished from the blank value (matrix response+ 10x the standard deviation of the matrix). S-20

21 Lipid standard Method Polarity MS1 Adduct MS2 LOQ Linear range [Min - Max] R 2 Matrix response Cer 18:1;2/12:0;0 CerP 18:1;2/12:0;0 GlcCer 18:1;2/12:0;0 LacCer 18:1;2/12:0;0 SM 18:1;2/12:0;0 LCB 17:1;2 LCB 17:0;2 LCBP 17:0;2 LCBP 17:1;2 LPA 17:1 LPC 13:0 LPG 17:1 LPS 17:1 PA 17:0/14:1 PC 17:0/14:1 PE 17:0/14:1 PG 17:0/14:1 PI 17:0/14:1 PS 17:0/14:1 CL 15:0(3)/16:1 DAG 17:0/17:0 d5 TAG 17:0/17:1/17:0 d5 HPLC Negative [+HCOO] NLC Negative [+HCOO] HPLC Positive [+H] NLC Positive [+H] yes HPLC Negative [-H] yes NLC Negative [-H] HPLC Positive [+H] NLC Positive [+H] HPLC Negative [+HCOO] NLC Negative [+HCOO] HPLC Positive [+H] NLC Positive [+H] HPLC Negative [+HCOO] NLC Negative [+HCOO] HPLC Positive [+H] NLC Positive [+H] HPLC Negative [+HCOO] NLC Negative [+HCOO] HPLC Positive [+H] NLC Positive [+H] yes HPLC Negative [+HCOO] - not detected n.d. n.d. n.d. n.d. NLC Negative [+HCOO] - precursor only yes HPLC Positive [+H] NLC Positive [+H] HPLC Negative [+HCOO] - not detected n.d. n.d. n.d. n.d. NLC Negative [+HCOO] - precursor only yes HPLC Positive [+H] yes NLC Positive [+H] HPLC Negative [-H] - precursor only HPLC Negative [-H] yes NLC Negative [-H] - precursor only yes NLC Negative [-H] - MS2 wrong window HPLC Positive [+H] + precursor only yes HPLC Positive [+H] yes NLC Positive [+H] + precursor only yes NLC Positive [+H] + MS2 wrong window HPLC Negative [-H] - precursor only yes HPLC Negative [-H] yes NLC Negative [-H] - precursor only yes NLC Negative [-H] - MS2 wrong window HPLC Positive [+H] + precursor only yes HPLC Positive [+H] yes NLC Positive [+H] + precursor only yes NLC Positive [+H] + MS2 wrong window HPLC Negative [-H] NLC Negative [-H] yes HPLC Positive [+NH4] NLC Positive [+NH4] yes HPLC Negative [+HCOO] yes NLC Negative [+HCOO] HPLC Positive [+H] yes NLC Positive [+H] yes HPLC Negative [-H] NLC Negative [-H] HPLC Positive [+NH4] > 2000 n.d. n.d. n.d. NLC Positive [+NH4] HPLC Negative [-H] NLC Negative [-H] HPLC Positive [+H] NLC Positive [+H] HPLC Negative [-H] NLC Negative [-H] HPLC Positive [+NH4] yes NLC Positive [+NH4] yes HPLC Negative [+HCOO] NLC Negative [+HCOO] yes HPLC Positive [+H] yes NLC Positive [+H] yes HPLC Negative [-H] NLC Negative [-H] HPLC Positive [+H] yes NLC Positive [+H] yes HPLC Negative [-H] NLC Negative [-H] HPLC Positive [+NH4] NLC Positive [+NH4] yes HPLC Negative [-H] NLC Negative [-H] yes HPLC Positive [+NH4] yes NLC Positive [+NH4] yes HPLC Negative [-H] NLC Negative [-H] yes HPLC Positive [+H] NLC Positive [+H] yes HPLC Negative [-H] NLC Negative [-H] (10000) (0.9575) yes HPLC Positive [+NH4] (16) 400 (16) (0.9964) NLC Positive [+NH4] yes HPLC Negative [+HCOO] - not detected n.d. n.d. n.d. n.d. NLC Negative [+HCOO] - precursor only yes HPLC Positive [+NH4] NLC Positive [+NH4] (80) (0.9188) yes HPLC Negative [+HCOO] - unknown n.d. n.d. n.d. n.d. NLC Negative [+HCOO] - unknown n.d. n.d. n.d. n.d. HPLC Positive [+NH4] NLC Positive [+NH4] yes S-21

22 Figure S9. Benchmark of the linear range (comprehensive view). The sensitivity and the linear dynamic range of the nano-lc-ms (NLC) method was benchmarked against a corresponding narrow bore HPLC method (HPLC) in both positive and negative ionization mode for the indicated lipid standards within a yeast matrix. The plots show the peak area (n=3) of different lipid standards dependent on the injection amount of the respective standard. M refers to the yeast lipid extract serving as a constant sample matrix. S-22

23 Figure S10. Detection of PE (31:1) in the yeast lipid extract using the nlc method. The chromatograms of fatty acid product ions are shown for different subspecies of PE (31:1) in the blank yeast matrix, and, when 80 fmol of PE (17:0/14:1) were spiked into the matrix. Notably, the artificial standard shows both a pre-separated double-peak and a slight retention time shift compared to the natural endogenous yeast lipids. S-23

24 Area [log10] A) PA PE PC PG PI PS #Carbon 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB *** B) PA [%] PE [%] PC [%] PG [%] PI [%] PS [%] #Carbon 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB *** Area [%] Isomers on msl / species C) PA isomers PE isomers PC isomers PG isomers PI isomers PS isomers #Carbon 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB 0 DB 1 DB 2 DB 3 DB total sl ID 47 total sl ID 68 total sl ID 22 total sl ID 64 total sl ID 22 total sl ID Figure S11. The 8 yeast putative sl phospholipidome ID 17 putative sl ID measured 9 putative on sl ID the 2 nlc. putative A) Log sl ID scale 10 of putative peak sl ID areas 5 on species putative sl ID 22 approved sl ID 30 approved sl ID 59 approved sl ID 20 approved sl ID 54 approved sl ID 17 approved sl ID level. The IDs are distinguished by cumulated carbon amount in FA chains and degree of unsaturation. 39 approved msl ID 62 approved msl ID 137 approved msl ID 38 approved msl ID 134 approved msl ID 24 approved msl ID 1.77 Isomers / Species 2.07 Isomers / Species 2.32 Isomers / Species 1.90 Isomers / Species 2.48 Isomers / Species 1.41 Isomers / Species Area_max [log10] Area_max [log10] Area_max [log10] 9.23 Area_max [log10] Area_max [log10] 8.98 Area_max [log10] B) Percentage of the indicated lipids relative to the most abundant species for each class. C) Number 3 Max_isomers 4 Max_isomers 6 Max_isomers 4 Max_isomers 5 Max_isomers 3 Max_isomers of identified isomers on molecular species level per lipid identification on species level. S-24

25 Figure S12. Lipid class distribution in yeast. Lipid class distribution in yeast based on summarized total intensities of individual lipid species. Table S8. Intensity comparison of 3 lipids of different classes and retention times Lipid Method Adduct Precursor RT Intensity [%] Nano Area [%] Nano FWHM PI 26:1 Nano [M+NH4] E E PI 26:1 HPLC [M+NH4] E E PI 26:1 Nano [M-H] E E PI 26:1 HPLC [M-H] E E PG 34:1 Nano [M+NH4] E E PG 34:1 HPLC [M+NH4] E E PG 34:1 Nano [M-H] E E PG 34:1 HPLC [M-H] E E PC 42:2 Nano [M+H] E E PC 42:2 HPLC [M+H] E E PC 42:2 Nano [M+HCOO] E E PC 42:2 HPLC [M+HCOO] E E S-25

26 Table S9. Comparison of lipid identifications in the different chromatographic conditions. Species level: sl; molecular species level: msl SL MSL PA Nano HPLC 25x HPLC Total: 38 IDs Total: 41 IDs (sl) ID (sl) putative ID (sl) not detected (msl) ID (msl) not detected SL MSL PE Nano HPLC 25x HPLC (sl) ID Total: 48 IDs (sl) putative ID (sl) not detected Total: 67 IDs (msl) ID (msl) not detected SL MSL PC Nano HPLC 25x HPLC (sl) ID Total: 68 IDs (sl) putative ID (sl) not detected Total: 137 IDs (msl) ID (msl) not detected SL MSL PG Nano HPLC 25x HPLC (sl) ID Total: 24 IDs (sl) putative ID (sl) not detected Total: 39 IDs (msl) ID (msl) not detected SL MSL PI Nano HPLC 25x HPLC (sl) ID Total: 55 IDs Total: 139 IDs (sl) putative ID (sl) not detected (msl) ID (msl) not detected SL MSL PS Nano HPLC 25x HPLC (sl) ID Total: 22 IDs (sl) putative ID (sl) not detected Total: 24 IDs (msl) ID (msl) not detected Summary of Identifications: Nano HPLC 25x HPLC (sl) ID SL Total: 255 IDs putative (sl) ID MSL Total: 447 IDs (msl) ID S-26

27 Figure S13. Chromatograms and MS/MS spectra for PL 30:1. The chromatograms on the left depict nlc-ms based extracted ion chromatograms (< 3ppm) of A) PC 30:1; B) PA 30:1; C) PG 30:1, D) PS 30:1, E) PI 30:1 and F) PE 30:1. The respective principal adduct is shown for negative (red) and positive ion mode (black) ion mode. The upper chromatograms were obtained by analysis of a yeast lipid extract (compare Figure 4/5) while the lower chromatograms refer to a subsequent was wash blank. Exemplary MS/MS spectra (yeast extract analysis) are shown on the right for both positive and negative ionization mode. The retention times, where the MS/MS experiment were triggered are marked by a blue dots within the chromatograms. Often observed MS/MS fragments in negative mode were: m/z : PO3- ; m/z : H2PO4-; m/z : G3P-H2O- S-27

28 Figure S13. (continued) S-28

29 Figure S13. (continued) S-29

30 Figure S14. Chromatograms and MS/MS spectra of various yeast lipids. The chromatograms on the left depict nlc-ms based extracted ion chromatograms (< 3ppm) of A) CDPDG 34:2 B) MAG 18:1 C) IPC 44:0;4 D) TAG 48:2; E) SE 28:2/16:1; F) MIPC 44:0;4 and G) LPA 16:0. The respective principal adduct is shown for negative (red) and positive ion mode (black) ion mode. Exemplary MS/MS spectra are shown on the right for both positive and negative ionization mode. The retention times, where the MS/MS experiments were triggered are marked by blue dots. S-30

31 Figure S14. (continued) S-31

32 Figure S14. (continued) (*: Mannosyl-inositolphosphate) S-32

33 Figure S14. (continued) S-33

34 Supplementary References 1. Aicheler, F.; Li, J.; Hoene, M.; Lehmann, R.; Xu, G.; Kohlbacher, O., Retention Time Prediction Improves Identification in Nontargeted Lipidomics Approaches. Anal Chem 2015, 87 (15), Ovcacikova, M.; Lisa, M.; Cifkova, E.; Holcapek, M., Retention behavior of lipids in reversedphase ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry. J Chromatogr A 2016, 1450, Foster, J. M.; Moreno, P.; Fabregat, A.; Hermjakob, H.; Steinbeck, C.; Apweiler, R.; Wakelam, M. J.; Vizcaino, J. A., LipidHome: a database of theoretical lipids optimized for high throughput mass spectrometry lipidomics. PLoS One 2013, 8 (5), e Pauling, J. K.; Hermansson, M.; Hartler, J.; Christiansen, K.; Gallego, S. F.; Peng, B.; Ahrends, R.; Ejsing, C. S., Proposal for a common nomenclature for fragment ions in mass spectra of lipids. PLoS One 2017, 12 (11), e Appendix S-34

35 Additional reproducibility plots associated to Figure S8 and Table S6 S-35

36 Additional reproducibility plots associated to Figure S8 and Table S6 S-36

37 Additional reproducibility plots associated to Figure S8 and Table S6 S-37

38 Additional reproducibility plots associated to Figure S8 and Table S6 S-38