Supporting Information

Ohno et al. 10.1073/pnas.1503491112 SI Materials and Methods hexane. Methanol/water and hexane phases were separated by Cell Culture and Transfection. HeLa and HEK 293T cells were cul- centrifugation, and the hexane (upper) phase was recovered. tured in DMEM (Sigma) supplemented with 10% FBS, 100 U/mL After hexane extraction was repeated three times, the extracted penicillin, and 100 μg/mL streptomycin. HEK 293T cells were lipids were combined, dried, and suspended in chloroform/ grown in dishes precoated with 0.3% collagen. Transfections methanol (2:1, vol/vol). Lipids were separated by reverse-phase were performed using Lipofectamine Reagent and Plus Re- TLC with Silica Gel 60 RP-18 F254s TLC plates (Merck Milli- agent (Life Technologies), according to the manufacturer’s pore) with chloroform/methanol/water (15:15:2, vol/vol/vol) and protocols. Normal human epidermal keratinocytes isolated detected as described above. from juvenile donors were purchased from CELLnTEC and grown in PCT Epidermal Keratinocyte Medium (CELLnTEC). Lipid Analysis by MS. To examine ceramide and FA species pre- Keratinocyte differentiation was induced by exchanging the pared from cultured cells, lipids were analyzed by UPLC coupled medium to Epidermal Keratinocyte 3D Prime Medium with ESI tandem triple quadrupole MS (Xevo TQ-S; Waters). (CELLnTEC) after the cell confluency reached ≥80%. Cells were washed twice with 1 mL of PBS, suspended in 1 mL of The Saccharomyces cerevisiae strain BY4741 (MATa his3Δ1 PBS, and detached from culture dishes by pipetting. Cell sus- leu2Δ0 met15Δ0 ura3Δ0) (36) was used. Cells were grown in pensions were transferred to silicon-coated plastic tubes. After × synthetic complete (SC) medium (0.67% yeast nitrogen base and centrifugation (400 g at room temperature for 3 min), cells μ 2% D-glucose) containing 0.5% casamino acids, 20 mg/mL ade- were suspended in 100 L of PBS, followed by the successive μ nine, and 20 mg/mL tryptophan but lacking uracil (SC-URA). addition and mixing of 375 L of chloroform/methanol/12 M formic acid (100:200:1, vol/vol/vol), 2 μLof25μM C17:0 ce- Plasmids. The pCE-puro 3xFLAG-ELOVL4 plasmid encoding ramide (internal standard; Avanti Polar Lipids), 125 μLofchlo- N-terminally 3xFLAG-tagged ELOVL4 has been described pre- roform, and 125 μL of water. After centrifugation (9,000 × g at viously (29). Human CYP4F subfamily genes and the human room temperature for 1 min), the organic phase was recovered CERS3 gene were amplified by PCR using their respective for- and dried. Lipids were suspended in 200 μL of chloroform/ ward and reverse primers listed in Table S2. The amplified DNAs methanol (1:2, vol/vol) and were mixed with 6 μLof4MKOH. first were cloned into pGEM-T Easy Vector (Promega) and then After incubation for 1 h at 37 °C, lipids were mixed with 7 μLof were transferred to the pCE-puro 3xFLAG-1 plasmid, a mam- 4 M formic acid, 66.6 μL of chloroform, and 133.3 μL of water. malian expression vector designed to produce an N-terminal After centrifugation (9,000 × g at room temperature for 1 min), 3xFLAG-tagged protein. The CYP4F22 gene was also transferred the organic phase was recovered, dried, and dissolved in 50 μLof to the pAK1017 plasmid (URA3 marker, CEN), a yeast expression chloroform/methanol (1:1, vol/vol). As controls, epidermal lipids vector designed to produce an N-terminal, tandemly oriented were prepared from mice as described previously (29, 38). Lipids His6-, Myc-, and 3xFLAG-tagged protein under the control of the were resolved by UPLC on a reverse-phase column (ACQUITY TDH3 (glyceraldehyde 3-phosphate dehydrogenase) promoter, UPLC BEH C18 column, length 150 mm; Waters) at 45 °C and creating the pNS29 plasmid. Ichthyosis mutations were introduced were detected by MS. The flow rate was 0.1 mL/min in the binary into the CYP4F22 gene using the QuikChange Site-Directed gradient system using a mobile phase A [acetonitrile/water/metha- Mutagenesis Kit (Agilent Technologies), using the primers de- nol (4:4:2, vol/vol/vol) containing 0.1% formic acid and 0.025% scribed in Table S2. The N-glycosylation site cassette was inserted ammonia] and a mobile phase B [2-propanol/methanol (4:1, vol/vol) into the CYP4F22 gene as described previously (37), using the containing 0.1% formic acid and 0.025% ammonia]. The elution T (topology)-series primers listed in Table S2. gradient steps were as follows: 0 min, 5% B; 0–10 min, gradient to 60% B; 10–50 min, gradient to 80% B; 50–55 min, gradient to 100% [3H]Sphingosine Labeling Assay. HEK 293T cells were transfected B; 55–65 min, 100% B; 65–66 min, gradient to 5% B; 66–80 min, with appropriate plasmids. Twenty-four hours after transfection, 5% B. The ESI capillary voltage was set at 3.0 kV; the sampling cells were labeled for 4 h at 37 °C with 2 μCi [3-3H]sphingosine cone was set at 30 V; and the source offset was set at 50 V in (20 Ci/mmol; PerkinElmer Life Sciences). Cells were washed positive ion mode. Each ceramide species was detected by MRM by + + twice with 1 mL of PBS and suspended in 100 μL of PBS. Lipids selecting the m/z ([M−H2O+H] and [M+H] ) of specific ceramide were extracted by successive addition and mixing of 375 μLof species at Q1 and the m/z 264.2 at Q3 (Table S3). Data analysis and chloroform/methanol/HCl (100:200:1, vol/vol/vol), 125 μL of chlo- quantification were performed using MassLynx software (Waters). roform, and 125 μL of 1% KCl. Phases were separated by centri- For quantitation of FAs in keratinocytes, lipids were extracted fugation (20,000 × g at room temperature for 3 min), after which and treated with an alkali as described above. As an internal stan- the organic (lower) phase was recovered, dried, and subjected to dard, C13:0 FA (0.2 pmol; Sigma) was added. Extracted lipids were + normal-phase TLC (Silica Gel 60 TLC plates; Merck Millipore) dried, derivatized by an AMP Mass Spectrometry Kit (Cayman with the following solvent systems: (i) chloroform/methanol/water Chemical) according to the manufacturer’s protocol, and resolved (40:10:1, vol/vol/vol), developed to 2 cm from the bottom of the by UPLC-ESI MS essentially as described above except as outlined TLC plate, dried, and then developed again to 5 cm from below. The flow rate was set at 0.15 mL/min and the elution gra- the bottom; (ii) chloroform/methanol/acetic acid (47:2:0.5, vol/vol/vol), dient steps were set as follows: 0 min, 5% B; 0–3min,gradientto developed to the top; and (iii) hexane/diethylether/acetic acid 60% B; 3–10 min, gradient to 80% B; 10–11 min, gradient to 100% B; (65:35:1, vol/vol/vol), developed to the top twice. Labeled lipids 11–20 min, 100% B; 20–21 min, gradient to 5% B; 21–30 min, were visualized by spraying the plate with a fluorographic re- 5% B. Hydroxy FA species were detected by MRM by selecting the + agent [2.8 mg/mL 2,5-diphenyl-oxazole in 2-methylnaphthalene/ m/z ([M+H] ) of the derivatized hydroxy FA species at Q1 and the 1-buthanol (1:3.3, vol/vol)]. The TLC plate was exposed to X-ray m/z 238.9 at Q3, corresponding to the fragment cleaved between C3 film at −80 °C. and C4 of derivatized FAs (Table S3). For reverse-phase TLC analysis, extracted lipids were sus- To examine ceramide species in the stratum corneum of human pended in 150 μL of 90% methanol and mixed with 150 μLof subjects, tape stripping was performed by pressing an acryl film tape

Ohno et al. www.pnas.org/cgi/content/short/1503491112 1of11 (465#40; Teraoka Seisakusho) to the skin of the forearm. Five (39), using anti-FLAG M2 (10 μg/mL; Sigma), anti-calnexin 4F10 strips, measuring 25 mm × 50 mm each, were obtained from a (10 μg/mL; Medical & Biological Laboratories), or anti-HA HA-7 single person. The tapes were immersed in 3.0 mL methanol with (50 μg/mL; Sigma) antibody as a primary antibody and Alexa μ 60 L 500 nM C17:0 ceramide as an internal standard. After 10 min Fluor 488-conjugated anti-rabbit antibody or Alexa Fluor 594- of sonication, the lipid extracts were dried under a nitrogen stream conjugated anti-mouse antibody (each at 5 μg/mL; Molecular and then were dissolved in chloroform/methanol/2-propanol Probes, Life Technologies) as a secondary antibody. Coverslips (10:45:45, vol/vol/vol) so that the final concentration of the in- ternal standard was 50 nM. This lipid solution was subjected to were mounted with Prolong Gold Antifade Reagent (Molecular reversed-phase LC/MS. An Agilent 1100 Series LC/MSD SL Probes, Life Technologies) and observed under a Leica DM5000B system equipped with a multi-ion source, ChemStation software, microscope (Leica Microsystems). a 1,100-well plate autosampler (Agilent Technologies), and an L-column ODS (2.1 mm i.d. × 150 mm; Chemicals Evaluation and Deglycosylation. Protein deglycosylation was performed using Research Institute) was used. Chromatographic separation of endoglycosidase H (New England Biolabs), according to the ’ the lipids was achieved at a flow rate of 0.2 mL/min using a bi- manufacturer s instructions. nary gradient solvent system of mobile phase C [methanol/ water ω (1:1, vol/vol) containing 5 mM acetic acid and 10 mM ammo- In Vitro FA -Hydroxylase Assay. Total membrane fractions were nium acetate] and mobile phase D (2-propanol containing 5 mM prepared from yeast cells as described previously (40) and were acetic acid and 10 mM ammonium acetate). The mobile phases suspended in assay buffer [100 mM potassium phosphate (pH 7.4), were consecutively programmed as follows: 0–1 min, 20% D; 1–2 10% glycerol, 1× Complete protease inhibitor mixture (EDTA- min, gradient to 60% D; 2–30 min, gradient to 100% D; 30–35 free; Roche Diagnostics), 1 mM phenylmethylsulfonyl fluoride, min, 100% D; 35–45 min, 20% D. The injection volume was and 1 mM DTT]. The substrate C30:0 FA (1 mM, 0.5 μL; Tokyo 20 μL. The column temperature was maintained at 40 °C. Mass Chemical Industry) was dissolved in chloroform, aliquoted into spectrometry parameters were as follows: polarity, negative ion plastic tubes, dried, and then suspended in a mixture of 25 μLof mode; flow of heated dry nitrogen gas, 4.0 L/min; nebulizer gas yeast membrane fractions (50 μgprotein),23μL of assay buffer, pressure, 60 psi; heater temperature of nitrogen gas, 350 °C; va- and 1 μL of 10% digitonin by sonication. After addition of 1 μLof porizer temperature, 200 °C; capillary voltage, 4,000 V; charging 50 mM NADPH or water, samples were incubated for 1 h at voltage, 2,000 V; fragmenter voltage, 200 V. Each ceramide spe- + − 37 °C. Lipids were extracted by the successive addition and mixing cies was detected by selected ion monitoring as m/z [M CH3COO] μ μ μ (Table S4). of 33.15 L of 5 M HCl, 91.85 Lofwater,100 Lofethanol,and 700 μL of hexane. After centrifugation, the organic phase was + Immunofluorescence Microscopy. Indirect immunofluorescence recovered, dried, derivatized by an AMP Mass Spectrometry Kit, microscopy was performed essentially as described previously and resolved by UPLC-ESI MS, as described above.

Fig. S1. Structures of the epidermis, the stratum corneum, acylceramide, and protein-bound ceramide. Acylceramides are produced mainly in the stratum granulosum and partly in the stratum spinosum and are stored in lamellar bodies as glucosylated forms (acyl glucosylceramides). At the interface of the stratum granulosum and stratum corneum, the lamellar bodies fuse with the plasma membrane and release their contents into the extracellular space, where acyl glucosylceramides are converted to acylceramides. Thus, released acylceramides, FAs, and cholesterol form lipid lamellae in the stratum corneum. Some acylceramide is hydrolyzed to ω-hydroxyceramide, followed by covalent binding to corneocyte surface proteins to create corneocyte lipid envelopes. Acyl- ceramide contains ULCFAs with carbon chain lengths of C28–C36. The FA elongase ELOVL1 produces VLCFAs, which are further elongated to ULCFAs by ELOVL4 (29). The ceramide synthase CERS3 creates an amide bond between ULCFA and LCB (17). ω-Hydroxylation of ULCFA is required for acylceramide production. However, the responsible ω-hydroxylase had not been identified previously; its identification is the subject of this research.

Ohno et al. www.pnas.org/cgi/content/short/1503491112 2of11 Fig. S2. Structure and synthetic pathways of ceramides in mammals. (A) Structure and nomenclature of epidermal ceramides. Epidermal ceramides are classified into 12 classes depending on their differences in the LCB and FA moieties. N-type and A-type ceramides contain C16–C30 FAs (n = 1–15), whereas EO- type ceramides contain C28–C36 FAs (n = 13–21) (6, 7). (B) FA elongation and ceramide synthesis in mammals. The FA elongation pathways of saturated and monounsaturated FAs and the ceramide-synthetic pathways are illustrated. E1–E7 and C1–C6 indicate the ELOVL (ELOVL1–7) and CERS (CERS1–6) isozymes involved in each step, respectively. The differences in the letter size of E1–E7 reflect their enzyme activities in each FA elongation reaction. Cer, ceramide; MUFA, monounsaturated FA; SFA, saturated FA.

Ohno et al. www.pnas.org/cgi/content/short/1503491112 3of11 Fig. S3. MRM chromatogram of ceramides produced by combined ELOVL4 and CERS3 expression. Lipids were extracted from HEK 293T cells transfected with control vector (A) or pCE-puro 3xFLAG-ELOVL4 and pCE-puro 3xFLAG-CERS3 (B) or mouse epidermis (C) and subjected to UPLC/ESI-MS using a triple quadrupole + + mass spectrometer (Xevo TQ-S; Waters). Each ceramide was detected by MRM by setting the appropriate [M+H] and [M+H−H2O] values at Q1 and m/z 264.2 (corresponding to C18:0 sphingosine) at Q3. Each MRM peak was overlaid using MassLynx software. Insets show enlarged views of the indicated areas of the original chromatograms.

Ohno et al. www.pnas.org/cgi/content/short/1503491112 4of11 Fig. S4. TLC chromatogram of ceramides. The acylceramide EOS and ω-hydroxyceramide were prepared as follows. Lipids were prepared from mouse epi- dermis and separated by normal-phase TLC. Silica containing EOS ceramide was scraped from the TLC plate and eluted with chloroform/methanol (1:2, vol/vol). A portion of EOS ceramide was converted to ω-hydroxyceramide by hydrolysis of the ester bond connecting ω-hydroxyceramide and linoleic acid with 0.1 M NaOH. The prepared EOS ceramide and ω-hydroxyceramide, as well as C24:0 ceramide (Avanti Polar Lipids), C16:0 ceramide (Avanti Polar Lipids), and glu- cosylceramide (Avanti Polar Lipids), were separated by normal-phase TLC and visualized by cupric acetate/phosphoric acid staining. Cer, ceramide; GlcCer, glucosylceramide; ω-OH Cer, ω-hydroxyceramide.

Ohno et al. www.pnas.org/cgi/content/short/1503491112 5of11 Fig. S5. MRM chromatogram of ω-hydroxyceramide species produced by CYP4F22. Lipids were extracted from HEK 293T transfected with pCE-puro 3xFLAG-ELOVL4 and pCE-puro 3xFLAG-CERS3, together with control vector (A) or pCE-puro 3xFLAG-CYP4F22 (B). EOS from mouse epidermis was treated with an alkali to liberate ω-hydroxyceramides (C). Lipids were subjected to UPLC/ESI-MS. Each ceramide was detected by MRM by setting the appropriate + + [M+H] and [M+H−H2O] values at Q1 and m/z 264.2 at Q3. Each MRM peak was overlaid using MassLynx software. ωhC30:1, ω-hydroxyceramide with a chain length of C30:1. IS, internal control (C17:0 ceramide). The Inset in C shows an enlarged large view of the indicated area of the original chromatogram.

Ohno et al. www.pnas.org/cgi/content/short/1503491112 6of11 Fig. S6. Working model for the acylceramide synthesis in the ER. Palmitoyl-CoA is elongated to ULCFA-CoA on the cytosolic side of the ER membrane. During FA elongation, ULCFA (C30–C36) portions of ULCFA-CoAs may be bent in the cytosolic leaflet of the ER membrane. After conversion of ULCFA-CoA to ULCFA, the ω-carbon of ULCFA is hydroxylated by CYP4F22. ω-Hydroxy-ULCFA then is converted to ω-hydroxy-ULCFA-CoA by acyl-CoA synthetase, followed by synthesis of ω-hydroxyceramide by CERS3. Finally, an acyltransferase catalyzes the formation of an ester bond between linoleic acid and the ω-hydroxy group of ω-hydroxyceramide, producing acylceramide.

Table S1. Ceramide levels in the stratum corneum Ceramide level, ng/μg protein

Ceramide class Control Patient Mother Father

NDS 1.10 0.82 2.63 2.17 NS 1.94 3.58 5.16 3.34 NH 3.21 1.82 6.55 4.27 NP 5.07 2.06 12.77 9.07 ADS 0.64 0.46 0.81 0.90 AS 1.36 4.20 3.18 2.61 AH 2.64 2.94 4.53 4.88 AP 2.59 2.33 4.57 7.51 EOS 0.41 0.02 0.61 0.44 EOH 0.37 0.02 0.65 0.45 EOP 0.10 0.01 0.27 0.24 Total 19.44 18.26 41.73 35.86

Control, wild type; patient, R243H/D380T fs2X; patient’smother, WT/D380T fs2X; patient’s father, WT/R243H.

Ohno et al. www.pnas.org/cgi/content/short/1503491112 7of11 Table S2. Primers used in this study Primer Sequence

CYP4F2-F 5′-GGATCCATGTCCCAGCTGAGCCTGTCCTGG-3′ CYP4F2-R 5′-TCAGCTCAGGGGCTCCACCCGCAGC-3′ CYP4F3-F 5′-AGATCTATGCCACAGCTGAGCCTGTCCTCGC-3′ CYP4F3-R 5′-TCAGCTCAGGGGCTCCACCCGCAGC-3′ CYP4F11-F 5′-AGATCTATGCCGCAGCTGAGCCTGTCCTGGC-3′ CYP4F11-R 5′-TCACTGTGAGTTCGCACCCAGGGGC-3′ CYP4F12-F 5′-AGATCTATGTCGCTGCTGAGCCTGCCCTGGC-3′ CYP4F12-R 5′-TCACTGCAAGCTTACATTCAGGGGC-3′ CYP4F8-F 5′-GGATCCATGTCGCTGCTGAGCCTGTCTTGGC-3′ CYP4F8-R 5′-TCAGCCCAGGGGTTCTACTCGCAGC-3′ CYP4F22-F 5′-GGATCCATGCTGCCCATCACAGACCGCCTGC-3′ CYP4F22-R 5′-TCAGGCCCGCGGAGGCAGCGGCTCC-3′ CERS2-F 5′-TGTCGACATGCTCCAGACCTTGTATGATTACTTC-3′ CERS2-R 5′-TCAGTCATTCTTACGATGGGTTGTATTG-3′ CERS3-F 5′-GGATCCATGTTTTGGACGTTTAAAGAATGGTTC-3′ CERS3-R 5′-CTAATGGCCATGCTGGCCATTGGGAAT-3′ F59L-F 5′-GCGGCTGCGCTGCTTGCCCCAGCCTCCCCGG-3′ F59L-R 5′-CCGGGGAGGCTGGGGCAAGCAGCGCAGCCGC-3′ R243H-F 5′-GCTGTCTGTCCGGCACCAGTATCGCTTGCAC-3′ R243H-R 5′-GTGCAAGCGATACTGGTGCCGGACAGACAGC-3′ R372W-F 5′-GAAGTCATGAAAGGCTGGGAGCTGGAGGAGC-3′ R372W-R 5′-GCTCCTCCAGCTCCCAGCCTTTCATGACTTC-3′ H435Y-F 5′-CAGCATCTATGGAACCTACCACAACCCCACAG-3′ H435Y-R 5′-CTGTGGGGTTGTGGTAGGTTCCATAGATGCTG-3′ H436D-F 5′-CATCTATGGAACCCACGACAACCCCACAGTG-3′ H436D-R 5′-CACTGTGGGGTTGTCGTGGGTTCCATAGATG-3′ D380T fsX2-F 5′-GAGCTGGAGGAGCTGGAGTGGACGATCTGACTCAGCTGCCC-3′ D380T fsX2-R 5′-GGGCAGCTGAGTCAGATCGTCCACTCCAGCTCCTCCAGCTC-3′ T-E85/K-F 5′-GAGGCGGGCCTTCAAGATGAGGGATCCAAGAAGGTACTGGACAACATG-3′ T-E85/K-R 5′-CATGTTGTCCAGTACCTTCTTGGATCCCTCATCTTGAAGGCCCGCCTC-3′ T-H155/R-F 5′-GGTGACAAGTGGAGCCGGCACGGATCCCGTCGCCTGCTGACACCCGCC-3′ T-H155/R-R 5′-GGCGGGTGTCAGCAGGCGACGGGATCCGTGCCGGCTCCACTTGTCACC-3′ T-A285/L-F 5′-CCAGGAACGGCGGCGGGCAGGATCCCTGCGTCAGCAGGGGGCCGAG-3′ T-A285/L-R 5′-CTCGGCCCCCTGCTGACGCAGGGATCCTGCCCGCCGCCGTTCCTGG-3′ T-C361/R-F 5′-CCGGAATACCAGGAGAAATGCGGATCCCGAGAAGAGATTCAGGAAGTC-3′ T-C361/R-R 5′-GACTTCCTGAATCTCTTCTCGGGATCCGCATTTCTCCTGGTATTCCGG-3′ T-D455/N-F 5′-CCCTACCGCTTTGACCCGGACGGATCCAACCCACAGCAGCGCTCTCC-3′ T-D455/N-R 5′-GGAGAGCGCTGCTGTGGGTTGGATCCGTCCGGGTCAAAGCGGTAGGG-3′ T-R508/K-F 5′-CGAACGCGCAAGGTGCGGCGGGGATCCAAGCCGGAGCTCATACTGCGC-3′ T-R508/K-R 5′-GCGCAGTATGAGCTCCGGCTTGGATCCCCGCCGCACCTTGCGCGTTCG-3′

Ohno et al. www.pnas.org/cgi/content/short/1503491112 8of11 Table S3. Selected m/z values for ceramide and FA species in MS analysis Lipid species Precursor ion, Q1 Product ion, Q3 Collision energy, V

d18:1/C16:0 Cer 520.2, 538.2 264.2 20 d18:1/C18:0 Cer 548.2, 566.2 264.2 20 d18:1/C20:0 Cer 576.3, 594.3 264.2 20 d18:1/C22:0 Cer 604.3, 622.3 264.2 25 d18:1/C24:1 Cer 630.3, 648.3 264.2 25 d18:1/C24:0 Cer 632.3, 650.3 264.2 30 d18:1/C26:1 Cer 658.4, 676.4 264.2 30 d18:1/C26:0 Cer 660.4, 678.4 264.2 30 d18:1/C28:1 Cer 686.4, 704.4 264.2 30 d18:1/C28:0 Cer 688.4, 706.4 264.2 30 d18:1/C30:1 Cer 714.4, 732.4 264.2 35 d18:1/C30:0 Cer 716.4, 734.4 264.2 35 d18:1/C32:1 Cer 742.5, 760.5 264.2 35 d18:1/C32:0 Cer 744.5, 762.5 264.2 40 d18:1/C34:1 Cer 770.5, 788.5 264.2 40 d18:1/C34:0 Cer 772.5, 790.5 264.2 40 d18:1/C36:1 Cer 798.5, 816.5 264.2 45 d18:1/C36:0 Cer 800.5, 818.5 264.2 45 d18:1/ωhC24:0 Cer 648.3, 666.3 264.2 30 d18:1/ωhC26:1 Cer 674.4, 692.4 264.2 30 d18:1/ωhC26:0 Cer 676.4 694.4 264.2 30 d18:1/ωhC28:1 Cer 702.4, 720.4 264.2 30 d18:1/ωhC28:0 Cer 704.2, 722.4 264.2 30 d18:1/ωhC30:1 Cer 730.4, 748.4 264.2 35 d18:1/ωhC30:0 Cer 732.4, 750.4 264.2 35 d18:1/ωhC32:1 Cer 758.5, 776.5 264.2 35 d18:1/ωhC32:0 Cer 760.5, 778.5 264.2 40 d18:1/ωhC34:1 Cer 786.5, 804.5 264.2 40 d18:1/ωhC34:0 Cer 788.5, 806.5 264.2 40 d18:1/ωhC36:1 Cer 814.5, 832.5 264.2 40 d18:1/ωhC36:0 Cer 816.5, 834.5 264.2 40 ωhC26:1 FA 577.2 238.9 55 ωhC26:0 FA 579.2 238.9 55 ωhC28:1 FA 605.2 238.9 55 ωhC28:0 FA 607.2 238.9 55 ωhC30:1 FA 633.2 238.9 60 ωhC30:0 FA 635.2 238.9 60 ωhC32:1 FA 661.2 238.9 60 ωhC32:0 FA 663.2 238.9 60 ωhC34:1 FA 689.2 238.9 60 ωhC34:0 FA 691.2 238.9 60 ωhC36:1 FA 717.2 238.9 60 ωhC36:0 FA 719.2 238.9 60

Cer, ceramide; ωh, ω-hydroxy.

Ohno et al. www.pnas.org/cgi/content/short/1503491112 9of11 Table S4. Selected m/z values for ceramide species in the MS analysis of the stratum corneum of human subjects m/z value Ceramide species

570.5 NDS C32 584.5 NDS C33 NH C32 AS C32 598.5 NDS C34 NH C33 AS C33 612.5 NDS C35 NH C34 AS C34 626.5 NDS C36 NH C35 AS C35 640.5 NDS C37 NH C36 AS C36 654.5 NDS C38 NH C37 AS C37 668.6 NDS C39 NH C38 AS C38 682.6 NDS C40 NH C39 AS C39 696.6 NDS C41 NH C40 AS C40 710.6 NDS C42 NH C41 AS C41 724.6 NDS C43 NH C42 AS C42 738.6 NDS C44 NH C43 AS C43 752.6 NDS C45 NH C44 AS C44 766.7 NDS C46 NH C45 AS C45 780.7 NDS C47 NH C46 AS C46 794.7 NDS C48 NH C47 AS C47 808.7 NDS C49 NH C48 AS C48 822.7 NDS C50 NH C49 AS C49 836.7 NDS C51 NH C50 AS C50 850.7 NDS C52 NH C51 AS C51 864.8 NDS C53 NH C52 AS C52 878.8 NDS C54 NH C53 AS C53 892.8 NH C54 AS C54 602.5 AP C32 616.5 AP C33 630.5 AP C34 644.5 AP C35 658.5 AP C36 672.6 AP C37 686.6 AP C38 700.6 AP C39 714.6 AP C40 728.6 AP C41 742.6 AP C42 756.6 AP C43 770.7 AP C44 784.7 AP C45 798.7 AP C46 812.7 AP C47 826.7 AP C48 840.7 AP C49 854.7 AP C50 868.8 AP C51 882.8 AP C52 896.8 AP C53 910.8 AP C54 568.5 NS C32 582.5 NS C33 596.5 NS C34 610.5 NS C35 624.5 NS C36 638.5 NS C37 652.5 NS C38 666.6 NS C39 680.6 NS C40 694.6 NS C41 708.6 NS C42 722.6 NS C43 736.6 NS C44 750.6 NS C45 764.7 NS C46

Ohno et al. www.pnas.org/cgi/content/short/1503491112 10 of 11 Table S4. Cont. m/z value Ceramide species

778.7 NS C47 792.7 NS C48 806.7 NS C49 820.7 NS C50 834.7 NS C51 848.7 NS C52 862.8 NS C53 876.8 NS C54 586.5 NP C32 ADS C32 600.5 NP C33 ADS C33 AH C32 614.5 NP C34 ADS C34 AH C33 628.5 NP C35 ADS C35 AH C34 642.5 NP C36 ADS C36 AH C35 656.5 NP C37 ADS C37 AH C36 670.6 NP C38 ADS C38 AH C37 684.6 NP C39 ADS C39 AH C38 698.6 NP C40 ADS C40 AH C39 712.6 NP C41 ADS C41 AH C40 726.6 NP C42 ADS C42 AH C41 740.6 NP C43 ADS C43 AH C42 754.6 NP C44 ADS C44 AH C43 768.7 NP C45 ADS C45 AH C44 782.7 NP C46 ADS C46 AH C45 796.7 NP C47 ADS C47 AH C46 810.7 NP C48 ADS C48 AH C47 824.7 NP C49 ADS C49 AH C48 838.7 NP C50 ADS C50 AH C49 852.7 NP C51 ADS C51 AH C50 866.8 NP C52 ADS C52 AH C51 880.8 NP C53 ADS C53 AH C52 894.8 NP C54 ADS C54 AH C53 908.8 AH C54 1072.9 EODS C66 1086.9 EODS C67 EOH C66 1100.9 EODS C68 EOH C67 1114.9 EODS C69 EOH C68 1128.9 EODS C70 EOH C69 1142.9 EODS C71 EOH C70 1156.9 EODS C72 EOH C71 1170.9 EOH C72 1070.9 EOS C66 1084.9 EOS C67 1098.9 EOS C68 1112.9 EOS C69 1126.9 EOS C70 1140.9 EOS C71 1154.9 EOS C72 1088.9 EOP C66 1102.9 EOP C67 1116.9 EOP C68 1130.9 EOP C69 1144.9 EOP C70 1158.9 EOP C71 1172.9 EOP C72

Ohno et al. www.pnas.org/cgi/content/short/1503491112 11 of 11