SUPPLEMENTAL MATERIAL. A novel truncated form of Apolipoprotein A-I transported by dense LDL is increased in. diabetic patients

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1 SUPPLEMENTAL MATERIAL A novel truncated form of Apolipoprotein A-I transported by dense LDL is increased in diabetic patients Cubedo et al: ApoA-I truncated form in diabetes By Judit Cubedo a, Teresa Padró a, Maisa García-Arguinzonis a, Gemma Vilahur a, Inka Miñambres b, Jose María Pou b, Juan Ybarra c, Lina Badimon a,d * From a Cardiovascular Research Center (CSIC-ICCC) and Biomedical Research Institute Sant Pau (IIB-Sant Pau); b Endocrinology Department, Hospital de la Santa Creu i Sant Pau; c Teknon Medical Center; d Cardiovascular Research Chair UAB; Barcelona, Spain. 7 Supplemental Figures and 1 Supplemental Table Correspondence to: Prof. Lina Badimon Cardiovascular Research Center, c/sant Antoni MªClaret 167, Barcelona, Spain. Phone: (34) Fax: (34) lbadimon@csic-iccc.org 1

2 LEGENDS TO SUPPLEMENTAL FIGURES Supplemental Figure 1. Representative serum 2-DE image Apo A-I spots profile in different ph and MW ranges (pi 4-7, 10% PAGE; and pi , 15% PAGE) and 3D image of the 5 detected forms of 28kDa. Supplemental Figure 2. Representative images of Apo A-I profile in VLDL samples (N=3/group) showing only the 28 kda form by 2-DE (pi range 4-7, 10% PAGE) and 1-DE followed by WB Apo A-I detection. Supplemental Figure 3. Representative 2-DE image (pi range 4-7, 10% PAGE) of LDL. The main detected proteins are Apo A-I and Apo E. Supplemental Figure 4. Bar diagrams showing differences in lipidomic profile in LDL fractions between DM (N=10) and non-dm-patients (N=8): (A) Free cholesterol and triglycerides in LDL assessed by TLC; (B) Cholesterol esters (CE), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) analyzed by LC-MS. *p<0.05 Supplemental Figure 5. Representative 2-DE image (pi range 4-7, 10% PAGE) of Apo A-I in HDL subclasses. Apo A-I Δ(1-38) is not detected in HDL2 nor in HDL3. Supplemental Figure 6. (A) Representative 2-DE image (pi range 4-7, 10% PAGE) of Apo A-I in rat serum showing both Apo A-I forms of 28 and 26 kda. (B) Box-plot diagram of the intensity of the 26 kda Apo A-I form in hepatic tissue of control and diabetic rats (N=3/group; *p<0.05). (C) Human and rat Apo A-I aminoacid sequences showing in silico cathepsin D cleavage site. 2

3 Supplemental Figure 7. (A) 1-DE profile of cathepsin D digestion of purified Apo A-I. Hemoglobin was used as a possitive control for cathepsin D activity. (B) Box-plot diagram of cathepsin D activity in hepatic tissue of control (N=8) and diabetic rats (N=7; *p<0.05). (C) Representative 2-DE, followed by Apo A-I WB, image (pi range 4-7, 10% PAGE) of Apo A-I in rat liver extracts showing both Apo A-I forms of 28 and 26 kda. (D) Box-plot diagram showing the intensity of the 26 kda Apo A-I form in hepatic tissue of control (N=4) and diabetic rats (N=4; *p<0.05). 3

4 SUPPLEMENTAL TABLE 1: Peak list obtained in the simulated tryptic digestion of Apo A-I using Sequence Editor tool of BioTools (Bruker Daltonics, BioTools 3.02) Nº Range Mono Partials Sequence 44 [ ] QKVEPLR 49 [ ] AELQEGAR 55 [ ] LHELQEK 29 [75-82] DLEEVKAK 77 [ ] LAARLEALK 1 [1-9] ATVYVDVLK 65 [ ] ARAHVDALR 91 [ ] AKPALEDLR 58 [ ] LSPLGEEMR 50 [ ] AELQEGARQK 53 [ ] QKLHELQEK 80 [ ] LEALKELGGAR 88 [ ] ATEHLSTLSEK 94 [ ] QGLLPVLESFK 34 [83-92] VQPYLDDFQK 75 [ ] QRLAARLEALK 63 [ ] DRARAHVBALR 40 [94-102] WQEEMELYR 70 [ ] THLAPYSDELR 59 [ ] LSPLGEEMRDR 23 [64-74] ETEGLRQEMSK 26 [70-80] QEMSKDLEEVK 35 [83-93] VQPYLDDFQKK 97 [ ] VSFLSALEEYTK 7 [14-26] DYVSQFEGSALGK 38 [93-102] KWQEEMELYR 83 [ ] ENGGARLAEYHAK 2 [1-13] ATVYVDVLKDSGR 32 [81-92] AKVQPYLDDFQK 47 [ ] VEPLRAELQEGAR 98 [ ] VSFLSALEEYTKK 60 [ ] LSPLGEEMRDRAR 27 [70-82] QEMSKDLEEVKAK 41 [94-104] WQEEMELYRQK 78 [ ] LAARLEALKENGGAR 33 [81-93] AKVQPYLDDFQKK 71 [ ] THLAPYSDELRQR 13 [32-45] LLDNVDSVTSTFSK 39 [93-104] KVQEEMELYRQK 45 [ ] QKVEPLRAELQEGAR 48 [ ] VEPLRAELQEGARQK 5 [10-26] DSGRDYVSQFEGSALGK 4

5 Nº Range Mono Partials Sequence 14 [32-47] LLDNWDSVTSTFSKLR 56 [ ] LHELQELSPLGEEMR 19 [48-63] EQLGPVTQEFWDNLEK 81 [ ] LEALKENGGARLAEYHAK 99 [ ] VSFLSALEEYTKKLNTQ 8 [14-31] DYVSQFEGSALGKQLNLK 72 [ ] THLAPYSDELRQRLAAR 51 [ ] AELQEGARQKLHELQEK 24 [64-80] ETEGLRQEMSKDLEEVK 86 [ ] LAEYHAKATEHLSTLSEK 68 [ ] AHVDALRTHLAPYSDELR 42 [94-109] WQEEMELYRQKVEPLR 30 [75-92] DLEEVKAKVQPYLDDFQK 54 [ ] QKLHELQEKLSPLGEEMR 57 [ ] LHELQEKLSPLGEEMRDR 17 [46-63] LREQLGPVTQEFWDNLEK 11 [27-45] QLNLKLLDNWDSVTSTFSK 89 [ ] ATEHLSTLSEKAKPALEDLR 92 [ ] AKPALEDLRQGLLPVLESFK 66 [ ] ARAHVDALRTHLAPYSDELR 69 [ ] AHVDALRTHLAPYSDELRQR 6 [10-31] DSGRDYVSQFEGSALGKQLNLK 12 [27-47] QLNLKLLDNWDSVTSTFSKLR 95 [ ] QGLLPVLESFKVSFLSALEEYTK 84 [ ] ENGGARLAEYHAKATEHLSTLSEK 20 [48-69] EQLGPVTQEFWDNLEKETEGLR 36 [83-102] VQPYLDDFQKKWQEEMELYR 96 [ ] QGLLPVLESFKVSFLSALEEYTKK 3 [1-26] ATVYVDVLKDSGRDYVSQFEGSALGK 18 [46-69] LREQLGPVTQEFWDNLEKETEGLR 5

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Suppl. Table 1: CV of pooled lipoprotein fractions analysed by ESI-MS/MS

$Suppl. Table 1: CV of pooled lipoprotein fractions analysed by ESI-MS/MS$ Supplement VLDL LDL HDL PC 3.3 1.77 1.3 LPC 4.82 2.5.35 SM 3.1 4.6 1.92 CER 2.17 6.3 4.15 PE 3.18 1.93 2.79 PE-pl 13.18 1.9 2.32 CE 2.9.65.4 FC.36 3.5 2.54 Suppl. Table 1: CV of pooled lipoprotein fractions