Supplemental Figure S1-3, Table S1-4

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1 Supplemental Figure S1-3, Table S1-4 File format: PDF Title: Identification of Blood Brain Barrier-Permeable Proteins Derived from a Peripheral Organ: In Vivo and in Vitro Evidence of Blood-to-Brain Transport of Creatine Kinase AUTHOR NAMES Kazuki Sato, Masanori Tachikawa, Michitoshi Watanabe, Eisuke Miyauchi, Yasuo Uchida, and Tetsuya Terasaki * AUTHOR AFFILIATIONS Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan * Corresponding author: Professor Tetsuya Terasaki, Ph.D. 1

2 Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki, Aoba, Sendai , Japan. Voice: , FAX: , 2

3 Figure S1. Synthesis of mouse creatine kinase B-type (a) and M-type (b) recombinant proteins BL21-CodonPlus(DE3)-RIPL E. coli were transfected with pet17b-ck B-type and -CK M-type, and cultured under IPTG induction (30 C, 3 h). Synthesized proteins were solubilized in 8 M urea, 100 mm Na2HPO4, 10 mm Tris-HCl, ph 8.0. The solution was centrifuged at 15,000 g, 4 C, 10 min and the supernatant and pellet were collected as soluble fraction (S) and insoluble fraction (I), respectively. Protein synthesis was confirmed by SDS-PAGE, followed by staining with Coomassie Brilliant Blue. Theoretically, CK B-type (a) and CK M-type (b) should be detected in 49.8 and 50.1 kda, respectively. 3

4 Figure S2. Plasma concentration-time profile of rabbit CK-MM after intravenous administration to mice Rabbit CK-MM (589 nmol/kg) was intravenously administered to mice, and plasma was prepared at 1, 5, 15, 30, 60, 180, 360 and 720 min. The plasma was digested with Lys-C and trypsin, and the target peptide of rabbit CK-MM in the digest was measured by LC- MS/MS to determine the concentration. Serum was collected from 15 mice. 4

5 Figure S3. BBB integrity in mice treated with biotinylated liver cytosol proteins Biotinylated liver cytosolic proteins (320 mg/kg) or saline were intravenously administered to mice. The mice were transcardially perfused with 100 ml of 0.5% trypan blue in PBS containing 2.5 U/mL heparin at 2 hours after the administration (Reynolds DS and Morton AJ, J Neurosci Methods, 79(1):115-21, 1998), and the cerebrums were extracted and fixed in 4% PFA/PB. Sections (50 μm in thickness) were prepared with a cryostat (Leica, CM1900) and photographed through a microscope (Olympus, IX71). Scale bar = 100 μm. 5

6 Table S1. Details of SWATH-MS-identified peptides-derived from in vivo-bbb permeable cytosolic proteins in cerebrum Precursor Accession Protein name Peptide sequence Charge ions number m/z LVLGDNSLAIR P05201 Aspartate aminotransferase, cytoplasmic IVAATLSDPELFK Q04447 Creatine kinase B-type DLFDPIIEER P Pyruvate kinase isoform M1 LLFEELVR P16125 L-lactate dehydrogenase B chain LIASVADDEAAVPNNK Peak area of product ions (counts) Product Biotin Product ions labeledcerebrum Control Ratio ion type m/z cytosol administrated y y y y y y y y y y y y y y y Average ratio ± S.E.M ± ± ± ± 0.59 Mouse cerebrum cytosolic proteins were biotinylated with biotin-ss-sulfo Osu, and the biotin-labeled cerebrum cytosolic proteins were intravenously administered to mice. Cerebrum cytosolic proteins were extracted from the administered mice at 1 hour, after PBS perfusion 6

7 (20 ml) to wash out proteins in the vascular space. Non-treated normal mouse cerebrum cytosolic proteins were also extracted as a control. Biotin-labeled proteins were collected on streptavidin beads, and the collected proteins were eluted from the beads and digested with Lys- C and trypsin. The peptide digest was examined by LC-MS/MS with SWATH-MS. Peak identification and calculation of peak areas of the product ions of identified peptides were done by PeakView software. Among the detected peptides, reliable peptides were extracted based on the peptide selection criteria as described in materials and methods. The peak area ratio of product ions of identified peptides was calculated between biotin-labeled cerebrum cytosol protein-administered mice and the control. The source proteins of peptides showing at least 2-fold increase in the administered mice are listed. 7

8 Table S2. Details of SWATH-MS-identified peptides-derived from in vivo-bbb permeable cytosolic proteins in liver Precursor Accession Protein name Peptide Charge ions number m/z Q99KI0 Aconitate hydratase, mitochondrial TDIANLAEEFK NLDYVATSIHEAVTK Aspartate P05201 aminotransferase, LVLGDNSLAIR cytoplasmic IVAATLSDPELFK P06745 Glucose-6-phosphate isomerase HFVALSTNTAK P15105 Glutamine synthetase LVLC[CAM]EVFK Peak area of product ions (counts) Product Product Biotin ions ion type labeled-liver m/z Control Ratio cytosol administrated y y y y y y y y y y y y b y b y y y Average ratio ± S.E.M ± ± ±

9 P Pyruvate kinase isoform M1 LLFEELVR LIASVADDEAAVPNNK P16125 L-lactate dehydrogenase B chain LIASVADDEAAVPNNK Q04447 Creatine kinase B-type DLFDPIIEER Q9QZZ4 Unconventional myosin- XV GLFSSVPAR P10649 Glutathione S-transferase Mu 1 YIATPIFSK y y y y y y y y y y y y y y y y y y ± ± ± ± ± ± 0.33 Mouse liver cytosolic proteins were biotinylated with biotin-ss-sulfo Osu, and the biotin-labeled liver cytosolic proteins were intravenously administered to mice. Cerebrum cytosolic proteins were extracted from the administered mice at 1 hour, after PBS perfusion (20 ml) to wash out proteins in the vascular space. Non-treated normal mouse cerebrum cytosolic proteins were also extracted as a control. Biotin-labeled proteins were collected on streptavidin beads, eluted from the beads, and digested with Lys-C and trypsin. The peptide 9

10 digest was examined by LC-MS/MS with SWATH-MS. Peak identification and calculation of peak areas of the product ions of identified peptides were done by PeakView software. Among the detected peptides, reliable peptides were extracted based on the peptide selection criteria as described in materials and methods. The peak area ratios of product ions of identified peptides were calculated between biotinlabeled liver cytosol protein-administered mice and the control. The source proteins of peptides showing at least 2-fold increase in the administered mice are listed. 10

11 Table S3. Target peptide transitions in LC-MS/MS analysis Accession number P00563 P02787 P SRM/MRM transitions (m/z) Molecular name Probe Sequence St/Is Q1 Q3-1 Q3-2 Q3-3 Q3-4 Rabbit creatine SEEEYPDLSK St kinase muscle-type SEEEYPDLSK Is Human EGYYGYTGAFR St serotransferrin EGYYGYTGAFR Is Bovine serum AEFVEVTK St albumin AEFVEVTK Is Reference VIAPVLGR St peptide^vi VIAPVLGR Is Reference LFGPSIPLAR St peptide^lf LFGPSIPLAR Is These SRM/MRM transitions were used in the measurements with the micro LC- QTRAP5500 system. Amino acids indicated in bold were labeled with 13 C and 15 N. 11

12 Table S4. Compartmental pharmacokinetic parameters of rabbit CK-MM intravenously administered to mice CL tot (ml/min/kg) A 14.4 (μmol/l) α (min -1 ) B 7.45 (μmol/l) β (min -1 ) k el (min -1 ) k (min -1 ) k (min -1 ) AUC (min μmol/l) V (ml/kg) V (ml/kg) V ss 35.3 (ml/kg) Rabbit CK-MM (589 nmol/kg) was intravenously administered to mice, and the plasma was prepared at 1, 5, 15, 30, 60, 180, 360 and 720 min. The concentration was determined by LC-MS/MS in the SRM/MRM mode. The plasma concentration profile (as shown in Fig. S2) was fitted with a two-compartment model and the kinetic parameters were calculated. 12