Evaluation of Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl Ether ("compound A") Effects on Urine Protein Excretion in Rats Using Mass Poster No. Spectrometry P27-T Keling Dong 1, Marjorie S Minkoff 1, Matthew Willetts 1, James E Carlson 1, Jeffrey D Miller 1, Evan. D. Kharasch 2 1Applied Biosystems, 500 Old Connecticut Path, Framingham, MA, United States; 2Department of Anesthesiology, Washington University, St. Louis, MO, United States.

Table 1. List of identified proteins with expression level ABSTRACT RESULTS Figure 3. Protein expression pie chart change after FDVE treatment Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE Figure 1. Ion exchange chromatography or "compound A"), a haloalkene degradant of the volatile N AccessionName # Trend N AccessionName # Trend Unchange #66 spt|P13635|CERU_RATCeruloplasmin precursor Down fractionation #6 spt|P17475|A1AT_RATAlpha-1-antiproteinase precursorUp anesthetic sevoflurane, is nephrotoxic in rats. FDVE Corticosteroid-binding globulin 79% precursor Down #7 spt|P12346|TRFE_RATSerotransferrin precursor Up #67 spt|P31211|CBG_RAT bioactivation mediates the toxicity, but the molecular and Ezrin-radixin-moesin-binding Hemopexin precursor fraction A67- #8 spt|P20059|HEMO_RAT Up phosphoprotein 50 Down cellular mechanisms of toxification are unknown. FDVE #71 spt|Q9JJ19|NHERF_RAT 1 68 Regenerating islet-derived protein 3 gamma precursor Down caused rapid and brisk changes in kidney gene expression, #12 spt|P24090|FETUA_RATAlpha-2-HS-glycoprotein precursorUp #82 spt|P42854|REG3G_RAT #95 spt|Q6QLM7|KIF5A_RATKinesin heavy chain isoformDown 5A fraction A69- #13 spt|P08932|KNT2_RATT-kininogen 2 precursor Up providing potential insights into mechanisms of toxicity, and Leukemia inhibitory factor receptor 2 70 Contrapsin-like protease inhibitor 6 precursor Down #171 spt|O70535|LIFR_RAT precursor Up potential biomarkers for nephrotoxicity[1]. Nevertheless, it is #16 spt|P09006|CPI6_RAT #173 spt|Q9R1J4|MYOC_RATMyocilin precursor Down fraction A71- #17 spt|Q9QX79|FETUB_RATFetuin-B precursor Up Tyrosine-protein phosphatase non- receptor type 7 Down unknown whether gene expression changes are reflected in 3 72 #20 spt|P02767|TTHY_RATTransthyretin precursor Up #194 spt|P49445|PTN7_RAT #204 spt|Q8K3M6|ERC2_RATERC protein 2 Down protein expression, or whether such tissue changes would be #27 spt|P06866|HPT_RATHaptoglobin precursor Up fraction A73- Human immunodeficiency virus #34 spt|P02625|PRVA_RATParvalbumin alpha Up type I enhancer-binding protein 2 reflected in excreted urine proteins. This investigation was to 4 75 homolog Down #209 spt|Q00900|ZEP2_RAT #218 spt|Q8R500|MFN2_RATTransmembrane GTPase MFN2Down evaluate FDVE effects on urine protein excretion using mass fraction A76- #38 spt|P04276|VTDB_RATVitamin D-binding protein precursorUp Double-stranded RNA-specific 5 77 editase 1 Up-Down spectrometry and 8-plex iTRAQ® reagents for relative Down #41 spt|P02770|ALBU_RATSerum albumin precursor Up #107 spt|P51400|RED1_RAT Serum amyloid P-component Up-Down Afamin precursor Up Dow n-Up #44 spt|P36953|AFAM_RAT Up precursor Up-Down quantitiation. Results demonstrate that FDVE causes certain fraction 11% #124 spt|P23680|SAMP_RAT Transmembrane emp24 domain- 6 A78 4% containing protein 1 precursorUp-Down alterations in urine protein/peptide excretion. Multiple 5% 1% #49 spt|P00502|GSTA1_RATGlutathione S-transferase alpha-1Up #140 spt|Q5BK85|TMED1_RAT #175 spt|Q62889|NLGN3_RATNeuroligin-3 precursor Up-Down fraction Serine protease inhibitor A3M components were differentially expressed in a time-dependent In this experiment there are 231 proteins identified from the FDVE treated rat urine precursor Up #217 spt|P00763|TRY2_RATAnionic trypsin-2 precursorUp-Down 7 A79 #50 spt|Q63556|SPA3M_RAT manner. samples. As would be expected, the majority of the identified proteins (79%) show #226 spt|Q62824|EXOC4_RATExocyst complex component 4 fraction A80- no change in protein expression levels after FDVE treatment. The proteins which do #53 spt|P02764|A1AG_RATAlpha-1-acid glycoprotein precursorUp show expression level changes are categorized into 4 groups: There are 11% 8 81 #60 spt|P20760|GCA_RATIg gamma-2A chain C regionUp showing downward trend; 5% upward; 4% up first then down; and 1% down first and Up-Down #73 spt|P62898|CYC_RATCytochrome c, somatic Up INTRODUCTION fraction A82- then up (see Figure 4 for examples). #78 spt|P01048|KNT1_RATT-kininogen 1 precursor Up Seminal vesicle protein 2 precursor 9 85 #87 spt|P02783|SVP2_RAT Up-Down #97 spt|P11232|THIO_RATThioredoxin Up Extracellular superoxide dismutase [Cu-Zn] precursor Up-Down Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE #145 spt|Q08420|SODE_RAT #105 spt|Q64268|HEP2_RATHeparin cofactor 2 precursorUp or "compound A"), a haloalkene degradant of the volatile #120 spt|Q62671|EDD1_RATUbiquitin-protein ligase EDD1Up #84 spt|P15205|MAP1B_RATMicrotubule-associated proteinUp-Down 1B anesthetic sevoflurane, is nephrotoxic in rats. FDVE Zinc fingers and homeoboxes Figure 4. Examples of rat urine protein expression level protein 1 Up #127 spt|P06238|A2MG_RATAlpha-2-macroglobulin precursorDown-Up #166 spt|Q8R515|ZHX1_RAT bioactivation mediates the toxicity, but the molecular and changes after FDVE treatment. #169 spt|Q5U2Y9|CF152_RATProtein C6orf152 homolog Up Interferon-induced guanylate- #228 spt|Q63663|GBP2_RAT cellular mechanisms of toxification are unknown. FDVE binding protein 2 Down-Up Figure 2. Example of MSMS caused rapid and brisk changes in kidney gene expression, 3.5 spectrum. CONCLUSIONS providing potential insights into mechanisms of toxicity, and potential biomarkers for nephrotoxicity[1]. Nevertheless, it is 3
The results obtained demonstrate /suggest? that FDVE causes certain alterations in urine protein/peptide excretion. unknown whether gene expression changes are reflected in
Multiple components were differentially expressed in a time-dependent manner. Excretion 2.5 Day1/Day0 protein expression, or whether such tissue changes would be of several endogenously excreted proteins was rapidly decreased by FDVE. Day2/Day0
Other proteins showed increased excretion following FDVE, and then gradually decreased reflected in excreted urine proteins. This investigation was to 113.1228 to pre-dose levels. 114.1124 Day3/Day0 evaluate FDVE effects on urine protein excretion using mass 2
Excretion of a third set of proteins, minimally or not detectable in controls, was upregulated 115.1075 117.1334 Day4/Day0 following FDVE. spectrometry and 8-plex iTRAQ Reagents for relative 119.1117
With the 8-plex iTRAQ® Reagents, relative protein excretion levels can be determined Day5/ Day0 R1.5 a tio quantitiation. quantitatively, demonstrating that it is an ideal tool for time-course studies. Day6/Day0 MATERIALS AND METHODS 1 Day7/ Day0 REFERENCES

After Animal Use Committee approval, Male Fisher 344 rats 0.5 1. E. D. Kharasch, J. L. Schroeder, T. Bammler, R. Beyer, and S. Srinouanprachanh, Toxicological Sciences, 90, 419-431 (2006) (250-300g) housed in individual metabolic cages received a 2. P. Sheffels, J. L. Schroeder, T. G. Altuntus, H. D. Liggitt, and E. D. Kharasch, Chem. Res. single intraperitoneal injection of 0.25 mmol/kg FDVE, and all 0 Toxicol. 17, 1177-1189 (2004) urine was collected daily for one week, as described Dow n Up Up-Dow n DowACKNOWLEDGEMENTS n-Up previously[2]. y1 y3 y4 y5 y7 y10 y11 y12 Equal volumes of 6 replicate time points were pooled to create 1719.83 Protein This project was supported by NIH DK53765 assay time point samples. 150 uL of each pool was brought to b1 b2 b3 b4 b6b7 b8 b9b10 b11 b12 2mls PBS. Each sample was applied to an anti-HSA column 1633.78 Example Protein TRADEMARKS/LICENSING (POROS® Affinity Depletion Cartridges). The flow-through 114.11 732.50 451.32 Dow n Haptoglobin precursor Applera Corporation is committed to providing the world’s leading technology and information (albumin depleted) was desalted on a POROS R150 column. for life scientists. Applera Corporation consists of the Applied Biosystems and Celera The protein was eluted and dried. Samples were then Up Protein-tyrosine phosphatase LC-PTP Genomics businesses. Applied Biosystems/MDS SCIEX is a joint venture between Applera Corporation and MDS Inc. reconstituted in 1M TEAB. 50ug of each sample (from day 0 to m/z, Da Up-Dow n Neuroligin-3 precursor Applied Biosystems, Applera and AB (design) are registered trademarks of Applera day 7) was processed with the 8-plex iTRAQ® reagents Corporation or its subsidiaries in the U.S. and/or certain other countries. MIDAS, Paragon, Dow n-Up Alpha-2-macroglobulin precursor Pro Group, Tempo and ProteinPilot are trademarks and Q TRAP and NanoSpray are according to manufacturer's instructions. registered trademarks of Applied Biosystems/MDS Sciex. The iTRAQ® reagent labeled sample was then subjected to Information subject to change without notice. iTRAQ labeled peptide fragmentation spectrum is © 2007 Applera Corporation and MDS Inc. All rights reserved strong cation exchange chromatoghapy separation and nine There are generally 4 types of trends in the protein level changes: 1) protein level decreases shown. after initial FDVE treatment and slowly levels off; 2) protein level increases after initial FDVE fractions were collected. Six of the fractions were analyzed ADLSGITEDAPLK[IT8] treatment and reaches maximum at the end of sampling; 3) protein level increases initially using LC-MALDI on the 4800 MALDI TOF/TOF™ Analyzer after FDVE treatment then slowly decreases after reaches maximum; 4) protein level Alpha-1-antiproteinase precursor (AB/MDS SCIEX). MS and MS/MS data were processed and decreases initially after FDVE treatment and slowly levels off then turns upward. searched against the IPI rat database (ipi.RAT.v3.26.fasta) using ProteinPilot™ Software (ABSciex).