The effect of cigarette smoke exposure on the proteomic composition of human bronchial epithelial cell airway surface liquid Linsey E Haswell 1, Wanda Fields 2, Laetitia Cortes 3, Pascal Croteau 3, Laura McIntosh 3, Daniel Chelsky 3 Clive Meredith 1 and Gary Phillips 1 1British American Tobacco, Group Research and Development, Southampton, SO15 8TL, UK, 2R. J. Reynolds Tobacco Co, Abstract Nº: 1530 3 Poster Board Nº: 146 Research & Development, Winston-Salem, North Carolina, USA and Caprion Proteomics Inc, Montréal, Québec, Canada Corresponding email: [email protected]

Introduction Misc. Lipid metabolism Translation 5% Extracellular region 3% 2% DI A B Protein DI A B Protein DI A B 14% The conducting airway epithelium is covered by a thin layer of liquid known as the airway surface Nuclear organization - Transcription HNRNPD 1491.81 X AKR1C2 3.20 X X PSMA7 2.66 X X 4% liquid (ASL). The ASL plays an important defensive role against inhaled particles and chemicals CD109 4.39 X X S100A9 3.12 X X PSMA8 2.66 X X Protein degradation such as cigarette smoke. Human bronchial epithelial cells (HBECs) cultured in vitro at the air- 5% SERPINA3 4.35 X X S100P 3.04 X X CALM1 2.61 X X Inflammation - FUT3 3.85 X X KRT4 3.01 X X S100A11 2.58 X Immune response FUT5 3.85 X X CTSD 2.80 X X CCL20 2.55 X liquid interface secrete and other from their apical surface which are thought to Calcium ion binding 13% 7% FUT6 3.85 X X SERPINA1 2.77 X X PRSS3 2.36 X X mimic the ASL observed in vivo . Mass spectrometry (MS) based proteomics is a technique that is PLUNC 3.70 X X PSME1 2.75 X X KRT9 2.36 X X S100A8 3.51 X X ANXA5 2.69 X X FTH1 2.35 X capable of detecting patterns of secreted proteins in either in vitro or in vivo samples, such as Redox homeostasis - S100A6 3.40 X X KLK13 2.68 X X YWHAQ 2.30 X Stress response ASL or sputum. To verify and quantify selected proteins that could be differentially secreted in the 7% CAPN1 3.27 X X IL8 2.67 X X LDHA 2.30 X Signaling ASL of cultured HBECs, a quantitative mass spectrometry based, multiple reaction monitoring 13% Energy metabolism Table 2. Top 30 proteins differentially secreted following smoke exposure at 48 hours in the low dose vs. air 8% (MRM) assay was developed. This assay offers a fast and cost-effective alternative to Cell adhesion - Wound control comparison. [A] 40% significant peptides (q-value < 0.1) with DI in the same direction (all above or Cytoskeleton healing immunoassays for following panels of potential protein biomarkers. The aim of the current study 8% 11% below 1). [B] 80% of the peptides with DI in the same direction and median DI greater than 1.4. X indicates the was to develop an MRM assay and to identify changes in the protein composition of HBEC ASL specified criteria are met. Figure 1. Biological processes ( GO) associated with ASL proteins in all treatments groups following exposure to cigarette smoke. Effect of cigarette smoke on the ASL proteome Gene Gene Gene Gene Exposure to smoke resulted in 1,113 unique peptide sequences being differentially secreted (q- Protein name Protein name Protein name Protein name Protein name name name name name name Methods 60 kDa heat shock protein, value < 0.1 and DI > 2) in at least one treatment comparison. At the protein-level, 376 distinct ACTN1 Alpha-actinin-1 CRNN Cornulin HSPD1 MUC16 -16 S100A9 Protein S100-A9 mitochondrial Anterior gradient protein 2 10 kDa heat shock protein, Cell culture: Primary HBECs (Lonza) from 6 non-smoking donors were grown at the air-liquid AGR2 CSTB Cystatin-B HSPE1 MUC20 Mucin-20 S100P S100P-binding protein proteins were considered significant, with the greatest secretion changes occurring 48 hours homolog mitochondrial ® Plasminogen activator inhibitor interface on 6.5 mm Transwells (Corning) for 30 days, by which time the cells had undergone AHNAK2 Protein AHNAK2 CTSD Cathepsin D IL1B Interleukin-1 beta MUC4 Mucin-4 SERBP1 after low dose smoke exposure as compared to the air control (Table 1). The majority of the 1 RNA-binding protein 1 Aldo-keto reductase family 1 Interleukin 1 receptor mucocilliary differentiation . AKR1B10 CTSZ Cathepsin Z IL1RN MUC5AC Mucin-5AC SERPINA1 Alpha-1-antitrypsin differentially secreted proteins are related to inflammation and immune response, calcium member B10 antagonist protein Aldo-keto reductase family 1 AKR1C2 CTTN Src substrate cortactin IL6 IL-6 MUC5B Mucin-5B SERPINA3 Alpha-1-antichymotrypsin binding and homeostasis, squamous cell differentiation and keratinocytes, protein metabolism member C2 Aldehyde dehydrogenase, ALDH3A1 CXCL1 C-X-C motif chemokine 1 IL8 IL-8 NCL Nucleolin SERPINB1 Leukocyte elastase inhibitor Smoke exposure: A RM20S Smoking Machine (Borgwaldt) was used to generate and dilute dimeric NADP-preferring and oxidative stress (Figure 2). The top 30 proteins that were positively differentially secreted are NAD(P)H dehydrogenase ANXA1 Annexin A1 DKK2 Dickkopf-related protein 2 JRKL Jerky protein homolog-like NQO1 SERPINB13 Serpin B13 smoke from 3R4F cigarettes (University of Kentucky) under the ISO smoking regime (35 ml puff [quinone] 1 listed in Table 2. ANXA5 Annexin A5 EIF5A2 Elongation factor 1-alpha 1 KDM3A Lysine-specific demethylase 3A NUCB1 Nucleobindin-1 SERPINB3 Serpin B3 Nuclear ubiquitous casein and volume drawn over 2 seconds once every minute). The HBECs were transferred to exposure Eukaryotic translation initiation Far upstream element-binding ASAH1 Acid ceramidase ENO1 KHSRP NUCKS1 -dependent SERPINB4 Serpin B4 factor 5A-2 protein 2 substrate chambers and exposed at the air surface interface (ALI) for 30 minutes to a low or (1:160, ATP synthase subunit O, Pigment epithelium-derived ATP5O ENSA Alpha-endosulfine KLK11 Kallikrein-11 NUDC Nuclear migration protein nudC SERPINF1 2 Time post exposure Number of proteins mitochondrial factor Comparison Protein C and casein smoke:air), high dose (1:60, smoke:air) of cigarette whole smoke or filtered-air alone . ASL was DNA repair protein (hours) ATRN Isoform 2 of Attractin ERCC5 KLK13 Kallikrein-13 PACSIN2 kinase substrate in neurons SFN 14-3-3 protein sigma AB complementing XP-G cells protein 2 collected from the exposed cultures at 4, 12, 24 and 48 hours after exposure by adding 150 µl 4 LD vs AC 0 32 Secreted frizzled-related B2M Beta-2-microglobulin ERO1L ERO1-like protein alpha KRT10 Keratin, type I cytoskeletal 10 PGAM1 Phosphoglycerate mutase 1 SFRP1 protein 1 4 HD vs AC 0 48 phosphate buffered saline to the apical surface, incubating at 37ºC for 10 minutes before Polymeric immunoglobulin Sodium-dependent phosphate BASP1 acid soluble protein 1 EZR Ezrin KRT4 Keratin, type II cytoskeletal 4 PIGR SLC34A2 4 HD vs LD 0 39 receptor transport protein 2B removal. 12 LD vs AC 0 74 3'(2'),5'-bisphosphate Fumarylacetoacetate hydrolase BPNT1 FAHD1 KRT9 Keratin, type I cytoskeletal 9 PIP Prolactin-inducible protein SNAP23 Antileukoproteinase 12 HD vs AC 0 62 nucleotidase 1 domain-containing protein 1 12 HD vs LD 0 92 Peptidyl-prolyl cis-trans -type plasminogen Synaptosomal-associated C3 Complement C3 FKBP3 KYNU Kynureninase PLAU SNRPD3 isomerase FKBP3 activator protein 23 Proteomics analysis: ASL samples were concentrated, digested with and desalted. 24 LD vs AC 0 37 Fibrous sheath-interacting Small nuclear ribonucleoprotein C4B Complement C4-B FSIP2 LAMB2 Laminin subunit beta-2 PLUNC Protein Plunc SPP1 24 HD vs AC 0 70 protein 2 Sm D3 Processed samples were analysed by reversed phase liquid chromatography (LC) (nanoAcquity, Neutrophil gelatinase- CALM1 Calmodulin FSTL1 Follistatin-related protein 1 LCN2 PRDX1 Peroxiredoxin-1 SSBP1 Osteopontin 24 HD vs LD 0 104 associated lipocalin Succinyl-CoA ligase [GDP- Waters) using a water/acetonitrile/formic acid gradient. The LC was coupled by nanospray to an 48 LD vs AC 123 192 L-lactate dehydrogenase A CAPN1 Calpain-1 catalytic subunit FTH1 Ferritin heavy chain LDHA PRDX2 Peroxiredoxin-2 SUCLG2 forming] subunit beta, chain 48 HD vs AC 0 106 mitochondrial Orbitrap XL (Thermo Fisher) mass spectrometer. Survey scan (LC-MS) and tandem mass Thioredoxin-dependent 48 HD vs LD 177 179 Isoform 2 of Alpha-(1,3)- L-lactate dehydrogenase B Transforming growth factor CAPS Calcyphosin FUT6 LDHB PRDX3 peroxide reductase, TGFB2 fucosyltransferase chain beta-2 spectrometry (MS/MS) data were acquired in the same run. Protein identification was mitochondrial Table 1. Protein summary in treatment effect comparisons. [A] 40% significant peptides (q-value < 0.1) with CAT Catalase GAA Lysosomal alpha-glucosidase LGALS3 Galectin-3 PRDX5 Peroxiredoxin-5, mitochondrial TIMP1 Metalloproteinase inhibitor 1 accomplished using data acquired by LC-MS/MS. The MS/MS spectra were matched to the Glyceraldehyde-3-phosphate CCL20 C-C motif chemokine 20 GAPDH LGALS3BP Galectin-3-binding protein PRSS3 Trypsin-3 TNF TNF-α differential intensity (DI) in the same direction (all above or below 1). [B] 80% of the peptides with DI in the same dehydrogenase Proteasome subunit alpha corresponding peptide sequences found in the UniProt human protein database using Mascot CD109 CD109 antigen GCN1L1 Translational activator GCN1 LPLUNC1 LPLUNC1 PSMA1 TPI1 Triosephosphate isomerase direction and median DI greater than 1.4. type-1 Monocyte differentiation Lipolysis-stimulated lipoprotein Proteasome activator complex Thioredoxin reductase 1, CD14 GDF15 Growth/differentiation factor 15 LSR PSME1 TXNRD1 (Matrix Science) software. antigen CD14 receptor subunit 1 cytoplasmic Microtubule-associated protein CEL Bile salt-activated lipase GPX2 Glutathione peroxidase 2 MAP4 RORB Nuclear receptor ROR-beta TYMP Thymidine phosphorylase Lipid metabolism Translation Misc. 4 7% 3% 0% Macrophage migration UTP--glucose-1-phosphate Extracellular region CFB GRN Granulins MIF RPS12 40S ribosomal protein S12 UGP2 Nuclear organization - inhibitory factor uridylyltransferase 17% Biological process annotation: (GO) and Ingenuity Systems Pathway Analysis Transcription Vascular endothelial growth CFD Complement GSN Gelsolin MMP1 Matrix metalloproteinase-1 RPSA 40S ribosomal protein SA VEGFA 0% factor A Vacuolar protein sorting- were utilised to annotate the biological processes associated with the proteins identified in the Protein degradation CFH Complement factor H GSTP1 Glutathione S-transferase P MMP10 Stromelysin-2 S100A11 Protein S100-A11 VPS37B 7% associated protein 37B Charged multivesicular body Hydroxyacylglutathione CHMP4B HAGH MMP11 Stromelysin-3 S100A16 Protein S100-A16 XKR8 XK-related protein 8 dataset. The over-represented biological processes were identified and grouped into categories protein 4b hydrolase, mitochondrial Beta-hexosaminidase subunit CLU HEXB MMP3 Stromelysin-1 S100A2 Protein S100-A2 YWHAQ 14-3-3 protein theta and the number of proteins represented in each category determined. beta Heterogeneous nuclear CORO2B Coronin-2B HNRNPA2B1 MMP9 Matrix metalloproteinase-9 S100A6 Protein S100-A6 YWHAZ 14-3-3 protein zeta/delta ribonucleoproteins A2/B1 Inflammation - Heat shock 70 kDa protein Immune response CP Ceruloplasmin HSPA1A MUC1 Mucin-1 S100A8 Protein S100-A8 20% 1A/1B MRM assay: A quantitative mass spectrometry MRM assay was developed using synthetic Calcium ion binding 27% peptides unique to the proteins of interest (where possible 5 peptides per target) and a Signaling Table 3. List of proteins included in the MRM assay for determining the impact of cigarette smoke on the protein 0% NanoAquity HPLC (Waters) coupled to a QTRAP 5500 mass spectrometer (AB Sciex). Based on Cytoskeleton composition of the apical surface liquid. 3% the proteomics and published data, 690 peptides corresponding to 158 proteins of interest were Redox homeostasis - Cell adhesion - Wound Stress response Energy metabolism healing selected with which to develop the MRM assay. 0% 10% 6% Figure 2. Biological processes (Entrez GO) associated with top 30 ASL proteins differentially secreted Summary and conclusions Statistics: For the proteomics data, peptide peaks were aligned across all samples and following smoke exposure. differential intensity ratios were calculated using the peak intensity data. Paired t-tests were • The analysis of ASL proteins has been shown to be compatible with a proteomic approach, used to determine whether there was an effect of smoke exposure. For the MRM data, the peak with no significant interference caused by the culturing conditions. area of each transition (peptide fragment ion) was integrated. Differential intensity ratios were Development of a MRM assay • Cigarette smoke had the greatest effect on protein secretion 48 hours after a low dose then calculated and statistics were performed. All statistical test p-values were adjusted for Proteins were selected for the MRM assay based on differential secretion following smoke exposure. This is the longest time point after smoke exposure and appears to allow for a multiple testing by conversion to q-values using Storey’s method. exposure in this study, previous studies using HBECs or in the ASL/sputum in the published robust response of protein synthesis/secretion. literature. Based on the optimisation results, 624 peptides corresponding to 154 protein targets • Many of the proteins identified in this study have been previously detected in sputum samples, were included in the final assay (Table 3). The MRM assay detected 133 proteins in at least 50% indicating HBEC ASL may be used to model smoke exposure in vitro . Results of the samples from one treatment group. Over 100 proteins were differentially secreted with • Construction of a MRM assay specific for smoke exposure enables the quantitation of over 100 Characterisation of the ASL proteome most of those proteins being up-regulated 48 hours after low dose smoke exposure when bronchial epithelial cell proteins in a single sample which may be used to further investigate Proteomic analysis of the ASL resulted in the identification of 2,414 unique peptide sequences compared to air control. Qualification results were highly consistent with previous proteomic the effects of smoke exposure in both in vitro ASL and in vivo sputum samples. corresponding to 487 ASL proteins with high confidence and related to key biological processes. analysis, as judged by the DI values. As in previous results most of the significant changes were Among the proteins identified were the mucins, MUC1, MUC4, MUC5B, MUC16, and MUC20. observed in the 48 hour low dose vs. air control comparison. Additionally, there were significant References Non-mucin proteins included those from the cytoskeletal, signalling, proteolysis, calcium binding changes in the 48 hour high dose vs. air control comparison, although the changes were slightly 1. Gray TE, et al ., 1996, Am J Respir Cell Mol Biol , 14:104-112 and immune system pathways (Figure 1). lower in magnitude. This drop in magnitude may be an indication of toxicity. 2. Phillips J, et al ., 2005, Altern Lab Anim, 33(3):239-48 www.bat-science.com