PROTEOMICS ANALYSIS OF PRO-INFLAMMATORY CYTOKINE-STIMULATED HUMAN LUNG FIBROBLASTS AND BRONCHIAL EPITHELIAL CELLS LIAO WUPENG (BSc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS This thesis is the product of much more than five years of hard work, worries and concerns, changing minds and directions, and a great amount of external assistance First of all, I would like to gratefully acknowledge the enthusiastic supervision of Dr Fred Wong during this work I will never forget our numerous discussions, his passion for research, his stringent scientific attitude, and his perseverant spirits, which will benefit me throughout my career and life Without help and suggestions from my colleagues and friends, Jasmine, Hui Hwa, Zhao Jing, Bao Zhang, Cheng Chang, Kai Ling, Richard Betts, Khai Nee, Zhu Hua, Chui Hong, I would never have finished these laborious work smoothly Thanks to Ms Wang Xianhui and Ms Michelle Lim from the Protein and Proteomics Center for their earnest service for protein identification I would also like to express my sincere appreciation to the National University of Singapore for providing me years of scholarship, and the staff and students from the Department of Pharmacology Finally, I am forever indebted to my dearest parents and wife for their understanding, endless love and encouragement when it was most required LIAO Wupeng Oct 2008 ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii SUMMARY viii LIST OF TABLES xi LIST OF FIGURES xii LIST OF ABBREVIATIONS xiv LIST OF PUBLICATIONS AND CONFERENCE PAPERS xvii 1 INTRODUCTION 1.1 Lung Structural Cells in Pulmonary Immune Response 1.2 Lung Structural Cells in Airway Inflammation 1.2.1 Epithelial cells 1.2.2 Fibroblasts 1.3 Lung Structural Cells in Airway Tissue Repair 1.4 Tumor Necrosis Factor (TNF)-α 13 1.4.1 Cellular sources of TNF-α 14 1.4.2 TNF-α receptors 14 1.4.3 TNF-α cellular signaling 14 1.4.4 Role of TNF-α in pulmonary pathophysiology 18 1.4.5 TNF-α as a therapeutic target for pulmonary diseases 19 1.5 Interferon (IFN)-γ 20 iii 1.5.1 Cellular sources of IFN-γ 20 1.5.2 IFN-γ receptors 21 1.5.3 IFN-γ cellular signaling 21 1.5.4 Role of IFN-γ in pulmonary pathophysiology 24 1.6 Proteomics 1.6.1 Separation of proteins 26 27 1.6.1.1 Two-dimensional gel electrophoresis 28 1.6.1.2 Alternatives to electrophoresis 30 1.6.2 Mass spectrometry (MS) 33 1.6.2.1 Principles of MS 33 1.6.2.2 Types of mass spectrometers 38 1.6.3 Protein database and protein identification 40 1.6.4 Application of proteomics 42 1.6.4.1 Expression proteomics 43 1.6.4.2 Structural proteomics 43 1.6.4.3 Functional proteomics 44 1.7 Rationale and Purpose of Study METHODS AND MATERIALS 44 47 2.1 Cell culture 48 2.2 Proteomics analysis 49 2.2.1 Sample preparation 51 2.2.2 Two-dimensional gel electrophoresis 51 iv 2.2.3 Silver staining and imaging 53 2.2.4 Image analysis 53 2.2.5 In-gel digestion and MALDI-TOF MS or MS/MS analysis 54 2.3 RT-PCR 2.4 Real-time PCR 58 2.5 Antibodies and immunoblots 58 2.6 MTT assay 59 2.7 Transfection with antisense oligodeoxynucleotides 60 2.8 Transfection with siRNA 61 2.9 Lipopolysaccharide-induced acute lung injury in mice 62 2.10 Influenza A virus stock and inoculation of mice 63 2.11 Acute asthma model 64 2.12 Bronchoalveolar lavage fluid cell counts 64 2.13 Enzyme-linked immunosorbent assay (ELISA) 64 2.14 Statistical methods 65 56 DENDRITIC CELL-DERIVED IFN-γ-INDUCED PROTEIN (DCIP) MEDIATES TNF-α STIMULATION OF NHLF 3.1 Results 66 67 3.1.1 Proteomics analysis of DCIP induction by TNF-α 67 3.1.2 Verification of proteomics data by RT-PCR 75 3.1.3 Regulation of TNF-α-induced DCIP expression by IRF-1 77 3.1.4 Characterization of DCIP-selective ASO 79 v 3.1.5 Regulation of TNF-α-responsive gene targets by DCIP 81 3.1.6 Up-regulation of DCIP by pro-inflammatory cytokines 83 3.1.7 Up-regulation of MG11 in a murine acute lung injury model 85 3.1.8 Up-regulation of MG11 in an influenza A virus-infected lung 87 injury mouse model 3.2 Discussion 90 3.2.1 91 3.2.2 Remodeling related protein 93 3.2.3 Function unknown protein DCIP 95 PROTEOMICS STUDIES OF TNF-α AND/OR IFN-γ-STIMULATED NHBE Cell protective protein 100 4.1 Results 101 4.1.1 Proteomics analysis of apoL2 induction by IFN-γ 101 4.1.2 Up-regulation of the whole apoL family members by IFN-γ 109 4.1.3 Selective induction of apoL2 by IFN-γ 111 4.1.4 siRNA characterization 113 4.1.5 The effect of apoL2 siRNA on TNF-α or IFN-γ-induced 115 cytotoxicity 4.1.6 Up-regulation of apoLs in an influenza A virus-infected lung 118 injury mouse model 4.2 Discussion 121 4.2.1 Apolipoproteins 122 4.2.2 NDRG-1 128 4.2.3 SERPINB1 129 vi 4.2.4 Tapasin 131 4.2.5 Tryptophanyl-tRNA synthetase 132 CONCLUSIONS 135 REFERENCES 139 APPENDIX 166 vii SUMMARY Airway resident cells like epithelia and fibroblasts function not only as physical barriers for tissue integrity, but also regulators of airway pathophysiology Pulmonary diseases such as asthma, chronic obstructive pulmonary disease, acute lung injury and respiratory viral infection are associated with airway resident cell activation, contributing to airway immunity, inflammation and remodeling The present study investigated global protein profilings of normal human bronchial epithelial cells (NHBE) and normal human lung fibroblasts (NHLF) stimulated with pro-inflammatory cytokines tumor necrosis factor (TNF)-α and/or interferon (IFN)-γ Total proteins from cell lysates and culture media were separated by two-dimensional gel electrophoresis (2-DE), and differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS TNF-α was found for the first time to alter the expression levels of myxovirus resistance protein A (MxA), interferon-stimulated gene (ISG)-15, plasminogen activator inhibitor (PAI)-2, lysyl hydroxylase (isoform a) and prolyl 4-hydroxylase (α subunit) in human lung fibroblasts In particular, dendritic cell-derived IFN-γ-induced protein (DCIP) was up-regulated by TNF-α in lung fibroblasts, and its biological function is at present unknown In addition, we found that TNF-α-induced DCIP expression is dependent on the transcription factor interferon regulatory factor viii (IRF)-1 DCIP-selective antisense oligodeoxynucleotide (ASO) inhibited the expression of TNF-α-responsive gene targets including VCAM-1, ICAM-1, IL-6, IL-8, IP-10 and thymic stromal lymphopoietin Lastly, in a LPS-induced and an influenza A virus-infected lung injury mouse model, DCIP mRNA level was elevated together with that of TNF-α NHBE cells treated with either TNF-α or IFN-γ alone did not show any significant cell loss even after 72 h To our surprise, IFN-γ showed more substantial effects on the proteome alteration of NHBE cells as compared with TNF-α Several proteins were identified for the first time to be altered by IFN-γ in NHBE cells in our proteomics study, which include metastasis suppressor N-myc downstream-regulated gene-1 (NDRG-1), serine protease inhibitor, clade B, member (SERPINB1), cytoskeleton protein keratin10, tropomyosin isoform and γ-actin Interestingly, apolipoprotein L2 (apoL2) was found for the first time ever to be up-regulated by IFN-γ in epithelial cells In addition, we found almost the whole apoL family members except apoL5 were dramatically up-regulated by IFN-γ using RT-PCR analysis Furthermore, bronchial epithelial cell number significantly decreased in response to TNF-α or IFN-γ, respectively, only when the cells were pretreated with apoL2 siRNA, but not apoL1, or siRNAs Lastly, we verified the increased mRNA expressions of mouse apoL2, and 6, SERPINB1a, NDRG-1, WARS and tapasin in an influenza A virus-infected mouse lung injury model ix In summary, these studies not only reveal that proteomics is an efficient way to study the underlying molecular mechanisms after cytokine stimulation, but also demonstrate for the first time that DCIP is up-regulated by TNF-α and mediates TNF-α stimulation of human lung fibroblasts; and apoL2 is stimulated by IFN-γ, which in turn affects TNF-α or IFN-γ regulation of human bronchial epithelial cell proliferation, leading to airway immunity, inflammation and remodeling x Mascot Search Results Protein View Match to: gi|13489087 Score: 538 Expect: 6.5e-048 serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member [Homo sapiens] Nominal mass (Mr): 42829; Calculated pI value: 5.90 NCBI BLAST search of gi|13489087 against nr Unformatted sequence string for pasting into other applications Fixed modifications: Carbamidomethyl (C) Variable modifications: Oxidation (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 52% Matched peptides shown in Bold Red MEQLSSANTR FALDLFLALS ENNPAGNIFI SPFSISSAMA MVFLGTRGNT 51 AAQLSKTFHF NTVEEVHSRF QSLNADINKR GASYILKLAN RLYGEKTYNF 101 LPEFLVSTQK TYGADLASVD FQHASEDARK TINQWVKGQT EGKIPELLAS 151 GMVDNMTKLV LVNAIYFKGN WKDKFMKEAT TNAPFRLNKK DRKTVKMMYQ 201 KKKFAYGYIE DLKCRVLELP YQGEELSMVI LLPDDIEDES TGLKKIEEQL 251 TLEKLHEWTK PENLDFIEVN VSLPRFKLEE SYTLNSDLAR LGVQDLFNSS 301 KADLSGMSGA RDIFISKIVH KSFVEVNEEG TEAAAATAGI ATFCMLMPEE 351 NFTADHPFLF FIRHNSSGSI LFLGRFSSP Start - End - 10 57 - 69 57 - 69 70 - 80 70 - 80 97 - 110 97 - 110 111 - 129 score 68) 111 - 129 match) 111 - 130 match) 130 - 137 131 - 137 131 - 137 138 - 158 (No match) 144 - 158 144 - 158 (M) (No match) 178 - 186 178 - 186 197 - 202 match) 203 - 213 204 - 213 204 - 213 245 - 254 255 - 275 (No match) Observed Mr(expt) Mr(calc) Delta Miss Sequence 1005.51 1004.51 1004.49 1602.84 1601.84 1601.76 1602.84 1601.84 1601.76 1305.76 1304.75 1304.68 1305.76 1304.75 1304.68 1686.95 1685.94 1685.87 1686.95 1685.94 1685.87 2053.02 2052.02 2051.92 0.02 0.08 0.08 0.07 0.07 0.08 0.08 0.10 M.EQLSSANTR.F (No match) K.TFHFNTVEEVHSR.F (Ions score 27) K.TFHFNTVEEVHSR.F (No match) R.FQSLNADINKR.G (Ions score 32) R.FQSLNADINKR.G (No match) K.TYNFLPEFLVSTQK.T (Ions score 40) K.TYNFLPEFLVSTQK.T (No match) K.TYGADLASVDFQHASEDAR.K (Ions 2053.02 2052.02 2051.92 0.10 K.TYGADLASVDFQHASEDAR.K (No 2181.13 2180.12 2180.01 0.11 K.TYGADLASVDFQHASEDARK.T (No 1016.64 888.54 888.54 2219.21 1015.64 887.53 887.53 2218.20 1015.58 887.49 887.49 2218.10 0.05 0.05 0.05 0.10 R.KTINQWVK.G (No match) K.TINQWVK.G (Ions score 39) K.TINQWVK.G (No match) K.GQTEGKIPELLASGMVDNMTK.L 1618.89 1634.88 1617.88 1633.87 1617.81 1633.81 0.07 0.07 0 1006.55 1006.55 860.32 1005.54 1005.54 859.31 1005.49 1005.49 859.39 0.06 0.06 -0.08 1346.76 1218.66 1218.66 1230.75 2536.44 1345.75 1217.66 1217.66 1229.75 2535.44 1345.69 1217.60 1217.60 1229.69 2535.31 0.06 0.06 0.06 0.06 0.12 K.IPELLASGMVDNMTK.L (No match) K.IPELLASGMVDNMTK.L Oxidation K.EATTNAPFR.L (Ions score 19) K.EATTNAPFR.L (No match) K.MMYQKK.K Oxidation (M) (No 0 K.KFAYGYIEDLK.C (No match) K.FAYGYIEDLK.C (Ions score 54) K.FAYGYIEDLK.C (No match) K.KIEEQLTLEK.L (No match) K.LHEWTKPENLDFIEVNVSLPR.F 176 276 - 290 1785.99 1784.98 1784.89 0.08 R.FKLEESYTLNSDLAR.L (Ions score 276 - 290 278 - 290 291 - 301 302 - 311 302 - 311 match) 364 - 375 364 - 375 1785.99 1510.82 1207.69 964.51 980.45 1784.98 1509.81 1206.69 963.50 979.45 1784.89 1509.73 1206.62 963.44 979.44 0.08 0.08 0.06 0.06 0.01 R.FKLEESYTLNSDLAR.L (No match) K.LEESYTLNSDLAR.L (No match) R.LGVQDLFNSSK.A (No match) K.ADLSGMSGAR.D (No match) K.ADLSGMSGAR.D Oxidation (M) (No 1287.75 1287.75 1286.74 1286.74 1286.67 1286.67 0.07 0.07 0 36) R.HNSSGSILFLGR.F (Ions score 45) R.HNSSGSILFLGR.F (No match) 100 1602.8438 1287.7463 4700 Reflector Spec #1 MC[BP = 1287.8, 7601] 7600.8 90 80 799.0 2536.4404 2211.2021 2053.0227 2134.1196 1686.9490 1785.9857 1305.7561 1441.8 2035.0219 10 1708.9353 1768.9738 1813.9938 20 1516.8102 1558.8345 1618.8877 30 1440.7601 40 1102.6564 1144.6768 1207.6935 1270.7258 1333.7551 50 1218.6638 1178.6107 60 809.3004 842.5557 863.5002 888.5401 920.5321 964.5078 1006.5504 1011.6906 1016.6423 % Intensity 70 2084.6 2727.4 3370.2 4013.0 Mass (m/z) 177 Mascot Search Results Protein View Match to: gi|47419916 Score: 350 Expect: 4e-029 tryptophanyl-tRNA synthetase isoform a [Homo sapiens] Nominal mass (Mr): 53480; Calculated pI value: 5.83 NCBI BLAST search of gi|47419916 against nr Unformatted sequence string for pasting into other applications Fixed modifications: Carboxymethyl (C) Variable modifications: Oxidation (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 48% Matched peptides shown in Bold Red MPNSEPASLL ELFNSIATQG ELVRSLKAGN ASKDEIDSAV KMLVSLKMSY 51 KAAAGEDYKA DCPPGNPAPT SNHGPDATEA EEDFVDPWTV QTSSAKGIDY 101 DKLIVRFGSS KIDKELINRI ERATGQRPHH FLRRGIFFSH RDMNQVLDAY 151 ENKKPFYLYT GRGPSSEAMH VGHLIPFIFT KWLQDVFNVP LVIQMTDDEK 201 YLWKDLTLDQ AYSYAVENAK DIIACGFDIN KTFIFSDLDY MGMSSGFYKN 251 VVKIQKHVTF NQVKGIFGFT DSDCIGKISF PAIQAAPSFS NSFPQIFRDR 301 TDIQCLIPCA IDQDPYFRMT RDVAPRIGYP KPALLHSTFF PALQGAQTKM 351 SASDPNSSIF LTDTAKQIKT KVNKHAFSGG RDTIEEHRQF GGNCDVDVSF 401 MYLTFFLEDD DKLEQIRKDY TSGAMLTGEL KKALIEVLQP LIAEHQARRK 451 EVTDEIVKEF MTPRKLSFDF Q Start - End Observed Mr(expt) Mr(calc) 28 - 41 97 - 106 97 - 106 112 - 119 112 - 119 123 - 133 123 - 133 134 - 141 134 - 141 135 - 141 135 - 141 142 - 153 142 - 153 (No match) 142 - 162 (No match) 154 - 162 154 - 162 163 - 181 match) 254 - 264 257 - 264 257 - 264 278 - 298 match) 327 - 349 (No match) 350 - 366 match) 375 - 388 382 - 388 419 - 432 Delta Miss Sequence 1404.71 1191.69 1191.69 1000.60 1000.60 1319.71 1319.71 1019.56 1019.56 863.46 863.46 1439.67 1455.69 1403.70 1190.68 1190.68 999.59 999.59 1318.70 1318.70 1018.55 1018.55 862.45 862.45 1438.66 1454.68 1403.69 1190.67 1190.67 999.57 999.57 1318.70 1318.70 1018.55 1018.55 862.44 862.44 1438.64 1454.63 0.01 0.02 0.02 0.02 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.02 0.05 1 1 0 1 0 0 K.AGNASKDEIDSAVK.M (No match) K.GIDYDKLIVR.F (Ions score 24) K.GIDYDKLIVR.F (No match) K.IDKELINR.I (Ions score 35) K.IDKELINR.I (No match) R.ATGQRPHHFLR.R (No match) R.ATGQRPHHFLR.R (No match) R.RGIFFSHR.D (Ions score 16) R.RGIFFSHR.D (No match) R.GIFFSHR.D (Ions score 23) R.GIFFSHR.D (No match) R.DMNQVLDAYENK.K (No match) R.DMNQVLDAYENK.K Oxidation (M) 2565.33 2564.32 2564.24 0.08 R.DMNQVLDAYENKKPFYLYTGR.G 1144.62 1144.62 2068.15 1143.62 1143.62 2067.14 1143.61 1143.61 2067.06 0.01 0.01 0.08 0 K.KPFYLYTGR.G (Ions score 26) K.KPFYLYTGR.G (No match) R.GPSSEAMHVGHLIPFIFTK.W (No 1341.69 972.53 972.53 2325.32 1340.69 971.52 971.52 2324.31 1340.76 971.52 971.52 2324.20 -0.07 0.00 0.00 0.12 2485.44 2484.43 2484.35 0.08 R.IGYPKPALLHSTFFPALQGAQTK.M 1784.87 1783.86 1783.83 0.03 K.MSASDPNSSIFLTDTAK.Q (No 1611.79 899.43 1513.81 1610.78 898.43 1512.80 1610.75 898.41 1512.75 0.03 0.01 0.05 K.HAFSGGRDTIEEHR.Q (No match) R.DTIEEHR.Q (No match) K.DYTSGAMLTGELKK.A (No match) K.IQKHVTFNQVK.G (No match) K.HVTFNQVK.G (Ions score 21) K.HVTFNQVK.G (No match) K.ISFPAIQAAPSFSNSFPQIFR.D (No 178 433 - 448 1801.06 1800.05 1800.03 0.03 K.ALIEVLQPLIAEHQAR.R (Ions score 433 - 448 451 - 464 465 - 471 1801.06 1800.05 1800.03 1693.88 1692.88 1692.84 884.45 883.45 883.44 0.03 0.04 0.00 K.ALIEVLQPLIAEHQAR.R (No match) K.EVTDEIVKEFMTPR.K (No match) R.KLSFDFQ.- (No match) 11) 1319.7100 4700 Reflector Spec #1 MC[BP = 1319.7, 33672] 100 3.4E+4 863.4561 90 80 60 1441.8 2485.4470 2084.6 2565.3252 2325.3213 2225.1028 2068.1462 1801.0615 1929.1996 1611.7938 799.0 1693.8856 10 1302.7362 1357.6685 1416.6553 1475.8346 20 884.4526 30 972.5304 994.5232 40 1124.55441144.6226 1191.6890 50 806.4452 % Intensity 70 2727.4 3370.2 4013.0 Mass (m/z) 179 Mascot Search Results Protein View Match to: gi|47419918 Score: 316 Expect: 1e-025 tryptophanyl-tRNA synthetase isoform b [Homo sapiens] Nominal mass (Mr): 49163; Calculated pI value: 6.03 NCBI BLAST search of gi|47419918 against nr Unformatted sequence string for pasting into other applications Fixed modifications: Carbamidomethyl (C) Variable modifications: Oxidation (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 39% Matched peptides shown in Bold Red MLVSLKMSYK AAAGEDYKAD CPPGNPAPTS NHGPDATEAE EDFVDPWTVQ 51 TSSAKGIDYD KLIVRFGSSK IDKELINRIE RATGQRPHHF LRRGIFFSHR 101 DMNQVLDAYE NKKPFYLYTG RGPSSEAMHV GHLIPFIFTK WLQDVFNVPL 151 VIQMTDDEKY LWKDLTLDQA YSYAVENAKD IIACGFDINK TFIFSDLDYM 201 GMSSGFYKNV VKIQKHVTFN QVKGIFGFTD SDCIGKISFP AIQAAPSFSN 251 SFPQIFRDRT DIQCLIPCAI DQDPYFRMTR DVAPRIGYPK PALLHSTFFP 301 ALQGAQTKMS ASDPNSSIFL TDTAKQIKTK VNKHAFSGGR DTIEEHRQFG 351 GNCDVDVSFM YLTFFLEDDD KLEQIRKDYT SGAMLTGELK KALIEVLQPL 401 IAEHQARRKE VTDEIVKEFM TPRKLSFDFQ Start - End Observed Mr(expt) Mr(calc) 11 - 18 56 - 65 56 - 65 71 - 78 82 - 92 82 - 92 93 - 100 93 - 100 94 - 100 94 - 100 113 - 121 113 - 121 122 - 140 match) 180 - 190 213 - 223 216 - 223 216 - 223 224 - 236 224 - 236 286 - 308 (No match) 309 - 325 match) 392 - 407 6) 392 - 407 424 - 430 Delta Miss Sequence 824.32 1191.74 1191.74 1000.64 1319.77 1319.77 1019.61 1019.61 863.50 863.50 1144.68 1144.68 2068.16 823.31 1190.73 1190.73 999.63 1318.77 1318.77 1018.60 1018.60 862.49 862.49 1143.67 1143.67 2067.15 823.37 1190.67 1190.67 999.57 1318.70 1318.70 1018.55 1018.55 862.44 862.44 1143.61 1143.61 2067.06 -0.06 0.07 0.07 0.06 0.07 0.07 0.06 0.06 0.05 0.05 0.06 0.06 0.09 1 0 1 0 0 K.AAAGEDYK.A (No match) K.GIDYDKLIVR.F (Ions score 27) K.GIDYDKLIVR.F (No match) K.IDKELINR.I (No match) R.ATGQRPHHFLR.R (No match) R.ATGQRPHHFLR.R (No match) R.RGIFFSHR.D (Ions score 17) R.RGIFFSHR.D (No match) R.GIFFSHR.D (Ions score 17) R.GIFFSHR.D (No match) K.KPFYLYTGR.G (Ions score 19) K.KPFYLYTGR.G (No match) R.GPSSEAMHVGHLIPFIFTK.W (No 1265.69 1341.76 972.58 972.58 1416.72 1416.72 2485.49 1264.68 1340.75 971.57 971.57 1415.71 1415.71 2484.48 1264.61 1340.76 971.52 971.52 1415.64 1415.64 2484.35 0.07 -0.00 0.06 0.06 0.07 0.07 0.13 0 0 K.DIIACGFDINK.T (No match) K.IQKHVTFNQVK.G (No match) K.HVTFNQVK.G (Ions score 58) K.HVTFNQVK.G (No match) K.GIFGFTDSDCIGK.I (Ions score 49) K.GIFGFTDSDCIGK.I (No match) R.IGYPKPALLHSTFFPALQGAQTK.M 1784.92 1783.92 1783.83 0.09 K.MSASDPNSSIFLTDTAK.Q (No 1801.12 1800.11 1800.03 0.09 K.ALIEVLQPLIAEHQAR.R (Ions score 1801.12 884.50 1800.11 883.49 1800.03 883.44 0.09 0.05 K.ALIEVLQPLIAEHQAR.R (No match) R.KLSFDFQ.- (No match) 180 30 20 10 799.0 1441.8 2084.6 2565.3682 2485.4836 2211.2004 2068.1543 1927.1117 1978.1377 1794.9053 1801.1179 1693.9335 1611.8435 1513.8265 40 1144.6780 1319.7738 90 1265.6902 1325.7264 1376.7892 1416.7191 1439.7217 863.4998 100 1124.6034 1182.6689 1191.7396 806.4808 842.5585 855.1072 902.3679 954.3625 972.5818 1011.6937 1019.6087 1054.5791 % Intensity 4700 Reflector Spec #1 MC[BP = 863.5, 6326] 6325.9 80 70 60 50 Mass (m/z) 2727.4 3370.2 4013.0 181 Mascot Search Results Protein View Match to: gi|13325156 Score: 246 Expect: 9.9e-019 Apolipoprotein L, [Homo sapiens] Nominal mass (Mr): 37113; Calculated pI value: 6.28 NCBI BLAST search of gi|13325156 against nr Unformatted sequence string for pasting into other applications Fixed modifications: Carboxymethyl (C) Variable modifications: Oxidation (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 24% Matched peptides shown in Bold Red MNPESSIFIE DYLKYFQDQV SRENLLQLLT DDEAWNGFVA AAELPRDEAD 51 ELRKALNKLA SHMVMKDKNR HDKDQQHRQW FLKEFPRLKR ELEDHIRKLR 101 ALAEEVEQVH RGTTIANVVS NSVGTTSGIL TLLGLGLAPF TEGISFVLLD 151 TGMGLGAAAA VAGITCSVVE LVNKLRARAQ ARNLDQSGTN VAKVMKEFVG 201 GNTPNVLTLV DNWYQVTQGI GRNIRAIRRA RANPQLGAYA PPPHVIGRIS 251 AEGGEQVERV VEGPAQAMSR GTMIVGAATG GILLLLDVVS LAYESKHLLE 301 GAKSESAEEL KKRAQELEGK LNFLTKIHEM LQPGQDQ Start - End Observed Mr(expt) Mr(calc) Delta Miss Sequence 15 - 22 15 - 22 47 - 53 91 - 97 91 - 97 91 - 98 101 - 111 101 - 111 232 - 248 1042.50 1042.50 847.30 911.47 911.47 1039.57 1280.67 1280.67 1757.98 1041.49 1041.49 846.30 910.46 910.46 1038.56 1279.67 1279.67 1756.98 1041.49 1041.49 846.37 910.45 910.45 1038.55 1279.65 1279.65 1756.94 0.01 0.01 -0.08 0.01 0.01 0.02 0.01 0.01 0.04 0 0 0 K.YFQDQVSR.E (Ions score 21) K.YFQDQVSR.E (No match) R.DEADELR.K (No match) R.ELEDHIR.K (Ions score 39) R.ELEDHIR.K (No match) R.ELEDHIRK.L (No match) R.ALAEEVEQVHR.G (Ions score 27) R.ALAEEVEQVHR.G (No match) R.ANPQLGAYAPPPHVIGR.I (Ions score 232 - 248 match) 249 - 259 249 - 259 260 - 270 260 - 270 260 - 270 (Ions score 6) 260 - 270 match) 327 - 337 327 - 337 327 - 337 match) 1757.98 1756.98 1756.94 0.04 R.ANPQLGAYAPPPHVIGR.I 1174.59 1174.59 1144.59 1144.59 1160.58 1173.58 1173.58 1143.58 1143.58 1159.57 1173.56 1173.56 1143.57 1143.57 1159.57 0.02 0.02 0.01 0.01 0.01 0 0 R.ISAEGGEQVER.V (Ions score 19) R.ISAEGGEQVER.V (No match) R.VVEGPAQAMSR.G (Ions score 42) R.VVEGPAQAMSR.G (No match) R.VVEGPAQAMSR.G Oxidation (M) 1160.58 1159.57 1159.57 0.01 R.VVEGPAQAMSR.G 1295.61 1295.61 1311.60 1294.60 1294.60 1310.59 1294.60 1294.60 1310.59 0.00 0.00 -0.00 16) (No Oxidation (M) (No K.IHEMLQPGQDQ.- (Ions score 21) K.IHEMLQPGQDQ.- (No match) K.IHEMLQPGQDQ.- Oxidation (M) (No 182 20 10 70 60 50 40 30 799.0 1727.8550 1160.5791 1174.5861 1144.5922 1225.1639 1280.6752 1295.6071 90 80 1757.9833 1042.5017 100 994.1531 1066.4659 842.5195 911.4700 % Intensity 4700 Reflector Spec #1 MC[BP = 1042.5, 7073] 7073.3 1441.8 2084.6 Mass (m/z) 2727.4 3370.2 4013.0 183 Mascot Search Results Protein View Match to: gi|27436897 Score: 96 Expect: 0.0012 tapasin isoform precursor [Homo sapiens] Nominal mass (Mr): 44145; Calculated pI value: 6.35 NCBI BLAST search of gi|27436897 against nr Unformatted sequence string for pasting into other applications Fixed modifications: Carbamidomethyl (C) Variable modifications: Oxidation (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 21% Matched peptides shown in Bold Red MKSLSLLLAV ALGLATAVSA GPAVIECWFV EDASGKGLAK RPGALLLRQG 51 PGEPPPRPDL DPELYLSVHD PAGALQAAFR RYPRGAPAPH CEMSRFVPLP 101 ASAKWASGLT PAQNCPRALD GAWLMVSISS PVLSLSSLLR PQPEPQQEPV 151 LITMATVVLT VLTHTPAPRV RLGQDALLDL SFAYMPPTSE AASSLAPGPP 201 PFGLEWRRQH LGKGHLLLAA TPGLNGQMPA AQEGAVAFAA WDDDEPWGPW 251 TGNGTFWLPT VQPFQEGTYL ATIHLPYLQG QVTLELAVYK PPKVSLMPAT 301 LARAAPGEAP PELLCLVSHF YPSGGLEVEW ELRGGPGGRS QKAEGQRWLS 351 ALRHHSDGSV SLSGHLQPPP VTTEQHGARY ACRIHHPSLP ASGRSAEVTL 401 EVAGKSWELC GI Start - End Observed Mr(expt) Mr(calc) Delta 41 - 48 895.59 894.58 894.58 0.00 41 - 48 895.59 894.58 894.58 0.00 85 - 95 1212.53 1211.52 1211.52 0.01 96 - 104 929.56 928.55 928.54 0.01 105 - 117 1457.70 1456.70 1456.69 0.01 105 - 117 1457.70 1456.70 1456.69 0.01 294 - 303 1058.62 1057.62 1057.60 0.02 294 - 303 1074.61 1073.61 1073.59 0.02 match) 354 - 379 2731.34 2730.34 2730.32 0.01 R.HHSDGSVSLSGHLQPPPVTTEQHGAR.Y (No match) 384 - 394 1171.64 1170.63 1170.63 0.01 384 - 394 1171.64 1170.63 1170.63 0.01 Miss Sequence 0 0 0 0 K.RPGALLLR.Q (Ions score 16) K.RPGALLLR.Q (No match) R.GAPAPHCEMSR.F (No match) R.FVPLPASAK.W (No match) K.WASGLTPAQNCPR.A (Ions score 15) K.WASGLTPAQNCPR.A (No match) K.VSLMPATLAR.A (No match) K.VSLMPATLAR.A Oxidation (M) (No 0 R.IHHPSLPASGR.S R.IHHPSLPASGR.S (Ions score 17) (No match) 184 50 30 20 799.0 1441.8 1171.6412 1225.1526 1457.7035 1471.71401493.7429 10 2084.6 2731.3394 2383.9592 2211.1079 1707.7855 60 895.5879 825.1112 994.1457 80 1051.7112 1107.5541 1165.7412 1212.5303 1267.1283 1320.5858 1365.6536 40 882.5704 70 929.5580 842.5159 100 807.3068 % Intensity 4700 Reflector Spec #1 MC[BP = 842.5, 546] 545.9 90 Mass (m/z) 2727.4 3370.2 4013.0 185 Mascot Search Results Protein View Match to: gi|27436893 Score: 225 Expect: 1.4e-016 tapasin isoform precursor [Homo sapiens] Nominal mass (Mr): 47883; Calculated pI value: 6.50 NCBI BLAST search of gi|27436893 against nr Unformatted sequence string for pasting into other applications Fixed modifications: Carbamidomethyl (C) Variable modifications: Oxidation (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 22% Matched peptides shown in Bold Red MKSLSLLLAV ALGLATAVSA GPAVIECWFV EDASGKGLAK RPGALLLRQG 51 PGEPPPRPDL DPELYLSVHD PAGALQAAFR RYPRGAPAPH CEMSRFVPLP 101 ASAKWASGLT PAQNCPRALD GAWLMVSISS PVLSLSSLLR PQPEPQQEPV 151 LITMATVVLT VLTHTPAPRV RLGQDALLDL SFAYMPPTSE AASSLAPGPP 201 PFGLEWRRQH LGKGHLLLAA TPGLNGQMPA AQEGAVAFAA WDDDEPWGPW 251 TGNGTFWLPT VQPFQEGTYL ATIHLPYLQG QVTLELAVYK PPKVSLMPAT 301 LARAAPGEAP PELLCLVSHF YPSGGLEVEW ELRGGPGGRS QKAEGQRWLS 351 ALRHHSDGSV SLSGHLQPPP VTTEQHGARY ACRIHHPSLP ASGRSAEVTL 401 EVAGLSGPSL EDSVGLFLSA FLLLGLFKAL GWAAVYLSTC KDSKKKAE Start - End Observed Mr(expt) Mr(calc) Delta Miss Sequence 41 - 48 895.60 894.59 894.58 0.01 41 - 48 895.60 894.59 894.58 0.01 85 - 95 1212.54 1211.54 1211.52 0.02 85 - 95 1212.54 1211.54 1211.52 0.02 85 - 95 1228.54 1227.53 1227.51 0.02 match) 96 - 104 929.57 928.56 928.54 0.02 105 - 117 1457.72 1456.71 1456.69 0.02 105 - 117 1457.72 1456.71 1456.69 0.02 294 - 303 1058.63 1057.62 1057.60 0.02 294 - 303 1074.62 1073.61 1073.59 0.02 match) 354 - 379 2731.35 2730.35 2730.32 0.03 R.HHSDGSVSLSGHLQPPPVTTEQHGAR.Y (Ions score 63) 354 - 379 2731.35 2730.35 2730.32 0.03 R.HHSDGSVSLSGHLQPPPVTTEQHGAR.Y (No match) 384 - 394 1171.65 1170.64 1170.63 0.02 384 - 394 1171.65 1170.64 1170.63 0.02 429 - 441 1439.76 1438.75 1438.73 0.02 K.RPGALLLR.Q (Ions score 23) K.RPGALLLR.Q (No match) R.GAPAPHCEMSR.F (Ions score 28) R.GAPAPHCEMSR.F (No match) R.GAPAPHCEMSR.F Oxidation (M) (No R.FVPLPASAK.W (No match) K.WASGLTPAQNCPR.A (Ions score 38) K.WASGLTPAQNCPR.A (No match) K.VSLMPATLAR.A (No match) K.VSLMPATLAR.A Oxidation (M) (No R.IHHPSLPASGR.S (Ions score 31) R.IHHPSLPASGR.S (No match) K.ALGWAAVYLSTCK.D (No match) 186 20 10 799.0 1441.8 2084.6 2731.3572 2383.9639 2300.2261 2211.1196 1940.9570 1794.8414 1666.0256 1707.8031 30 1171.6505 1457.7152 40 1471.7322 1522.8627 50 1320.6036 1366.6902 60 895.5966 842.5240 100 822.2748 868.5612 885.5400 929.5643 996.6113 1045.5880 1109.5294 1151.7408 1212.5433 % Intensity 4700 Reflector Spec #1 MC[BP = 842.5, 2855] 2854.9 90 80 70 Mass (m/z) 2727.4 3370.2 4013.0 187 APPENDIX II List of Reagents and Solutions Sample Lysis Buffer (2-DE, -20oC) Urea Thiourea CHAPS Pharmalyte DTT Cocktail RNAase DNAase 8M 2M 4% 0.8% 65 mM 20 µl/ml µg/ml 20 µg/ml Rehydration Solution, pH 4-7 (-20oC) Urea CHAPS IPG buffer pH 4-7 1% bromophen blue DTT 8M 2% 0.5% 0.0005% 0.28 % Rehydration Solution, pH 6-11 (-20oC) Urea Thiourea CHAPS Isopropanol Glycerol IPG buffer pH 6-11 1% bromophen blue DTT 7M 2M 4% 10% 5% 0.5% 0.0005% 2.5 % Running Gel (2-DE, 12%, 80 ml/gel) 1.5 M Tris-Cl, pH8.8 10% SDS 30% Acrylamide (37.5:1) 10% APS TEMED Milli-Q H2O 20.0 ml 0.8 ml 32.0 ml 0.4 ml 40.0 µl 26.8 ml Equilibration Buffer (-20oC) 1.5 M Tris-HCl, pH 8.8 Urea Glycerol SDS 50 mM 6M 30% 2% 10 × Electrophoresis Buffer (R.T.) Tris-base Glycine SDS 250 mM 1.92 M 1% 188 Silver Staining (Prepare in fresh, 250 ml/gel) Fixation Ethanol Acetic acid, glacial Milli-Q H2O 100 ml 25 ml 125 ml Sensitizating Ethanol Sodium thiosulfate (5% w/v) Sodium acetate Milli-Q H2O 75 ml 0.5 g 17 g 175 ml Silver reaction Silver nitrate (2.5% w/v) Milli-Q H2O 0.625 g 250 ml Developing Sodium carbonate Formaldehyde (37% w/v) Milli-Q H2O 6.25 g 100 àl 250 ml Stopping EDTA-Na2 ì H2O Milli-Q H2O 3.65 g 250 ml Sample Lysis Buffer (Western blotting) Tris-HCl, pH 7.5 NaCl Triton X-100 SDS Na3VO4 PMSF Aprotinin Leupeptin 50 mM 150 mM 1% 0.1% mM mM 10 μg/ml 10 μg/ml × Sample Buffer (-20oC) M Tris-HCl, pH 6.8 Glycerol 2-Mercaptoethanol SDS 1% Bromophenol blue 312.5 mM 50% 25% 10% 0.0625% Stacking Gel (2.5 ml/gel) 0.5 M Tris-HCl, pH 6.8 10% SDS 30% Acrylamide (37.5:1) 10% Glycerol 10% APS TEMED Milli-Q H2O 0.625 ml 0.025 ml 0.3125 ml 0.025 ml 0.0075 ml 0.0025 ml 1.563 ml Running Gel (Western blotting, 10%, ml/gel) 1.5 M Tris-HCl, pH 8.8 10% SDS 1.865 ml 0.075 ml 189 30% Acrylamide (37.5:1) 10% Glycerol 10% APS TEMED Milli-Q H2O 2.465 ml 0.075 ml 0.0385 ml 0.00365 ml 3.05 ml 10 × Transfer Buffer for PVDF (4oC) Tris-HCl, pH 7.5 Glycine 1M 1.92 M 10 × TBS (Tris-buffered saline) Tris-HCl, pH 7.5 NaCl 1M 9% TTBS (Tris-buffered saline-Tween 20) 10 × TBS Tween 20 1× 0.05% AP (Alkaline phosphatase) Substrate Solution (7.5 ml/blot) 25 × AP buffer BCIP NBT Milli-Q H2O 300 µl 75 µl 75 µl 7.05 ml PBS (Phosphate-buffered saline, pH 7.2, per 1L) NaCl Na2HPO4 KH2PO4 KCl 8g 1.16 g 0.2 g 0.2 g Coating Buffer (Mouse TNF ELISA Set) 0.2 M Sodium phosphate, pH 6.5 To prepare 1L: Na2HPO4 NaH2PO4 11.8 g 16.1 g Coating Buffer (Mouse IFN-γ ELISA Set) 0.1 M Sodium carbonate, pH 9.5 To prepare 1L: NaHCO3 Na2CO3 7.13 g 1.59 g Assay Diluent (ELISA) PBS with 10% FBS (heat-inactivated) Mouse Sensitization Solution OVA (grade V) Al(OH)3 Saline 20 µg mg 0.1 ml 190 ... global protein profilings of normal human bronchial epithelial cells (NHBE) and normal human lung fibroblasts (NHLF) stimulated with pro- inflammatory cytokines tumor necrosis factor (TNF)-α and/ or... contribution of two major airway resident cells, bronchial epithelial cells and lung fibroblasts, to the inflammatory events will be reviewed and discussed 1.2.1 Bronchial Epithelial Cells Upon pro- inflammatory. .. IFN-γ and TNF-α 23 Mass spectrometry technology for proteomics 35 Scheme of proteomics workflow and intruments 50 Differential protein profiling of TNF-α -stimulated NHLF 69 MS spectrum and Mascot