SEARCH FOR UNIQUE MEMBRANE PROTEIN OF FIRST TRIMESTER PRIMITIVE ERYTHROBLAST ZHANG HUOMING A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF OBSTETRICS & GYNAECOLOGY NATIONAL UNIVERSITY OF SINGAPORE July 2007 Acknowledgements The work presented in this thesis describes the laboratory research undertaken by me at the Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore (NUS), from August 2003 to July 2007. Firstly, I would like to thank my supervisors, Dr Mahesh Choolani, Professor Ng Soon Chye for their scientific advice, guidance and support during the past five years. I am grateful to our lab postdoctors, Dr Ponnusamy Sukumar, Dr Kothandaraman Narasimhan for their advice, guidance and review of my documents. I would also like to extend my gratitude to Dr Lin Qingsong, Dr Shashikant Joshi, Mr Lim Teck Kwang who helped me with mass spectrometric experiments and analyses. I am grateful to the clinical staff and nurses in the Department of Obstetrics and Gynaecology for their help in getting sample. I am thankful to my colleagues in the Diagnostic Biomarker Discovery Laboratory: Dr Nuruddin Mohammed, Dr Qin Yan, Dr Sherry Ho Sze Yee, Dr Zhao Changqing, Fan Yi Ping, Aniza Mahyuddin, Liu Lin, and Ho Lai Meng, for their insightful discussions, technical and scientific advice, and friendship. I am very grateful to Ginny Chen Zhenzhi for her excellent administrative support and assistance. Finally, I am deeply indebted to my family for their consistent support, encouragement and inspiring. II Table of Contents PAGE ACKNOWLEDGEMENTS . II SUMMARY IX LIST OF TABLES XI LIST OF FIGURES .XII LIST OF ABBREVIATIONS XIV CHAPTER INTRODUCTION 1.1 Overview . 1.2 Current methods of prenatal diagnosis . 1.3 Disadvantages of current prenatal diagnostic tests 1.4 Developmental biology relevant to non-invasive prenatal diagnosis . 1.4.1 Placental development: the fetal-maternal interface . 1.4.2 Fetal-maternal trafficking 10 1.4.3 Ontological development of erythropoiesis . 12 1.5 Non-invasive prenatal diagnosis for chromosomal and single gene disorders 13 1.5.1 Transcervical samples 14 1.5.2 Fetal DNA in maternal blood 14 1.5.3 Fetal RNA in maternal blood 16 1.5.4 Fetal cells in maternal blood . 17 1.5.5 Candidate of fetal cells for non-invasive prenatal diagnosis . 20 1.5.6 Fetal NRBCs for non-invasive prenatal diagnosis: current state of the art . 24 1.5.6.1 Current enrichment of fetal NRBCs from maternal blood 24 III 1.5.6.2 Identification of fetal origin of enriched NRBCs from maternal blood 31 1.5.6.3 Diagnosis of chromosomal and monogenic disorders by analysing fetal NRBCs from maternal blood .33 1.6 Challenges to the use of fetal NRBCs in maternal blood for non-invasive prenatal diagnosis . 35 1.6.1 Inability to expand fetal cells from maternal blood in vitro 35 1.6.2 Lack of cell surface markers specific to fetal NRBCs . 37 1.7 Proteomics for protein identification and biomarker discovery39 1.7.1 Sample preparation for proteomic study . 39 1.7.1.1 Conventional sample fractionation 40 1.7.1.2 Affinity enrichment 44 1.7.2 Protein separation techniques 47 1.7.2.1 One-dimensional gel electrophoresis (1-DE) 48 1.7.2.2 Two-dimensional gel electrophoresis (2-DE) 48 1.7.2.3 Liquid chromatography .51 1.7.3 Mass spectrometry . 53 1.7.3.1 Ionisation techniques 53 1.7.3.2 Mass analysers 54 1.7.4 Database searching and bioinformatic analysis . 56 1.7.5 Proteomic strategies for membrane protein analysis 59 1.7.5.1 Gel-based approach .60 1.7.5.2 Shotgun approach 62 1.8 Adult RBC membrane proteins . 66 1.9 Experimental aims and hypotheses 70 1.9.1 Aims . 71 1.9.2 Hypotheses 71 CHAPTER MATERIALS AND METHODS 72 2.1 Materials . 72 2.1.1 Human tissue and blood samples 72 2.1.1.1 Ethical approval for use of human tissues and blood samples 72 2.1.1.2 First trimester placental tissues 72 2.1.1.3 Peripheral blood from healthy male and female volunteers 72 2.1.2 Antibodies, reagents, solutions and kits . 72 IV 2.1.2.1 Antibodies 73 2.1.2.2 Primers .73 2.1.2.3 Reagents 76 2.1.2.4 Water and solutions 77 2.1.2.5 Kits .77 2.1.3 IPG Strip, gels, membrane and film 78 2.1.4 Hardware 78 2.1.4.1 Pipettes, centrifuge tubes, freezing box and filters 78 2.1.4.2 Blood collection tubes, needles, slides, coverslips, haemocytometer and coplin jars .78 2.1.4.3 Immuno-cell sorting equipment .78 2.1.4.4 Centrifuges for polypropylene tubes, cytocentrifuge and speedvac 79 2.1.4.5 Water bath, thermo bath, thermo cycler and freeze dryer .79 2.1.4.6 Sonicator, electrophoresis system and supplements 79 2.1.4.7 Peptides desalting tip and columns 79 2.1.4.8 2-D LC, SELDI-TOF-MS and MALDI-TOF/TOF-MS .79 2.1.4.9 Microscope and spectrophotometer 80 2.1.4.10 Computer and software 80 2.2 Methods 81 2.2.1 Preparation of Percoll gradient . 81 2.2.2 Nucleated and red blood cell count 81 2.2.3 Cytospin preparation 82 2.2.4 Wright’s staining . 82 2.2.5 Separation of adult RBCs from whole blood . 82 2.2.6 Recovery of fetal NRBCs from placental tissues obtained from termination of pregnancy 83 2.2.7 Adult RBC Membrane preparation . 83 2.2.8 Fetal NRBC membrane preparation . 84 2.2.9 Protein estimation . 84 2.2.9.1 Bradford assay .84 2.2.9.2 RC DC protein assay 84 2.2.10 One-dimensional gel electrophoresis . 85 2.2.11 Two-dimensional gel electrophoresis . 85 2.2.12 Protein bands and spots visualisation 86 2.2.13 Western blotting . 86 2.2.14 In-gel digestion of proteins for MS analysis 87 V 2.2.14.1 In-gel digestion of proteins from stained gel .87 2.2.14.2 In-gel digestion of proteins from unstained gel 87 2.2.15 Peptide sample clean-up 88 2.2.16 Adult RBC membrane protein extraction and in solution digestion . 88 2.2.16.1 Protein extraction using aqueous MeOH and trypsin digestion .88 2.2.16.2 Protein extraction using aqueous TFE and trypsin digestion .89 2.2.16.3 Protein extraction using urea solution and trypsin digestion 89 2.2.17 Fetal NRBC membrane protein extraction and trypsin digestion . 89 2.2.18 SELDI-TOF analysis of proteins and tryptic peptides . 90 2.2.19 2-D LC separation of tryptic digests . 91 2.2.20 MALDI-TOF/TOF analysis of tryptic peptides . 92 2.2.21 Database searching and bioinformatics analysis 92 2.2.22 RT-PCR 93 2.2.22.1 RNA extraction and quantification .93 2.2.22.2 RT-PCR 94 2.2.23 Immunocytochemistry . 94 2.2.24 Measuring intensity of colour using Adobe Photoshop . 95 2.2.25 Immunomagnetic cell separation 95 2.2.25.1 Dynal system 95 2.2.25.2 MACS sorting .96 2.2.26 Fluorescence-activated cell sorting 97 2.2.27 Statistical Analysis 97 CHAPTER PROTEOMIC ANALYSIS OF RBC MEMBRANE PROTEINS USING GEL-BASED APPROACH AND SHOTGUN METHOD . 98 3.1 Introduction . 98 3.2 Optimisation of RBC membrane preparation 99 3.3 2-DE separation of RBC membrane proteins . 100 3.4 1-DE separation of RBC membrane proteins and MS analysis . 103 3.4.1 Protein identification from silver stained 1-DE gel 103 3.4.2 Protein identification from unstained 1-DE gel 105 VI 3.5 Shotgun proteomic analysis of RBC membrane proteins . 109 3.5.1 Membrane protein extraction and digestion 110 3.5.2 Mass spectrometric analysis of membrane protein digests 114 3.5.3 Differential recovery of hydrophobic and hydrophilic peptides . 122 3.5.4 Analysis of protein digests with longer LC elution gradient . 124 3.6 Comprehensive list of RBC membrane proteins 125 3.7 Conclusion 126 CHAPTER PROTEOMIC ANALYSIS OF FETAL NRBC MEMBRANE PROTEINS USING SHOTGUN APPROACH 129 4.1 Introduction . 129 4.2 Recovery of fetal NRBCs from placental tissues 130 4.3 Preparation of membrane protein digests for MS analysis . 132 4.4 Mass spectrometric and bioinformatic analyses . 134 4.4.1 Mass spectrometric analysis of fetal NRBC protein digests . 134 4.4.2 Subcellular and functional groups of identified proteins . 136 4.4.3 Hydropathy analysis of identified peptides and proteins . 139 4.5 Conclusion 140 CHAPTER IDENTIFICATION OF UNIQUE SURFACE PROTEIN(S) OF PRIMITIVE FETAL NRBCS BY COMPARING FETAL NRBC AND ADULT RBC MEMBRANE PROTEOMES . . 142 5.1 Introduction . 142 5.2 Identification of unique membrane proteins 142 5.3 Validation of unique fetal NRBC membrane proteins . 145 5.3.1 RT-PCR 146 5.3.2 Immunocytochemistry . 149 5.4 Functional annotation of unique fetal NRBC membrane proteins . 151 5.5 Conclusion 154 VII CHAPTER IMMUNOCYTOCHEMICAL SCREENING OF SURFACE MEMBRANE PROTEINS ON FETAL NRBCS AND ADULT RBCS AND SORTING OF THESE CELLS BASED ON POTENTIAL CANDIDATE IDENTIFIED 155 6.1 Introduction . 155 6.2 Immunocytochemical screening of surface antigens on fetal NRBCs and adult RBCs 156 6.3 Immunomagnetic cell sorting using anti-CD147 . 161 6.3.1 Dynal system 162 6.3.2 Magnetic-activated cell sorting . 163 6.4 Fluorescence-activated cell sorting with anti-CD147 165 6.5 Conclusion 170 CHAPTER GENERAL DISCUSSION . 172 7.1 Hypothesis 172 7.2 Research findings . 173 7.3 Implications and limitations of this research . 174 7.4 Directions of future study 175 7.5 Conclusion 177 REFERENCES . 179 APPENDIX TABLES . 210 PUBLICATIONS . 251 VIII Summary Current methods for obtaining fetal cells for prenatal diagnosis are invasive and carry a small but definitive risk of fetal loss. Recovery of first trimester fetal erythroblasts (NRBCs) in maternal blood represents an attractive and promising alternative for early non-invasive prenatal diagnosis. However, these cells are rare and it is technically challenging in recovering them from maternal blood due to the lack of a fetal specific surface marker that could be used to isolate fetal NRBCs from adult RBCs. This thesis investigated the membrane protein profiles of first trimester primitive fetal NRBCs and adult RBCs using proteomic approaches and immunocytochemical screening with an aim to identify unique surface marker(s). To enhance the recovery of membrane proteome from a limited amount of fetal NRBC sample, an efficient proteomic strategy was developed for membrane proteome analysis, that is, sequential use of organic solvents methanol (MeOH) and 2,2,2-trifluoroethanol (TFE) to recover both hydrophilic and hydrophobic peptides and identification of proteins using two-dimensional liquid chromatography coupled with matrix-assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (2-D LC-MALDI-TOF/TOF-MS). The use of this strategy to analyse fetal NRBC membrane enabled us to present its first relatively comprehensive membrane proteome, and to identify twentythree unique fetal NRBC membrane proteins when compared with adult RBC membrane proteome. In addition, three differentially/uniquely expressed surface antigens were identified using immunocytochemical screening. Of the twenty-six potentially useful markers, surface antigen CD147 was tested and demonstrated to be a very useful target for the separation of fetal NRBCs IX from adult RBCs in model mixture, by either immunomagnetic cell sorting or fluorescence-activate cell sorting. I envisage that CD147, and/or other potential targets after further investigation, would be useful for the development of an efficient protocol to isolate fetal NRBCs from maternal blood in the first trimester of pregnancy, for early non-invasive prenatal diagnosis. X Appendix table 4. Continued 22 23 IPI00647085 IPI00025039 34340.69 33763.42 9.73 10.18 0 3 131 94 72 72 Ribosomal Nuclear 24 25 26 27 28 29 30 Ribosomal protein L5 variant Fibrillarin NADH-ubiquinone oxidoreductase 39 kDa subunit, mitochondrial precursor PREDICTED: similar to ribosomal protein L18a 60S ribosomal protein L26 H2B/t variant Coproporphyrinogen III oxidase, mitochondrial precursor Splice isoform short of Heterogenous nuclear ribonucleoprotein U Cytochrome c oxidase polypeptide Va, mitochondrial precursor IPI00003968 IPI00176629 IPI00027270 IPI00454695 IPI00093057 IPI00386491 IPI00025086 42482.57 20753.89 17247.53 21458.16 50119.97 88890.16 16763.72 9.81 10.73 10.55 10.71 8.59 5.6 6.3 0 0 0 3 100 71 84 84 56 86 85 4 91 49 44 83 59 123 56 31 Lamin B receptor variant IPI00645733 70651.06 9.41 89 67 32 33 IPI00328415 IPI00552125 34081.68 27804.46 7.31 4.55 0 86 80 76 59 34 35 36 37 38 NADH-cytochrome b5 reductase HNRPC protein Solute carrier family (activators of dibasic and neutral amino acid transport), member ATP-binding cassette half-transporter Equilibrative nucleoside transporter Mitochondrial 2-oxoglutarate/malate carrier protein Nucleolar protein Nop56 Mitochondrial Ribosomal Ribosomal Nuclear Mitochondrial Nuclear MIM Nuclear inner membrane. ER; MOM; membrane; Cytosolic Nuclear IPI00554481 IPI00465442 IPI00550382 IPI00219729 IPI00411937 71079.20 99649.17 58924.58 33908.81 66194.78 4.84 9.26 8.49 9.92 9.21 11 0 2 85 52 79 84 86 2 64 59 52 61 61 39 40 41 42 43 44 45 46 47 48 49 Cytochrome c1, heme protein, mitochondrial precursor 60S ribosomal protein L27 Spectrin beta isoform a Ribosomal protein L27a Leukocyte elastase precursor Splice isoform of Reticulon 60S ribosomal protein L3 Membrane protein 40S ribosomal protein S4, X isoform H2A histone family, member Y isoform Ubiquinol-cytochrome c reductase complex 14 kDa protein IPI00029264 IPI00219155 IPI00216704 IPI00456758 IPI00027769 IPI00298289 IPI00550021 IPI00026111 IPI00217030 IPI00148096 IPI00220416 35367.00 15656.71 267589.56 16468.03 28499.79 40292.95 45948.72 21161.16 29448.01 39159.18 13390.94 9.15 10.56 5.24 11 9.71 4.71 10.19 9.77 10.16 9.83 8.75 0 0 2 0 2 2 1 2 110 68 90 76 123 117 73 110 73 103 59 1 3 75 63 62 93 101 52 96 65 56 97 81 PM PM PM MIM Nuclear Mitochondrial membrane Ribosomal PM Ribosomal PM ER membrane Ribosomal Unclassified Ribosomal Nuclear MIM 238 Appendix table 4. Continued 50 CAAX prenyl protease homolog IPI00027180 54777.53 7.12 97 93 51 52 Splice Isoform of Heat shock cognate 71 kDa protein 18 kDa protein IPI00037070 IPI00514123 53887.70 17639.75 5.74 10.86 0 2 51 61 46 58 53 54 55 56 57 IPI00005202 IPI00219486 IPI00646848 IPI00395887 IPI00303476 23803.73 15059.24 78334.00 31770.80 56524.60 4.76 10.89 8.54 4.92 5.26 0 1 1 94 91 91 85 82 1 1 44 79 84 65 49 IPI00024742 IPI00182533 9769.08 15606.63 10.08 12.02 0 1 82 81 1 42 74 MIM Ribosomal 60 61 62 Membrane associated progesterone receptor component Splice Isoform of 40S ribosomal protein S24 Growth-inhibiting protein 12 Thioredoxin domain containing protein precursor ATP synthase beta chain, mitochondrial precursor Ubiquinol-cytochrome c reductase complex ubiquinone-binding protein QP-C 60S ribosomal protein L28 Ubiquinol-cytochrome-c reductase complex core protein I, mitochondrial precursor RAB14, member RAS oncogene family Novel protein ER/Golgi; PM membrane; Cytosolic; Nuclear Ribosomal Microsome membrane Ribosomal PM ER membrane MOM IPI00013847 IPI00646415 IPI00640179 52585.42 20396.31 11673.18 5.94 5.94 10.21 0 1 48 78 74 1 58 38 69 63 Cathepsin G precursor IPI00028064 28819.07 11.19 73 45 64 65 66 IPI00006690 IPI00100247 IPI00216613 80989.13 38927.68 72217.75 10.31 4.31 9.26 1 38 70 68 1 41 90 72 67 68 69 70 71 Eosinophil peroxidase precursor Thioredoxin-like protein KIAA1162 precursor Splice Isoform short of Splicing factor, proline-and glutamine-rich Splice Isoform of Plasminogen activator inhibitor RNA-binding protein ATP synthase g chain, mitochondrial Cytochrome c GSTK1 protein Kell blood group glycoprotein MIM Universal Unclassified PM; Intermediate filament Lysosome; Nuclear ; PM Nuclear IPI00412714 IPI00027448 IPI00465315 IPI00440703 IPI00220459 42401.27 11421.24 11610.09 31545.51 82770.92 8.42 9.65 9.59 8.85 8.09 0 0 1 1 66 62 62 61 53 1 49 58 38 75 93 72 73 74 50 kDa protein Histone H1x DEK protein IPI00396485 IPI00021924 IPI00020021 50138.50 22473.53 42647.92 7.26 10.76 8.69 0 1 69 52 49 1 54 79 53 58 59 Unclassified Mitochondrial MIM Mitochondrion PM Nuclear; Cytosolic Nuclear Nuclear 239 Appendix table 4. Continued 75 76 77 Splice isoform of Vacuolar proton translocating ATPase 116 kDa subunit a isoform Olfactory receptor 11H4 Solute carrier family 43, member IPI00552514 IPI00168981 IPI00301100 96350.11 36861.19 54494.25 6.02 9.05 8.71 7 11 1 47 47 47 1 54 39 54 78 79 ATP-binding cassette sub-family D member PRA1 family protein IPI00002372 IPI00007426 75427.57 21600.41 9.41 9.77 3 1 44 43 64 49 80 Suppressor of actin IPI00022275 66908.01 6.66 45 39 81 82 83 84 85 HSPC051 Ribosomal protein S29 isoform H2A histone family, member J, isoform Histone H2A.q ADP/ATP translocase ATP-binding cassette sub-family B member 10, mitochondrial precursor IPI00100810 IPI00639942 IPI00220855 IPI00339274 IPI00007188 14391.31 8081.99 14010.93 13848.80 32743.13 10.96 10.08 10.9 10.9 9.76 0 0 1 7 41 39 132 121 101 1 40 39 IPI00015826 79048.95 9.91 72 B-cell receptor-associated protein BAP37 HP1-BP74 SAM50-like protein CGI-51 40S ribosomal protein S8 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 67 kDa subunit precursor Tubulin beta-2 chain Splice Isoform of Mitochondrial inner membrane protein Protoporphyrinogen oxidase Splice Isoform of Voltage-dependent anion-selective channel protein Splice Isoform of Voltage-dependent anion-selective channel protein PREDICTED: similar to ribosomal protein S3a ATP synthase alpha chain, mitochondrial precursor Splice Isoform of Protein 4.1 16 kDa protein IPI00027252 IPI00549250 IPI00412713 IPI00216587 33275.92 61169.27 51929.30 24059.12 9.83 9.69 6.44 10.32 0 0 3 56 111 93 88 MIM Mitochondrial membrane Nuclear MOM Ribosomal IPI00025874 IPI00011654 IPI00470829 IPI00031357 68526.81 49638.97 79977.51 50733.65 5.96 4.78 6.31 8.44 0 3 3 93 95 69 81 ER membrane Cytosolic MIM Mitochondrial IPI00294779 30770.33 8.85 100 MOM IPI00216024 IPI00472119 IPI00440493 IPI00218697 IPI00334432 35111.16 29951.83 59713.59 93181.33 15532.11 5.87 9.78 9.16 5.41 8.76 0 0 3 3 117 64 69 58 144 MOM Ribosomal MOM PM Cytosolic 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 Vesicle membrane; PM PM ER; PM Perixisome membrane ER Membrane ER membrane; Golgi Mitochondrial membrane Ribosomal Nuclear Nuclear MIM 240 Appendix table 4. Continued 107 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit b isoform Heterogeneous nuclear ribonucleoprotein M isoform b Mitochondrial substrate carrier family protein ATP synthase, H+ transporting, subunit f isoform 2a variant Splice Isoform of Retinol dehydrogenase 11 Probable DNA dC->dU editing enzyme APOBEC-3C Similar to Cytochrome b5 outer mitochondrial membrane isoform precursor 108 109 110 Splice Isoform A of Protein C20orf108 OTTHUMP00000028841 Hypothetical protein FLJ35097 IPI00096986 IPI00478327 IPI00554589 20411.22 22617.43 48583.91 10.45 10.43 8.96 0 2 89 96 133 111 112 Transmembrane protein 14C Tricarboxylate transport protein, mitochondrial precursor NADH-ubiquinone oxidoreductase 30 kDa subunit, mitochondrial precursor 60S ribosomal protein L10 21 kDa protein 50 kDa protein Rhesus blood group, CcEe antigens, isoform Mitochondrial carrier homolog HLA-B associated transcript variant Splice Isoform Heart of ATP synthase gamma chain, mitochondrial precursor 40S ribosomal protein S11 10 kDa protein 17 kDa protein IPI00009346 IPI00639810 11556.97 35972.12 9.87 10.12 93 60 MOM ER membrane; mitochondrial; Microsome Ribosomal Mitochondrial ER/Golgi membrane MIM IPI00025796 IPI00554723 IPI00398234 IPI00180730 IPI00465155 IPI00003833 IPI00641829 30222.71 24429.82 20886.74 50121.92 45421.20 33308.86 48987.98 6.99 10.11 9.46 9.06 9.4 8.25 5.51 0 0 12 0 2 2 2 74 65 60 79 61 55 55 MIM Ribosomal Ribosomal Ribosomal PM MIM Nuclear IPI00395769 IPI00025091 IPI00186338 IPI00642218 32860.24 18418.99 9733.91 16710.62 9.31 10.31 5.81 6.49 0 2 1 55 60 90 92 Mitochondrial transmembrane GTPase FZO-2 IPI00293073 Cytochrome c oxidase subunit IPI00017510 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform b IPI00456049 86938.53 25548.21 5.99 4.67 61 90 MIM Ribosomal Nuclear ER membrane Mitochondrial membrane MIM 15763.17 6.6 57 Mitochondrial 101 102 103 104 105 106 113 114 115 116 117 118 119 120 121 122 123 124 125 126 IPI00456746 IPI00383296 IPI00383240 IPI00220300 IPI00339385 IPI00555878 17451.13 73572.36 29874.04 10910.70 33981.68 22810.96 9.36 8.94 9.66 9.7 8.95 7.52 0 0 2 2 95 109 59 103 101 79 Mitochondrial Nuclear MIM Mitochondrial ER membrane Unclassified IPI00641334 14163.00 5.06 86 241 Appendix table 4. Continued 127 128 129 130 Myeloblastin precursor Steroid dehydrogenase homolog 15 kDa protein 40S ribosomal protein S26 IPI00027409 IPI00007676 IPI00477513 IPI00186712 27789.27 34328.24 15095.99 12876.04 8.72 9.34 7.9 11.01 0 52 87 134 87 131 132 SM-11044 binding protein Voltage-dependent anion-selective channel protein IPI00644458 IPI00216308 29918.72 30622.53 5.52 8.63 2 48 50 133 134 135 136 Ras-related protein Rab-7 Splice Isoform GN-1S of Glycogenin-1 Optic atrophy isoform 40S ribosomal protein S16 IPI00016342 IPI00216115 IPI00107750 IPI00221092 23474.84 29070.36 113445.91 16304.00 6.4 4.73 7.63 10.21 0 0 1 79 78 43 75 137 138 139 140 141 142 IPI00452747 IPI00172656 IPI00022246 IPI00007928 IPI00411968 IPI00166079 14159.28 52590.54 26868.65 273426.56 17775.75 19822.68 7.85 5.46 9.75 8.95 9.13 9.28 0 0 1 1 38 73 75 40 64 73 IPI00385976 IPI00470528 IPI00008167 IPI00215610 21254.36 24000.04 31492.09 52263.65 9.74 11.62 8.58 6.91 1 72 43 71 70 ER membrane Ribosomal PM PM 147 148 KIAA0102 protein Protein expressed in T-cells and eosinophils in atopic dermatitis Azurocidin precursor Pre-mRNA processing splicing factor Protein Vitamin K epoxide reductase complex subunit 1-like protein CDNA FLJ90376 fis, clone NT2RP2004794, highly similar to Butyrate- induced transcript 60S ribosomal protein L15 Sodium/potassium-transporting ATPase beta-3 chain 55 kDa erythrocyte membrane protein Splice isoform XB of Plasma membrane calcium-transporting ATPase Heterogeneous nuclear ribonucleoprotein L isoform b PM; Cytosolic Unclassified Cytosolic Ribosomal Endosome membrane MOM; PM Endosome; Golgi membrane Cytosolic Mitochondrial Ribosomal ER/Microsome membrane Cytosolic PM; Cytosolic Nuclear Nuclear Unclassified IPI00217169 IPI00465225 133845.70 50528.51 6.04 7.22 41 70 149 150 151 12 kDa protein NADH dehydrogenase PREDICTED: similar to hypothetical protein IPI00644559 IPI00643646 IPI00455976 12027.62 52526.70 27850.92 6.81 7.21 10.36 0 1 70 69 69 PM Nuclear Mitochondrial 152 153 154 Ras-related protein Rab-5A Neutral amino acid transporter B Splice isoform of Protein GPR107 precursor IPI00023510 IPI00019472 IPI00382815 23643.82 56576.27 61936.41 8.32 5.34 6.72 1 69 68 68 143 144 145 146 Mitochondrial Ribosomal Golgi membrane; PM PM 242 Appendix table 4. Continued 155 156 High mobility group protein PREDICTED: similar to RPL7L1 protein IPI00219097 IPI00455892 23887.68 59708.63 7.77 8.4 0 67 36 157 Golgi-associated plant pathogenesis-related protein IPI00007067 17076.48 9.44 66 158 159 IPI00016513 IPI00549893 22526.59 24735.88 8.59 10.09 1 65 65 160 Ras-related protein Rab-10 ATP synthase F0 subunit Thyroid hormone receptor-associated protein complex 150 kDa component IPI00104050 108629.04 10.16 65 161 Phosphatidylserine synthase IPI00010746 55491.10 8.71 33 162 Splice Isoform of Synaptophysin-like protein IPI00335277 26394.49 6.79 64 163 164 IPI00007755 IPI00376215 24201.21 465202.00 8.16 6.81 0 1 62 64 165 Ras-related protein Rab-21 Splice Isoform of DNA-dependent protein kinase catalytic subunit Splice Isoform of Translocon-associated protein alpha subunit precursor IPI00449669 29355.03 4.52 63 166 167 168 169 SLC27A2 protein PREDICTED: similar to ribosomal protein S2 Hypothetical protein DKFZp586C1924 Hypothetical protein DKFZp686L18234 IPI00447606 IPI00165486 IPI00031064 IPI00414000 64574.28 29940.93 21513.48 37566.79 8.73 10.01 9.36 9.45 1 1 61 61 61 59 170 171 Synaptosomal-associated protein 29 60S ribosomal protein L9 IPI00032831 IPI00031691 28952.59 21849.80 5.56 9.96 0 1 59 58 172 Zn-finger, RING domain containing protein IPI00414712 35574.19 8.54 57 173 174 Mitochondrial carrier homolog isoform b 13 kDa protein Splice Isoform of NADH-ubiquinone oxidoreductase 51 kDa subunit, mitochondrial precursor IPI00386258 IPI00374249 41517.29 12579.21 9.4 10.85 1 57 57 ER membrane ER/peroxisome membrane Ribosomal Unclassified Unclassified PM; Synaptosome Ribosomal Microsomal membrane Mitochondrial membrane; Cytosolic Ribosomal IPI00221298 49836.33 8.51 56 MIM 175 Nuclear Ribosomal Golgi membrane; Extracellular Endosome; Golgi membrane; MIM Nuclear Mitochondrial membrane Vesicle membrane; PM Endosome; Golgi membrane Nuclear 243 Appendix table 4. Continued 176 177 Hypothetical protein FLJ14938 FLJ00144 protein IPI00024642 IPI00383085 55838.31 28437.22 4.76 5.75 0 1 56 54 178 179 180 181 182 183 184 185 186 187 188 Prohibitin Aquaporin splice variant BCG induced integral membrane protein BIGM103 48 kDa protein Hypothetical protein FLJ90397 FLJ12442 protein Actin, cytoplasmic RCC2 protein Calreticulin precursor Similar to Translocon-associated protein, delta subunit precursor Proliferation-associated 2G4, 38kDa IPI00017334 IPI00428490 IPI00034208 IPI00471915 IPI00171459 IPI00100938 IPI00021439 IPI00465044 IPI00020599 IPI00644824 IPI00299000 29785.90 14913.58 49598.38 47536.39 36994.10 54142.43 41709.73 56049.20 48111.82 13270.66 43785.20 5.57 5.48 5.71 11.07 8.88 6.47 5.29 9.02 4.29 6.48 6.13 0 0 0 1 1 1 1 54 53 53 52 52 52 52 51 52 51 50 189 190 191 192 ATPase, H+ transporting, lysosomal V0 subunit a isoform Ubiquitin-specific protease 31 Der1-like domain containing protein Phytanoyl-coa hydroxylase IPI00419696 IPI00328815 IPI00643745 IPI00514332 98018.34 118956.15 21238.77 34049.12 6.18 5.75 9.69 9.02 1 1 49 48 48 49 193 194 130 kDa leucine-rich protein Hypothetical protein FLJ13940 IPI00329745 IPI00017454 158825.66 27533.68 5.91 7.71 0 1 48 47 195 196 197 198 ATP synthase e chain, mitochondrial PREDICTED: similar to dJ753D5.2 Erythrocyte band integral membrane protein Hypothetical protein MGC14288 IPI00218848 IPI00397441 IPI00219682 IPI00176708 7797.28 13749.95 31579.70 6595.25 9.34 9.36 7.9 9.58 1 1 1 47 47 47 46 199 200 Peroxisomal membrane protein ALEX3 protein variant IPI00221002 IPI00604615 22106.98 42443.90 10.58 8.75 1 46 46 201 202 Adaptor-related protein complex 2, mu subunit isoform b Mitochondrial aspartate-glutamate carrier protein IPI00619900 IPI00007084 49357.91 74256.70 9.57 8.79 0 1 46 45 Unclassified Unclassified mitochondrial membrane PM PM Ribosomal Unclassified Cytosolic Cytosolic Nuclear Cytosolic ER membrane Nuclear Vacuolar membrane; lysosome Nuclear ER membrane Peroxisome Nuclear; Cytosolic Cytosolic Mitochondrial membrane Ribosomal PM Unclassified Peroxisome membrane Unclassified Vesicle/Golgi membrane; Cytosolic; MIM; PM 244 Appendix table 4. Continued 203 204 205 206 207 NADH dehydrogenase subunit Isoform of Solute carrier family 22, member 11 Splice isoform Glycophorin D of Glycophorin C Isocitrate dehydrogenase [NADP], mitochondrial precursor Novel protein IPI00221391 IPI00418660 IPI00218128 IPI00011107 IPI00513768 66968.42 48269.11 11491.61 50876.86 52673.49 9.14 8.88 4.76 8.88 9.76 15 0 1 1 45 44 44 44 43 208 Heparan sulfate 2-O-sulfotransferase IPI00549891 41844.40 8.83 43 209 210 211 212 213 IPI00376529 IPI00220059 IPI00013678 IPI00290928 IPI00604532 9459.86 15068.03 51444.59 44021.66 17073.62 6.75 9.85 9.8 8.11 9.63 1 0 1 1 43 42 42 42 42 214 215 216 217 218 219 220 221 222 223 224 kDa protein NADH-ubiquinone oxidoreductase B15 subunit Metaxin Guanine nucleotide-binding protein, alpha-13 subunit 13kDa differentiation-associated protein Full-length cDNA clone CS0DC006YH13 of Neuroblastoma of Homo sapiens Splice isoform of Heterogeneous nuclear ribonucleoprotein D0 Hypothetical protein FLJ32830 Protein p65 46 kDa protein Cleft lip and palate transmembrane protein Splice isoform of Protein C9orf5 Novel protein Mitochondrial ribosomal protein L41 Splice isoform B of Chloride channel protein Splice isoform of Protein C20orf22 IPI00009247 IPI00028888 IPI00065287 IPI00014168 IPI00335130 IPI00396411 IPI00607576 IPI00642244 IPI00217553 IPI00305252 IPI00394779 8609.50 38410.30 65726.57 68575.97 46266.42 76048.46 100881.01 109351.93 15372.99 35910.30 45068.18 9.94 7.62 9.35 9.67 7.28 5.88 9.03 7.94 9.58 8.03 8.87 0 0 14 0 1 1 1 1 1 41 41 41 41 41 40 40 40 40 41 40 225 226 227 228 229 230 231 232 Kelch domain containing protein P450-like protein Histone H2A.o Histone H1.2 Histone H1.4 Histone H1.5 Ubiquitin and ribosomal protein S27a Histone H1.3 IPI00383231 IPI00419579 IPI00216457 IPI00217465 IPI00217467 IPI00217468 IPI00179330 IPI00217466 46661.73 52399.01 13955.85 21220.71 21720.96 22435.43 17893.44 22205.28 5.67 5.84 10.9 10.94 11.03 10.91 9.65 11.02 0 0 0 0 1 39 39 MIM PM PM Mitochondrial Unclassified Nuclear; Golgi; IMP Mitochondrial membrane MIM MOM; IMP PM Mitochondrial 5 5 125 111 113 112 159 103 Mitochondrial Nuclear Unclassified Nuclear Unclassified PM PM ER Ribosomal PM Unclassified Nuclear; Cytosolic Cytosolic Nuclear Nuclear Nuclear Nuclear Ribosomal Nuclear 245 Appendix table 4. Continued 233 234 235 236 237 238 Histone H2A Histone H2B.j Histone H1.1 Ribosomal protein S8 Hemoglobin gamma-2 chain Alpha globin variant IPI00031562 IPI00303133 IPI00217469 IPI00645201 IPI00554676 IPI00410714 14112.93 13752.50 21697.83 21866.01 15985.25 15270.94 11.05 10.32 10.99 10.37 6.71 8.72 0 0 0 4 121 128 112 104 146 106 239 240 241 242 243 Hemogen 60S ribosomal protein L19 HNRPR protein Ribosomal protein L10 variant 60S ribosomal protein L17 IPI00464963 IPI00025329 IPI00644055 IPI00641164 IPI00413324 55278.74 23451.25 71170.40 24526.87 21252.29 4.82 11.48 8.23 10.11 10.18 0 0 4 101 89 73 67 71 244 IPI00402185 62617.34 7.18 73 245 246 247 248 249 OTTHUMP00000016816 Splice Isoform of Bromodomain adjacent to zinc finger domain protein 1B ATP synthase, H+ transporting, mitochondrial F0 complex 25 kDa protein Novel protein similar to histone 2, H3c PREDICTED: similar to 60S ribosomal protein L32 IPI00216695 IPI00220487 IPI00413986 IPI00455457 IPI00455900 170340.38 18479.50 24735.28 15420.55 52270.86 8.7 5.21 9.8 11.27 10.1 0 0 3 55 90 81 47 118 250 251 252 253 Splice Isoform of Syntaxin-7 60S ribosomal protein L35 OTTHUMP00000016319 50 kDa protein IPI00552913 IPI00412607 IPI00176681 IPI00174849 27383.66 14411.52 12179.15 49540.60 5.02 11.04 9.3 4.75 0 0 2 66 108 76 80 254 255 256 257 258 259 260 Cytochrome c oxidase polypeptide VIb Hypothetical protein DKFZp564K247 ATP-dependent RNA helicase DDX18 Splice Isoform of U1 small nuclear ribonucleoprotein 70 kDa Histone H1.0 PREDICTED: similar to 60S ribosomal protein L21 18 kDa protein SWI/SNF related matrix associated actin dependent regulator of chromatin subfamily A me IPI00216085 IPI00295621 IPI00301323 IPI00219483 IPI00550239 IPI00374234 IPI00176755 10054.68 10137.19 75359.44 50587.32 20719.15 10973.01 17696.17 6.78 9.52 9.52 9.9 10.84 10.97 10.44 0 0 2 2 75 100 60 53 91 51 44 Nuclear MIM Ribosomal Nuclear Ribosomal Endosome membrane Ribosomal MOM Mitochondrial Mitochondrial Membrane; IMP Nuclear Nuclear Nuclear Ribosomal Ribosomal IPI00297211 121828.36 8.27 81 Nuclear 261 Nuclear Nuclear Nuclear Ribosomal Cytosolic Cytosolic Golgi; Nuclear Ribosomal Nuclear Ribosomal Ribosomal Nuclear; Cytosolic 246 Appendix table 4. Continued 262 263 264 265 266 267 268 269 270 Beta-globin gene from a thalassaemia patient, complete cds Hemoglobin delta chain CAG-ISL Hypothetical protein FLJ32119 Splice Isoform of H/ACA ribonucleoprotein complex subunit 60S ribosomal protein L7 60S ribosomal protein L22 HEAT repeat containing protein Ribosomal protein L15 IPI00382950 IPI00473011 IPI00069693 IPI00065554 IPI00607820 IPI00030179 IPI00219153 IPI00549664 IPI00375511 18918.59 15914.25 23417.02 62438.95 20821.40 29207.20 14646.76 105626.65 24071.05 6.28 7.97 10.94 9.43 10.45 10.66 9.22 9.45 11.62 0 0 0 0 2 1 1 50 50 45 73 71 102 70 69 36 271 272 Splice Isoform short of Prostaglandin G/H synthase precursor PREDICTED: similar to 40S ribosomal protein S16 IPI00298268 IPI00397701 64440.72 16401.85 7.9 10.05 0 39 64 273 274 NADH-ubiquinone oxidoreductase MWFE subunit Splice Isoform of Chromodomain helicase-DNA-binding protein IPI00005695 IPI00455210 8067.13 220694.56 8.93 5.7 1 64 64 275 276 277 278 279 280 281 282 283 284 285 286 NADH-ubiquinone oxidoreductase 15 kDa subunit High mobility group protein NADH dehydrogenase Stromal cell-derived receptor-1 beta EH-domain containing protein Protein C10orf70 Splice Isoform of Putative RNA-binding protein Luc7-like Antibacterial protein FALL-39 precursor Flap endonuclease-1 Hypothetical protein FLJ31842 40S ribosomal protein S15 Hypothetical protein FLJ14347 IPI00220063 IPI00217477 IPI00604684 IPI00018311 IPI00021458 IPI00020510 IPI00216804 IPI00292532 IPI00026215 IPI00043429 IPI00216153 IPI00022300 12378.35 22834.40 30141.58 44359.50 61857.09 12191.20 46396.71 19578.27 42566.10 30020.92 16898.13 28266.57 9.29 8.5 6.25 8.11 6.06 9.19 10.1 9.48 8.8 9.4 10.39 8.7 0 1 1 1 1 2 63 31 60 59 57 56 56 55 27 54 27 53 287 288 NADH-ubiquinone oxidoreductase B22 subunit Splice Isoform of N-acylneuraminate cytidylyltransferase NADH-ubiquinone oxidoreductase 23 kDa subunit, mitochondrial precursor IPI00255052 IPI00479958 21685.80 29543.15 8.59 8.59 0 1 54 52 IPI00010845 23689.62 51 289 Cytosolic Cytosolic Ribosomal Cytosolic Nuclear Ribosomal Ribosomal Nuclear Ribosomal Microsome membrane; Cytosolic; Ribosomal Mitochondrial Membrane Nuclear Mitochondrial Membrane Nuclear Mitochondrial PM Nuclear Mitochondrial Nuclear PM Nuclear PM Ribosomal Unclassified Mitochondrial Membrane Unclassified Mitochondrial Membrane 247 Appendix table 4. Continued 290 Dihydroorotate dehydrogenase isoform precursor IPI00607764 22006.30 9.27 50 291 292 Vesicle-associated membrane protein NLN protein IPI00553138 IPI00025564 12509.63 70259.06 7.82 8.42 1 50 50 293 Splice Isoform of Myb-binding protein 1A IPI00607584 149273.88 9.34 49 294 295 Splice Isoform A of Band 4.1-like protein 65 kDa protein IPI00032230 IPI00478631 120603.14 64667.14 5.09 9.8 0 1 48 47 296 297 298 IPI00641145 IPI00556310 IPI00013452 7972.15 57968.46 170483.09 9.93 10.11 7.02 0 1 47 47 45 299 300 301 ATPase subunit 58 kDa protein Hypothetical protein DKFZp313B047 Splice Isoform of Voltage-dependent anion-selective channel protein kDa protein TUBA6 protein IPI00031804 IPI00639803 IPI00166768 30639.28 8078.35 36624.66 8.85 9.01 8.2 1 46 45 45 302 303 304 305 306 307 Splice Isoform long of Dematin 40S ribosomal protein S19 Stomatin ATP synthase, H+ transporting, mitochondrial F1 complex Sec61 alpha subunit Ferritin heavy chain IPI00292290 IPI00215780 IPI00377081 IPI00549805 IPI00218466 IPI00419501 45486.22 15919.49 13466.03 59771.60 52230.51 25207.52 8.94 10.31 7.82 9.07 8.3 6.33 0 10 1 1 1 45 44 43 42 41 41 308 309 310 311 312 313 314 315 Vesicular integral-membrane protein VIP36 precursor Cell death-regulatory protein GRIM19 40S ribosomal protein S9 Pol protein Similar to Telomeric repeat binding factor Ras-related protein Rap-1A NDUFC2 protein Serine/threonine kinase 22B (Spermiogenesis associated) IPI00009950 IPI00219685 IPI00221088 IPI00386255 IPI00216552 IPI00019345 IPI00555919 IPI00289715 40203.10 25804.38 22446.51 97621.79 32277.72 20973.71 14164.40 40971.18 6.46 9.82 10.66 8.89 9.6 6.38 9.04 8.95 1 0 0 1 1 1 1 41 40 40 39 37 36 36 36 MIM Vesicle membrane; Synapse; Mitochondrial Mitochondrial Nucleus; Cytosolic PM protein; Cytoskeleton Nuclear Mitochondrial membrane Ribosomal Cytosolic MOM Unclassified Cytosolic PM; Cytoskeleton Ribosomal PM MIM ER membrane PM ER membrane; Golgi MIM Ribosomal Cytosolic Nuclear Unclassified MIM Cytosolic 248 Appendix table Total identified fetal NRBC membrane proteins with potential surface domain(s) No. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Protein Description Membrane associated progesterone receptor component PRA1 family protein Steroid dehydrogenase homolog Vesicular integral-membrane protein VIP36 precursor Stromal cell-derived receptor-1 beta Neutral amino acid transporter B Calnexin precursor Azurocidin precursor Suppressor of actin Hypothetical protein FLJ14347 Band anion transport protein Transferrin receptor protein Membrane protein CAAX prenyl protease homolog Leukocyte elastase precursor Cathepsin G precursor Hypothetical protein DKFZp586C1924 BCG induced integral membrane protein BIGM103 Hypothetical protein FLJ31842 Thioredoxin-like protein KIAA1162 precursor Vitamin K epoxide reductase complex subunit 1-like protein Olfactory receptor 11H4 Hypothetical protein MGC14288 Splice Isoform XB of Plasma membrane calcium-transporting ATPase Splice Isoform Glycophorin D of Glycophorin C Erythrocyte band integral membrane protein Solute carrier family 2, facilitated glucose transporter member Accession No. IPI00005202 IPI00007426 IPI00007676 IPI00009950 IPI00018311 IPI00019472 IPI00020984 IPI00022246 IPI00022275 IPI00022300 IPI00022361 IPI00022462 IPI00026111 IPI00027180 IPI00027769 IPI00028064 IPI00031064 IPI00034208 IPI00043429 IPI00100247 IPI00166079 IPI00168981 IPI00176708 IPI00217169 IPI00218128 IPI00219682 IPI00220194 TMD 3 1 11 7 1 12 Sub-cellular localisation Microsome membrane; Plasma membrane ER Membrane; Plasma membrane Unclassified ER/Golgi membrane; Plasma membrane Plasma membrane Plasma membrane ER membrane; Plasma membrane Plasma membrane ER/Golgi membrane; Plasma membrane Unclassified Plasma membrane Plasma membrane Unclassified ER/Golgi membrane; Plasma membrane Plasma membrane Plasma Membrane Unclassified Plasma membrane Plasma membrane Plasma membrane Unclassified Plasma membrane Unclassified Plasma membrane Plasma membrane Plasma membrane Plasma membrane 249 Appendix table 5. Continued 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Kell blood group glycoprotein Antibacterial protein FALL-39 precursor Hypothetical protein DKFZp564K247 Splice Isoform of Reticulon Solute carrier family 43, member Splice Isoform B of Chloride channel protein Splice Isoform of Synaptophysin-like protein Stomatin Splice Isoform of Protein GPR107 precursor Splice Isoform of Protein C20orf22 Thioredoxin domain containing protein precursor Cleft lip and palate transmembrane protein Isoform of Solute carrier family 22, member 11 Aquaporin splice variant Rhesus blood group, CcEe antigens, isoform ATP-binding cassette half-transporter Equilibrative nucleoside transporter Splice Isoform of Vacuolar proton translocating ATPase 116 kDa subunit a isoform Splice Isoform of Syntaxin-7 IPI00220459 IPI00292532 IPI00295621 IPI00298289 IPI00301100 IPI00305252 IPI00335277 IPI00377081 IPI00382815 IPI00394779 IPI00395887 IPI00396411 IPI00418660 IPI00428490 IPI00465155 IPI00465442 IPI00550382 1 2 11 3 12 11 Plasma membrane Plasma membrane Unclassified ER membrane; Plasma membrane ER membrane; Plasma membrane Plasma membrane Vesicle membrane; Plasma membrane Plasma membrane Plasma membrane Unclassified ER membrane; Plasma membrane Plasma membrane Plasma membrane Plasma membrane Plasma membrane Plasma membrane Plasma membrane IPI00552514 IPI00552913 Vesicle-associated membrane protein Solute carrier family (activators of dibasic and neutral amino acid transport), member ALEX3 protein variant Splice Isoform of Protein C9orf5 kDa protein Microsomal glutathione S-transferase 17 kDa protein 25 kDa protein IPI00553138 Vesicle membrane; Plasma membrane Endosome membrane; Plasma membrane Vesicle membrane; Integral membrane protein; Synapse; Mitochondria IPI00554481 IPI00604615 IPI00607576 IPI00639803 IPI00639812 IPI00642218 IPI00646289 1 14 3 Plasma membrane Unclassified Plasma membrane Unclassified ER/Microsome membrane; Plasma membrane ER membrane; Plasma membrane Unclassified 250 Publications Zhang HM, Lin QS, Ponnusamy S, Kothandaraman N, Lim TK, Zhao CQ, Arijit B, Rauff M, Hew CL, Chung MCM, Joshi SB, Choolani M. Differential recovery of membrane proteins after extraction by aqueous methanol and trifluoroethanol. PROTOEMICS. 2007, 7, 1654-1663 Zhao CQ, Annamalai L, Guo CF, Kothandaraman N, Koh SC, Zhang HM, Arijit B, Choolani M. Circulating haptoglobin is an independent prognostic factor in sera of patients with epithelial ovarian cancer. NEOPLASIA. 2007, 9(1), 1-7. Choolani M, SSY Ho, K Razvi, S Ponnusamy, S Baig, NM Fisk, A Biswas, Rapid Rapid Molecular Testing in Prenatal Diagnosis Group (Mohammed N, Zhang HM, Leena Gole, Wong PC, Kevin Tan). FastFISH: technique for ultrarapid fluorescene in situ hybridisation on uncultured amniocytes yielding results within two hours of amniocentesis. MOLECULAR HUMAN REPORDUCTION, 2007, 13(6), 355-359. Zhang HM, Mahyuddin AP, Ponnusamy S, Zhao CQ, Arijit B, Rauff M, Choolani M. CD147, a potential marker for the enrichment of first trimester erythroblasts from maternal blood. (In preparation) Zhang HM, Lin QS, Ponnusamy S, Kothandaraman N, Lim TK, Zhao CQ, Arijit B, Rauff M, Hew CL, Chung MCM, Joshi SB, Choolani M. Proteomic analysis of human primitive erythroblast membrane proteins. (In preparation) 251 Zhao CQ, Annalamai L, Kothandaraman N, Koh CL, Zhang HM, Singini B, Ilancheran A, Razvi K, Biswas A, Choolani M. Proteomics-based Identification of Haptoglobin in Cyst Fluid: A Potential Diagnostic Marker for Epithelial Ovarian Cancer. (In preparation) Zhang HM, Mahyuddin AP, Ponnusamy S, Zhao CQ, Arijit B, Rauff M, Choolani M. A new potential cell surface marker for enrichment of first trimester erythroblasts from maternal blood. National Healthcare Group Annual Scientific Congress, 30 Sep-1 Oct 2006. Ann Acad Med Singapore 2006; 35 (suppl) 10: S5. (Prize for Best Oral Presentation in the Basic Science Session) Zhao CQ, Annamalai L, Guo CF, Kothandaraman N, Zhang HM, Chua SE, Choolani M. Effect of serum haptoglobin level on survival in patients with epithelial ovarian cancer. National Healthcare Group Annual Scientific Congress, 30 Sep-1 Oct 2006. Ann Acad Med Singapore 2006; 35 (suppl) 10: S5. (Prize for Best Poster Presentation in the Clinical Research Session) Zhang HM, Lin QS, Ponnusamy S, Kothandaraman N, Zhao CQ, Arijit B, Rauff M, Hew CL, Choolani M. Identification of membrane protein signatures of human primitive erythroblasts using 2D-LC MALDI TOF/TOF tandem mass spectrometry. The Third Joint BSPS/EBI Annual Proteomics meeting, Hinxtion, Cambridge, United Kingdom, 12-14 Jul 2006. Zhao CQ, Annamalai L, Zhang HM, Kothandaraman N, Chua SE, Choolani M. Proteomics-based identification of differential expression of ceruloplasmin in 252 benign and malignant ovarian tumors. The Third Joint BSPS/EBI Annual Proteomics meeting, Hinxtion, Cambridge, United Kingdom, 12-14 Jul 2006. Xu GL, Chan-Park MB, Ponnusamy S, Zhang HM, Kothandaraman N, Ying JY, Choolani M. Dielectrophoretic characterisation of fetal nucleated red blood cell for non-invasive prenatal diagnosis. Combined SingHealth and National Healthcare Group Scientific Meeting, 4-6 Nov 2005. Ann Acad Med Singapore 2005; 34 (suppl) 9: S90. Mohammed N, Ponnusamy S, Zhang HM, Tang PM, Choolani M. Enrichment of epsilon-globin-positive first trimester fetal primitive erythroblasts from maternal circulation: novel protocol for early non-invasive prenatal diagnosis. National Healthcare Group Annual Scientific Congress, 9-10 Oct 2004. Ann Acad Med Singapore 2004; 33 (suppl) 5: S133. 253 [...]... showing the number of integral membrane proteins identified using 2-D LC-MALDI-TOF/TOF-MS from MeOH, TFE and urea recovered peptides 116 Figure 3-9 Comparison of total proteins and proteins identified from my studies 126 Figure 4-1 Wright stain of fetal NRBC sample from placental tissues of termination of pregnancy 131 Figure 4-2 Flow chart showing fetal NRBC membrane protein preparation,... Simplified diagram of mass spectrometer 53 Figure 3-1 Western blotting analysis of RBC membrane preparation with Band 3 and GAPDH monoclonal antibodies 100 Figure 3-2 2-DE separation of RBC crude membrane and purified membrane preparations 102 Figure 3-3 SDS-PAGE separation and silver stain of RBC membrane proteins 104 Figure 3-4 SDS-PAGE separation of RBC membrane proteins using... Prediction of transmembrane domains (TMDs) of the identified proteins 135 Figure 4-4 Subcellular classifications (a) and functional categories (b) of fetal NRBC proteins identified from membrane preparations 138 Figure 4-5 Hydropathy comparison of the identified proteins from MeOH and TFE sequential extraction and digestion of fetal NRBC membrane 140 XII Figure 5-1 RT-PCR of the...List of Tables PAGE Table 1-1 Comparison of performance characteristics for two common tandem MS 55 Table 1-2 Various tools for MS-based protein identification 58 Table 2-1 Primer pairs used for the amplification for individual gene 74 Table 3-1 Proteins identified from silver stained 1-DE gel 105 Table 3-2 Proteins identified from unstained 1-DE gel 108 Table 3-3 The proteins... gel 106 Figure 3-5 SDS-PAGE analysis of RBC membrane proteins and their tryptic digests show the effective digestion 111 Figure 3-6 SELDI-TOF analysis of RBC membrane proteins and their tryptic digests the effective digestion 112 Figure 3-7 Schematic graphs showing the number of total proteins identified using 2-D LC-MALDI-TOF/TOF-MS from MeOH, TFE and urea recovered peptides... amniotic fluid is often performed in the second trimester of pregnancy (15-18 gestational weeks) for at-risk pregnant women In this procedure, a thin needle is inserted through abdomen and uterus into amniotic sac with ultrasonic guidance and 10-20 ml of amniotic fluid is withdrawn The levels of hormones (e.g α-fetoprotein) in the supernatant of amniotic fluid can be measured directly for screening various... 18 of gestational age (Moore et al., 1970), which produces large (~25 µm), nucleated primitive erythroblasts expressing embryonic haemoglobin (ε, ζ) (Palis et al., 1998) and some primitive macrophages These large primitive erythroblasts are predominant cell type in the fetal circulation during the first trimester pregnancy (Choolani et al., 2003) and likely cross into maternal blood The primitive erythroblasts... 2 Comprehensive membrane protein list summarised from various studies on human adult RBCs 215 Appendix table 3 Comprehensive RBC membrane proteins with potential surface domain(s) 228 Appendix table 4 Total proteins identified from fetal NRBC membrane 237 Appendix table 5 Total identified fetal NRBC membrane proteins with potential surface domain(s) 249 XI List of Figures PAGE... primitive fetal NRBCs from maternal blood of first trimester pregnancy In this thesis, I aimed to identify the differential expression of membrane proteins between primitive fetal NRBCs and adult RBCs, which can be potentially useful for the development of an efficient protocol to enrich primitive fetal NRBCs from maternal blood 3 1.2 Current methods of prenatal diagnosis Currently, there are four prenatal... for progression into the definitive program, but are not required for the establishment of primitive erythropoiesis (Nocka et al., 1989) In addition, erythropoietin (EPO) was found to be essential for maturation of definitive erythroid lineage, but not for primitive erythropoiesis (Lee et al., 2001) These distinct features raise the possibility that they could have distinct cell surface antigen profiles . SEARCH FOR UNIQUE MEMBRANE PROTEIN OF FIRST TRIMESTER PRIMITIVE ERYTHROBLAST ZHANG HUOMING A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF OBSTETRICS. IDENTIFICATION OF UNIQUE SURFACE PROTEIN( S) OF PRIMITIVE FETAL NRBCS BY COMPARING FETAL NRBC AND ADULT RBC MEMBRANE PROTEOMES 142 5.1 Introduction 142 5.2 Identification of unique membrane proteins. analysis of RBC membrane proteins 109 3.5.1 Membrane protein extraction and digestion 110 3.5.2 Mass spectrometric analysis of membrane protein digests 114 3.5.3 Differential recovery of hydrophobic