IDENTIFYING DIFFERENTIALLY EXPRESSED CELL MEMBRANE PROTEINS ON FETAL PRIMITIVE ERYTHROBLASTS AND ADULT ANUCLEATE ERYTHROCTYES FOR NON-INVASIVE PRENATAL DIAGNOSIS QIN YAN A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF OBSTETRICS & GYNAECOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEDGEMENTS The work presented in this thesis describes laboratory research undertaken by me at the Department of Obstetrics & Gynaecology, National University of Singapore (NUS), from January 2002 to June 2004 Throughout this time, I was supported by a research scholarship from NUS Firstly, I would like to thank my supervisors, Dr Mahesh Choolani and Associate Professor Arijit Biswas, for their guidance and support I would also like to extend my gratitude to Dr Punnusamy Sukumar and Dr K Narasimhan, for their scientific advice and technical help It has been a great pleasure working in the Rare-Event Detection Laboratory, NUS I am very grateful to Dr Li Qiu-Tian and Dr Li Guodong, who welcomed me to use the ultracentrifuges I’m grateful to Dr Robert Yang for all his help, and to all those who have helped me over the past few years, including the clinical staff, patients and my colleagues, but especially to Mr Chua Wei Yong, Dr Tang Phua Mien, Dr Damayanti and Dr Ng Ying Woo Finally, I want to thank my parents and brother for their blessings in this endeavour; and friends here in Singapore for encouraging me to pursue i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii SUMMARY viii LIST OF TABLES x LIST OF FIGURES xii ABBREVIATIONS xiv CHAPTER 1: INTRODUCTION 1.1 Overview 1.2 Current methods of fetal genetic screening and prenatal diagnosis 1.2.1 Screening for chromosomal disorders 1.2.2 Screening for genetic diseases 1.2.3 Diagnosis of chromosomal and genetic disorders 1.2.3.1 Invasive procedures for diagnosis of chromosomal and genetic disorders 1.2.4 Preference for non-invasive and early prenatal diagnosis 1.3 Non-invasive prenatal diagnosis of chromosomal and single gene disorders 1.3.1 Transcervical sampling of fetal cells 10 1.3.2 Fetal DNA in maternal blood 10 1.3.3 Fetal cells in maternal blood 11 1.3.3.1 Prevalence of fetal cells in maternal blood 11 1.3.3.2 Candidate target cells for non-invasive prenatal diagnosis 12 ii 1.3.4 Clinical trials of non-invasive prenatal diagnosis and the place of fetal cells derived from maternal blood 14 1.4 Enrichment of fetal erythroblasts from maternal blood 16 1.4.1 State of the art 16 1.4.2 Replacing density gradient centrifugation with an immunosorting strategy 17 1.5 Adult anucleated RBCs and first trimester FNRBCs 18 1.5.1 Genealogy of erythroid Cells 18 1.5.2 Membrane composition of adult anucleated red blood cells 20 1.5.3 First trimester fetal primitive erythroblasts 28 1.6 Proteomics approach to interrogate cell membrane proteins 30 1.6.1 Plasma membrane protein extraction 30 1.6.2 Membrane protein resolution and identification 31 1.6.2.1 SELDI-TOF-MS 32 1.6.2.2 MALDI-TOF and MALDI-TOF/TOF 35 1.7 Experimental aims and hypotheses 1.7.1 Hypothesis 38 39 CHAPTER 2: MATERIALS & METHODS 40 2.1 Materials 40 2.1.1 Human tissue samples 40 2.1.1.1 Ethical approval for use of human tissues 40 2.1.1.2 Peripheral blood from non-pregnant female volunteers 40 2.1.1.3 First trimester fetal nucleated red blood cells 40 2.1.2 Reagents, solutions, protein standard marker and kits 40 iii 2.1.2.1 Reagents 40 2.1.2.2 Water and Solutions 41 2.1.2.3 Protein standard marker 42 2.1.2.4 Kits 42 2.1.3 Hardware 42 2.1.3.1 Pipettes, centrifuge tubes and filters 42 2.1.3.2 Blood collection tubes, needles, slides, coverslips, haemocytometer, coplin jars and Dounce Homogeniser 42 2.1.3.3 Centrifuges 42 2.1.3.4 Water baths and thermo bath 43 2.1.3.5 Sonicator 43 2.1.3.6 Electrophoresis system 43 2.1.3.7 Microscope and Spectrophotometers 43 2.1.3.8 SELDI-TOF-MS, MALDI-TOF/MS and MALDI-TOF/TOF tandem mass spectrometer 43 2.1.3.9 Computer 44 2.1.4 Computer software 44 2.2 Methods 45 2.2.1 Preparation of Percoll gradients 45 2.2.2 Isolation of AARBCs from peripheral blood 46 2.2.3 Isolation of FNRBCs from placental tissue collected at termination of pregnancy 46 2.2.4 Nucleated red blood cell count 46 2.2.5 Cytospin preparation 47 2.2.6 Wright’s staining 47 2.2.7 AARBC membrane protein extraction 47 2.2.8 FNRBC membrane protein extraction 47 iv 2.2.9 Protein quantification 48 2.2.10 SELDI-TOF-MS 49 2.2.10.1 SELDI-TOF-MS: preparation 49 2.2.10.2 SELDI-TOF-MS: analysis 50 2.2.11 SDS-PAGE 51 2.2.11.1 Gel staining after SDS-PAGE: Coomassie blue staining 52 2.2.11.2 Gel staining after SDS-PAGE: Silver staining 52 2.2.12 In-gel digestion for MALDI-TOF/MS and MALDI-TOF/TOF tandem mass spectrometry 52 2.2.13 MALDI-TOF analysis 53 2.2.14 MALDI-TOF/TOF analysis 54 2.2.15 Protein database search using MS-Fit and Mascot search engines 54 2.2.15.1 MS-Fit 54 2.2.15.2 MASCOT 56 2.2.15.3 Search parameters 58 2.2.16 Evaluation of search parameters and results 59 CHAPTER OPTIMISATION OF MEMBRANE PROTEIN EXTRACTION 62 3.1 Introduction 62 3.2 Adult anucleated RBC membrane protein extraction 64 3.3 Isolation of FNRBCs and extraction of their membrane protein extraction 66 3.3.1 Isolation of FNRBCs from placental trophoblast tissue 66 3.3.2 FNRBC membrane protein extraction 68 3.3.2.1 Lysis of FNRBCs to remove their nuclei for membrane protein preparation 69 v 3.3.2.2 Dounce homogenisation-sucrose gradient centrifugation method 71 3.3.2.3 Freeze-thawing-Dounce homogenisation method 72 3.3.2.4 Biotinylation of surface membrane proteins for extraction 73 3.3.2.5 Protein quantification of membrane protein preparations 76 3.4 Conclusions 78 CHAPTER 4: DETECTION OF UNIQUE FNRBC MEMBRANE PROTEIN USING SELDI-TOF-MS 80 4.1 Introduction 80 4.2 Suitability of different chips for profiling FNRBC and AARBC membrane proteins 81 4.3 Protein profiling using NP20 and H4 ProteinChip arrays 84 4.4 Protein profiling using SAX2 and WCX2 ProteinChip arrays 86 4.5 Protein profiling using IMAC ProteinChips arrays 88 4.6 Conclusion 93 CHAPTER 5: MEMBRANE PROTEIN IDENTIFICATION USING SDS-PAGE, MALDI-TOF, MALDI-TOF/TOF AND DATABASE SEARCH 96 5.1 Introduction 96 5.2 AARBC membrane protein identification using MALDI-TOF/MS 98 5.3 Identification of FNRBC and AARBC membrane proteins by SDS-PAGE, MALDI-TOF/TOF Tandem MS and database search AARBC membrane protein identification Group 1: Membrane proteins within AARBCs Group 2: Membrane associated proteins within AARBCs 103 119 125 vi Group 3: Cytoplasmic and nuclear proteins within AARBCs 128 Group 4: Proteins with uncharacterised location within AARBCs 131 FNRBC membrane protein identification 132 Group 1: Membrane proteins within FNRBCs 136 Group 2: Membrane associated proteins within FNRBCs 138 Group 3: Cytoplasmic and nuclear proteins within FNRBCs 140 Group 4: Proteins with uncharacterised location within FNRBCs 143 5.4 Comparison of FNRBC and AARBC membrane proteins 146 5.5 Conclusions 150 Additional notes on Erythrocyte membrane band 152 CHAPTER GENERAL DISCUSSION 155 6.1 Hypotheses 156 6.2 Implication of results in context of non-invasive prenatal diagnosis 158 6.3 Limitations of this research 159 6.4 Directions for future research 159 REFERENCES 162 APPENDIX A A1 APPENDIX B B1 vii SUMMARY Development of non-invasive methods for obtaining intact fetal cells would allow accurate prenatal diagnosis for aneuploidy and single gene disorders without the attendant risks associated with amniocentesis, chorion villus biopsy and fetal blood sampling Currently available methods are limited by the very low number of fetal cells enriched from maternal blood, which could be due to fetal cell loss during the density gradient centrifugation This thesis investigates differentially expressed cell membrane proteins of first trimester fetal nucleated red blood cells (FNRBCs) and adult anucleate red blood cells (AARBCs) Unique membrane proteins would be potential antigens for the development of monoclonal antibodies to separate FNRBCs from AARBCs Surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS) was first used to screen the unique membrane proteins on FNRBCs Three unique membrane protein peaks were detected using immobilized metal affinity capture (IMAC) chips pre-treated with iron Subsequent analysis however revealed that these proteins were unsuitable candidates due to their intracellular localisation Sodium dodecylsulphate polyacrylamide-gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization–time of flight/time of flight (MALDITOF/TOF) tandem mass spectrometry were used to identify and characterise the different membrane proteins These data showed that spectrin (alpha and beta viii chain), actin (beta and gamma), and ankyrin were membrane proteins common to both FNRBCs and AARBCs N-ethylmaleimide-sensitive factor attachment protein alpha was unique to FNRBCs Erythrocyte membrane protein band 3, bands 4.1, 4.2, p55, flotillin and 2, tropomodulin 1, aldolase A, glyceraldehydes-3-phosphate dehydrogenase and band 7.2 were membrane proteins unique to AARBCs Several putative membrane proteins (derived from functional domains) were also identified to be unique to FNRBCs and to AARBCs Of all the identified membrane proteins unique to FNRBCs or AARBCs, none of the unique FNRBC membrane proteins contained extracellular domains Interestingly, the unique AARBC membrane protein—band 3—contained extracellular domains If further experiments such as western blot could confirm that band is absent in FNRBC membranes, monoclonal antibodies to band extracellular domains could potentially be used to separate FNRBCs from AARBCs in first trimester of gestation, for non-invasive prenatal diagnosis ix R16 41321 R16 gi|1425040 gi|4501885 R16 gi|4501889 42249 R16* gi|4265643 60787 R17 gi|4507553 40658 R17 gi|1280365 gi|1427773 40657 R17 gi|4507021 102013 R17* gi|1173565 245291 R17* gi|1236759 256666 R17* gi|4758794 775749 R17* gi|4777733 77229 R17* gi|4266066 77768 R18 gi|1427773 42623 R17 42052 42623 IMAGE:353827 5) [Homo sapiens] actin, beta [Homo sapiens] beta actin; beta cytoskeletal actin [Homo sapiens] actin, gamma propeptide; actin, alpha-3 [Homo sapiens] similar to FKSG30 [Homo sapiens] tropomodulin [Homo sapiens] Tropomodulin [Homo sapiens] Chain P, Crystal Structure Of The Cytoplasmic Domain Of Human Erythrocyte Band-3 Protein solute carrier family 4, anion exchanger, member (erythrocyte membrane protein band 3, Diego blood group); anion exchange protein 1; Waldner blood group golgin-245 [Homo sapiens] 256 kD golgin [Homo sapiens] nebulin [Homo sapiens] FLJ40198 protein [Homo sapiens] similar to hypothetical protein FLJ36144 [Homo sapiens] Chain P, Crystal Structure Of The 49 20 48 20 24 13 16 37 12 32 11 19 11 11 11 24 23 39 19 13 17 13 17 B20 R18 gi|4507021 102013 R18 gi|4557305 39851 R18 gi|28614 39706 R18 gi|229674 39720 R18* gi|1992413 139145 R18* gi|2687853 155341 R18* gi|1992412 154823 R18* gi|4758794 775749 R18* gi|1173565 245291 R18* gi|4777733 77229 R18* gi|4266066 77768 R18* gi|7023190 127497 R18* gi|3187389 150192 Cytoplasmic Domain Of Human Erythrocyte Band-3 Protein solute carrier family 4, anion exchanger, member (erythrocyte membrane protein band 3, Diego blood group); anion exchange protein 1; Waldner blood group aldolase A; fructosebisphosphate aldolase; Aldolase A, fructosebisphosphatase [Homo sapiens] aldolase A [Homo sapiens] Aldolase A (E.C.4.1.2.13) RAD50 homolog isoform [Homo sapiens] RAD50 homologue hsRAD50 [Homo sapiens] RAD50 homolog isoform [Homo sapiens] nebulin [Homo sapiens] golgin-245 [Homo sapiens] FLJ40198 protein [Homo sapiens] similar to hypothetical protein FLJ36144 [Homo sapiens] unnamed protein product [Homo sapiens] hypothetical protein [Homo sapiens] 10 10 37 13 37 13 37 13 15 22 14 22 14 22 40 11 23 18 14 18 14 13 17 12 18 B21 R18* gi|3187481 150190 R18* gi|4247629 150320 R18* gi|1236759 256666 R18* gi|2052111 153856 R19 gi|4507021 102013 R19 gi|1427773 42623 R19 gi|1094704 207108 R19 gi|105337 207138 R19 gi|1845265 204538 R19 gi|1360744 204518 R19 gi|1094703 204488 R19 gi|1094703 207346 hypothetical protein [Homo sapiens] trinucleotide repeat containing 15; Grb10 interacting GYF protein [Homo sapiens] 256 kD golgin [Homo sapiens] KIAA0642 protein [Homo sapiens] solute carrier family 4, anion exchanger, member (erythrocyte membrane protein band 3, Diego blood group); anion exchange protein 1; Waldner blood group Chain P, Crystal Structure Of The Cytoplasmic Domain Of Human Erythrocyte Band-3 Protein ankyrin isoform 3; ankyrin-1, erythrocytic; ankyrin-R [Homo sapiens] ankyrin 1, erythrocyte splice form – human ankyrin [Homo sapiens] ankyrin 1, erythrocyte splice form – human ankyrin isoform 4; ankyrin-1, erythrocytic; ankyrin-R [Homo sapiens] ankyrin 13 18 12 18 11 23 12 18 16 16 20 10 18 10 18 17 17 17 17 B22 R19 gi|1094704 189977 R19* gi|4115165 110767 R19* gi|3419075 50755 R19* gi|4758794 775749 R19* gi|6683717 76358 R19* gi|3034869 141915 R19* 71171 R20 gi|1280447 gi|31645 R20 gi|7669492 36201 R20 gi|35053 35698 R21 gi|4507021 102013 R21 gi|1427773 42623 36202 isoform 1; ankyrin-1, erythrocytic; ankyrin-R [Homo sapiens] ankyrin isoform 2; ankyrin-1, erythrocytic; ankyrin-R [Homo sapiens] hypothetical protein FLJ12649 [Homo sapiens] LOC375251 protein [Homo sapiens] nebulin [Homo sapiens] KIAA0565 protein [Homo sapiens] hypothetical protein [Homo sapiens] NLN protein [Homo sapiens] glyceraldehyde -3-phosphate dehydrogenase [Homo sapiens] glyceraldehyde -3-phosphate dehydrogenase [Homo sapiens] uracil DNA glycosylase [Homo sapiens] solute carrier family 4, anion exchanger, member (erythrocyte membrane protein band 3, Diego blood group); anion exchange protein 1; Waldner blood group Chain P, Crystal Structure Of The Cytoplasmic Domain Of Human Erythrocyte 10 16 14 20 24 12 40 16 14 12 18 17 13 39 12 39 12 34 12 14 B23 R21 gi|7669492 36201 R21 gi|31645 36202 R21 gi|35053 35698 R22 gi|181184 31860 R22 gi|3801691 31882 R22 gi|1103842 32741 R22 gi|1471507 31958 R22 gi|3964546 33759 R22 gi|1703319 36088 R22 gi|189617 36262 R22 gi|4502105 36290 R22* gi|3851586 138662 Band-3 Protein glyceraldehyde -3-phosphate dehydrogenase [Homo sapiens] glyceraldehyde -3-phosphate dehydrogenase [Homo sapiens] uracil DNA glycosylase [Homo sapiens] stomatin peptide stomatin isoform a; erythrocyte membrane protein band 7.2 (stomatin) [Homo sapiens] band 7.2b stomatin Stomatin, isoform a [Homo sapiens] Unknown (protein for MGC:75105) [Homo sapiens] Annexin A4 (Annexin IV) (Lipocortin IV) (Endonexin I) (Chromobindin 4)(Protein II) (P32.5) (Placental anticoagulant protein II) (PAP-II) (PP4X) (35-beta calcimedin) (Carbohydratebinding protein P33/P41)(P33/ 41) protein PP4-X annexin IV; annexin IV (placental anticoagulant protein II); placental anticoagulant protein II [Homo sapiens] chromosomeassociated 24 24 20 37 13 37 13 35 12 32 11 36 11 33 11 33 11 33 11 15 20 B24 R22* gi|3356334 228889 R22* gi|1236759 256666 R22* gi|3736093 180845 R22* gi|1173565 245291 R22* gi|2687853 155341 R22* gi|1992412 154823 R22* gi|1992413 139145 R23 gi|181184 31860 R23 gi|3801691 31882 R23 gi|1103842 32741 R23 gi|1471507 31958 R23 gi|2168699 32343 R24 gi|181184 31860 R24 gi|3801691 31882 protein-C [Homo sapiens] myosin, heavy polypeptide 14; nonmuscle myosin heavy chain II-C; myosin heavy chain 14 [Homo sapiens] 256 kD golgin [Homo sapiens] KIAA2034 protein [Homo sapiens] golgin-245 [Homo sapiens] RAD50 homologue hsRAD50 [Homo sapiens] RAD50 homolog isoform [Homo sapiens] RAD50 homolog isoform [Homo sapiens] stomatin peptide stomatin isoform a; erythrocyte membrane protein band 7.2 (stomatin) [Homo sapiens] band 7.2b stomatin Stomatin, isoform a [Homo sapiens] stomatin-like 3; erythrocyte band integralmembra ne protein; protein 7.2B [Homo sapiens] stomatin peptide stomatin isoform a; erythrocyte membrane protein band 7.2 (stomatin) [Homo sapiens] 11 24 10 26 13 21 10 25 13 21 13 21 13 19 40 17 40 17 35 16 35 15 10 48 14 48 14 B25 R24 gi|1103842 32741 R24 gi|1471507 31958 R24* gi|2792395 593665 R24* gi|4266246 145199 R24* gi|1815765 593646 R24* gi|1173565 245291 R24* gi|1236759 256666 R24* gi|3457704 593806 R24* R24* gi|1205991 gi|2203567 350062 380605 R24* gi|2430815 163619 R24* gi|2052194 163545 R24* gi|4503613 232789 R25 gi|181184 31860 band 7.2b stomatin Stomatin, isoform a [Homo sapiens] Bullous pemphigoid antigen 1, isoforms 6/9/10 (Trabeculinbeta) (Bullous pemphigoid antigen) (BPA) KIAA0542 gene product [Homo sapiens] bullous pemphigoid antigen eA [Homo sapiens] golgin-245 [Homo sapiens] 256 kD golgin [Homo sapiens] bullous pemphigoid antigen isoform 1eA precursor; bullous pemphigoid antigen (230/240kD); dystonin; hemidesmoso mal plaque protein [Homo sapiens] Nebulin pericentrin B; kendrin; pericentrin-2 [Homo sapiens] Uveal autoantigen with coiled-coil domains and ankyrin repeats; uveal autoantigen with coiled coil domains and ankyrin repeats [Homo sapiens] KIAA1561 protein [Homo sapiens] envoplakin [Homo sapiens] stomatin 43 13 43 12 37 15 20 36 110 24 24 35 8 26 29 0 12 19 12 19 10 23 29 B26 R25 gi|3801691 31882 R25 gi|1103842 32741 R25 gi|1471507 31958 R26 gi|181184 31860 R26 gi|3801691 31882 R26 gi|1103842 32741 R26 gi|1471507 31958 R27* gi|4758794 775749 R28* gi|4758794 775749 R29* gi|1043603 62783 R29* gi|2104025 37515 R30 gi|181184 31860 R30 gi|3801691 31882 R30 gi|1103842 32741 R31* gi|4025514 128924 peptide stomatin isoform a; erythrocyte membrane protein band 7.2 (stomatin) [Homo sapiens] band 7.2b stomatin Stomatin, isoform a [Homo sapiens] stomatin peptide stomatin isoform a; erythrocyte membrane protein band 7.2 (stomatin) [Homo sapiens] band 7.2b stomatin Stomatin, isoform a [Homo sapiens] nebulin [Homo sapiens] nebulin [Homo sapiens] unnamed protein product [Homo sapiens] TruB pseudouridine (psi) synthase homolog [Homo sapiens] stomatin peptide stomatin isoform a; erythrocyte membrane protein band 7.2 (stomatin) [Homo sapiens] band 7.2b stomatin chromosome open reading frame 60 [Homo sapiens] 29 29 24 27 27 26 21 7 40 38 25 12 35 10 33 33 32 19 17 Protein bands with * are those identified with significant score but the MW of the proteins are larger than the range of the gel bands B27 Table B5.2 Proteins identified from FNRBC membrane pellet fraction by SDSPAGE and MALDI-TOF/TOF (All proteins list below are significant result p