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Antibody based plasma protein profiling Ulrika Qundos KTH, Kungliga Tekniska Högskolan Royal Institute of Technology School of Biotechnology Science for Life Laboratory Stockholm 2013 © Ulrika Qundos, Stockholm 2013 KTH, Royal Institute of Technology School of Biotechnology Division of Proteomics and Nanobiotechnology Science for Life Laboratory Tomtebodavägen 23 A SE-171 65 Solna, Sweden ISBN 978-91-7501-829-4 TRITA-BIO Report 2013:12 ISSN 1654-2312 Printed by US-AB 2013 Abstract This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (PapersI-III)andaprostatecancercandidatebiomarkeridentifiedandveri- fied(PapersIII-V).Furthermore,aperspectiveontheresearchareaaffinity proteomicsandits’employmentinbiomarkerdiscovery,forimprovedunder- standing and potentially improved disease diagnosis, is provided. Paper I presents the results of a comparative plasma and serum protein profilingstudy,withatargetedbiomarkerdiscoveryapproachinthecontext of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma sam- ples. Minoreffectswerefoundonthedetectedlevelsofmorethan300pre- dictedorknownplasmaproteins. InPaperIII,thedetectabilityofproteinsin plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was ob- served for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several an- tibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich im- munoassay,aspresentedinPapersIVandV.Furthermore,thedeveloped sandwichimmunoassaytargetingmultipledistinctCNDP1epitopesinmore than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V). Asanoutcomeofthepresentinvestigationsandinparalleltostudieswithin the Biobank profiling research group, valuable lessons from study design andmultiplexantibodyanalysisofplasmawithinbiomarkerdiscoverytoex- perimental, technical and biological verifications have been collected. keywords: proteinprofiling,plasma,antibody,affinityproteomics,biomark- ers, multiplex, assay development i ! ii Thesis defense ThisthesiswillbedefendedonSeptember6,2013at10.00a.minthegammahouselec- turehallatSciLifeLab,Stockholm,Sweden Respondent UlrikaQundos,M.Sci. inBiomedicine SciLifeLab,DivisionofProteomicsandNanobiotechnology,SchoolofBiotechnology KTH,RoyalInstituteofTechnology Facultyopponent Dr. UlrikeKorf,groupleaderatDKFZ DivisionofMolecularGenomeAnalysis,GermanCancerResearchCenter(DKFZ),Heidel- berg,Germany. Evaluationcommittee Prof. StefanSta˚hl DivisionofProteinTechnology,KTH,RoyalInstituteofTechnology,Stockholm,Sweden Prof. MatsNilsson SciLifeLab,DepartmentofBiochemistryandBiophysics,StockholmUniversity,Stockholm, Sweden Assoc. Prof. JennyForshed SciLifeLab,DepartmentofOncology-Pathology,KarolinskaInstitutet,Stockholm,Sweden Chairmanofthedefense Prof. MathiasUhle´n SciLifeLab,DivisionofProteomicsandNanobiotechnology,SchoolofBiotechnology KTH,RoyalInstituteofTechnology Supervisors MainsupervisorAssoc. Prof. JochenM.Schwenk Co-supervisorProf. PeterNilsson Co-supervisorProf. MathiasUhle´n SciLifeLab,DivisionofProteomicsandNanobiotechnology,SchoolofBiotechnology KTH,RoyalInstituteofTechnology iii ! iv List of publications Thepresentedthesisisbasedonthefollowingfivepapers, referredtobytheirromannu- merals(I-V).AllpapersareincludedintheAppendix. I JochenM.Schwenk,UlrikaIgel,BernetS.Kato,GeorgeNicholson,Fredrik Karpe,MathiasUhle´n&PeterNilsson(2010). Comparativeproteinprofiling of serum and plasma using an antibody suspension bead array approach. Proteomics, 10(3), 532-540. doi:10.1002/pmic.200900657 II Ulrika Qundos, Mun-Gwan Hong, Gunnel Tybring, Mark Divers, Jacob Odeberg, Mathias Uhle´n, Peter Nilsson & Jochen M. Schwenk (2013). Profilingpost-centrifugationdelayofserumandplasmawithantibodybead arrays. Journal of Proteomics, In Press. doi:10.1016/j.jprot.2013.04.020 III Jochen M. Schwenk, Ulrika Igel, Maja Neiman, Hanno Langen, Charlotte Becker, Anders Bjartell, Fredrik Ponten, Fredrik Wiklund, Henrik Gro¨nberg, PeterNilsson&MathiasUhle´n(2010). Towardnextgenerationplasmapro- filing via heat-induced epitope retrieval and array-based assays. Mol Cell Proteomics, 9(11), 2497-2507. doi:10.1074/mcp.M110.001560 IV Barbara Hjelm, Bjo¨rn Forsstro¨m, Ulrika Igel, Henrik Johannesson, Char- lotteStadler,EmmaLundberg,FredrikPonten,AnnaSjo¨berg,JohanRock- berg, Jochen M. Schwenk, Peter Nilsson, Christine Johansson, & Mathias Uhle´n(2011). Generationofmonospecificantibodiesbasedonaffinitycap- ture of polyclonal antibodies. Protein Science, 20(11), 1824-1835. doi:10.1002/pro.716 V Ulrika Qundos, Henrik Johannesson, Claudia Fredolini, Gillian O’Hurley, Rui Branca, Mathias Uhle´n, Fredrik Wiklund, Anders Bjartell, Peter Nils- son & Jochen M. Schwenk. Plasma levels of carnosine dipeptidase 1 de- crease in prostate cancer patients with lymph node metastasis. Submitted to Translational Proteomics (2013) v Related publications Burcu Ayoglu, Anna Ha¨ggmark, Maja Neiman, Ulrika Igel, Mathias Uhle´n, Jochen M. Schwenk, & Peter Nilsson (2011). Systematic antibody and antigen-basedproteomicprofilingwithmicroarrays. ExpertReviewofMolec- ular Diagnostics, 11(2), 219-234. doi:10.1586/erm.10.110 Anna Ha¨ggmark, Sanna Bystro¨m, Burcu Ayoglu, Ulrika Qundos, Mathias Uhle´n,MohsenKhademi,TomasOlsson,JochenM.Schwenk&PeterNils- son (2013). Antibody-based profiling of cerebrospinal fluid within multiple sclerosis. Proteomics, 13, 2256-2267. doi:10.1002/pmic.201200580 vi Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i Thesis defense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii List of publications . . . . . . . . . . . . . . . . . . . . . . . . . . . v Affinity proteomics 1 Proteins and Proteomes . . . . . . . . . . . . . . . . . . . . . . . . 1 About proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Principles of an immunoassay . . . . . . . . . . . . . . . . . . . . . 4 Protein microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Affinity reagents for protein capture . . . . . . . . . . . . . . . . . . 7 The Human Protein Atlas . . . . . . . . . . . . . . . . . . . . . . . 11 Assay Specificity and Sensitivity . . . . . . . . . . . . . . . . . . . 12 Antibody arrays on beads . . . . . . . . . . . . . . . . . . . . . . . 18 Mass spectrometry based proteomics . . . . . . . . . . . . . . . . 20 Bridging proteomic methods . . . . . . . . . . . . . . . . . . . . . . 20 Blood 23 Blood components . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Fighting infections with antibodies . . . . . . . . . . . . . . . . . . 25 Blood coagulation and plasma preparation. . . . . . . . . . . . . . 28 Biobanks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 The detectable plasma proteome . . . . . . . . . . . . . . . . . . . 31 Biomarker discovery in plasma 33 Sample selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Study size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Target selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Experimental design . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Statistical tools for data analysis . . . . . . . . . . . . . . . . . . . 37 Present Investigation 41 Pre-analytical sample preparation (I-III) . . . . . . . . . . . . . . . 42 Biomarker discovery and verification (I, III-V) . . . . . . . . . . . . 45 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . 50 51 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 vii ! viii

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Abstract. This thesis is about protein profiling in serum and plasma using antibody . protein profiling of blood preparations using suspension bead arrays. sue preparation step for IHC can also be implemented for sample prepa-.
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Antibody based plasma protein profiling Ulrika Qundos KTH, Kungliga Tekniska Högskolan Royal Institute of Technology School of Biotechnology Science for Life Laboratory Stockholm 2013 © Ulrika Qundos, Stockholm 2013 KTH, Royal Institute of Technology School of Biotechnology Division of Proteomics and Nanobiotechnology Science for Life Laboratory Tomtebodavägen 23 A SE-171 65 Solna, Sweden ISBN 978-91-7501-829-4 TRITA-BIO Report 2013:12 ISSN 1654-2312 Printed by US-AB 2013 Abstract This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (PapersI-III)andaprostatecancercandidatebiomarkeridentifiedandveri- fied(PapersIII-V).Furthermore,aperspectiveontheresearchareaaffinity proteomicsandits’employmentinbiomarkerdiscovery,forimprovedunder- standing and potentially improved disease diagnosis, is provided. Paper I presents the results of a comparative plasma and serum protein profilingstudy,withatargetedbiomarkerdiscoveryapproachinthecontext of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma sam- ples. Minoreffectswerefoundonthedetectedlevelsofmorethan300pre- dictedorknownplasmaproteins. InPaperIII,thedetectabilityofproteinsin plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was ob- served for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several an- tibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich im- munoassay,aspresentedinPapersIVandV.Furthermore,thedeveloped sandwichimmunoassaytargetingmultipledistinctCNDP1epitopesinmore than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V). Asanoutcomeofthepresentinvestigationsandinparalleltostudieswithin the Biobank profiling research group, valuable lessons from study design andmultiplexantibodyanalysisofplasmawithinbiomarkerdiscoverytoex- perimental, technical and biological verifications have been collected. keywords: proteinprofiling,plasma,antibody,affinityproteomics,biomark- ers, multiplex, assay development i ! ii Thesis defense ThisthesiswillbedefendedonSeptember6,2013at10.00a.minthegammahouselec- turehallatSciLifeLab,Stockholm,Sweden Respondent UlrikaQundos,M.Sci. inBiomedicine SciLifeLab,DivisionofProteomicsandNanobiotechnology,SchoolofBiotechnology KTH,RoyalInstituteofTechnology Facultyopponent Dr. UlrikeKorf,groupleaderatDKFZ DivisionofMolecularGenomeAnalysis,GermanCancerResearchCenter(DKFZ),Heidel- berg,Germany. Evaluationcommittee Prof. StefanSta˚hl DivisionofProteinTechnology,KTH,RoyalInstituteofTechnology,Stockholm,Sweden Prof. MatsNilsson SciLifeLab,DepartmentofBiochemistryandBiophysics,StockholmUniversity,Stockholm, Sweden Assoc. Prof. JennyForshed SciLifeLab,DepartmentofOncology-Pathology,KarolinskaInstitutet,Stockholm,Sweden Chairmanofthedefense Prof. MathiasUhle´n SciLifeLab,DivisionofProteomicsandNanobiotechnology,SchoolofBiotechnology KTH,RoyalInstituteofTechnology Supervisors MainsupervisorAssoc. Prof. JochenM.Schwenk Co-supervisorProf. PeterNilsson Co-supervisorProf. MathiasUhle´n SciLifeLab,DivisionofProteomicsandNanobiotechnology,SchoolofBiotechnology KTH,RoyalInstituteofTechnology iii ! iv List of publications Thepresentedthesisisbasedonthefollowingfivepapers, referredtobytheirromannu- merals(I-V).AllpapersareincludedintheAppendix. I JochenM.Schwenk,UlrikaIgel,BernetS.Kato,GeorgeNicholson,Fredrik Karpe,MathiasUhle´n&PeterNilsson(2010). Comparativeproteinprofiling of serum and plasma using an antibody suspension bead array approach. Proteomics, 10(3), 532-540. doi:10.1002/pmic.200900657 II Ulrika Qundos, Mun-Gwan Hong, Gunnel Tybring, Mark Divers, Jacob Odeberg, Mathias Uhle´n, Peter Nilsson & Jochen M. Schwenk (2013). Profilingpost-centrifugationdelayofserumandplasmawithantibodybead arrays. Journal of Proteomics, In Press. doi:10.1016/j.jprot.2013.04.020 III Jochen M. Schwenk, Ulrika Igel, Maja Neiman, Hanno Langen, Charlotte Becker, Anders Bjartell, Fredrik Ponten, Fredrik Wiklund, Henrik Gro¨nberg, PeterNilsson&MathiasUhle´n(2010). Towardnextgenerationplasmapro- filing via heat-induced epitope retrieval and array-based assays. Mol Cell Proteomics, 9(11), 2497-2507. doi:10.1074/mcp.M110.001560 IV Barbara Hjelm, Bjo¨rn Forsstro¨m, Ulrika Igel, Henrik Johannesson, Char- lotteStadler,EmmaLundberg,FredrikPonten,AnnaSjo¨berg,JohanRock- berg, Jochen M. Schwenk, Peter Nilsson, Christine Johansson, & Mathias Uhle´n(2011). Generationofmonospecificantibodiesbasedonaffinitycap- ture of polyclonal antibodies. Protein Science, 20(11), 1824-1835. doi:10.1002/pro.716 V Ulrika Qundos, Henrik Johannesson, Claudia Fredolini, Gillian O’Hurley, Rui Branca, Mathias Uhle´n, Fredrik Wiklund, Anders Bjartell, Peter Nils- son & Jochen M. Schwenk. Plasma levels of carnosine dipeptidase 1 de- crease in prostate cancer patients with lymph node metastasis. Submitted to Translational Proteomics (2013) v Related publications Burcu Ayoglu, Anna Ha¨ggmark, Maja Neiman, Ulrika Igel, Mathias Uhle´n, Jochen M. Schwenk, & Peter Nilsson (2011). Systematic antibody and antigen-basedproteomicprofilingwithmicroarrays. ExpertReviewofMolec- ular Diagnostics, 11(2), 219-234. doi:10.1586/erm.10.110 Anna Ha¨ggmark, Sanna Bystro¨m, Burcu Ayoglu, Ulrika Qundos, Mathias Uhle´n,MohsenKhademi,TomasOlsson,JochenM.Schwenk&PeterNils- son (2013). Antibody-based profiling of cerebrospinal fluid within multiple sclerosis. Proteomics, 13, 2256-2267. doi:10.1002/pmic.201200580 vi Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i Thesis defense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii List of publications . . . . . . . . . . . . . . . . . . . . . . . . . . . v Affinity proteomics 1 Proteins and Proteomes . . . . . . . . . . . . . . . . . . . . . . . . 1 About proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Principles of an immunoassay . . . . . . . . . . . . . . . . . . . . . 4 Protein microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Affinity reagents for protein capture . . . . . . . . . . . . . . . . . . 7 The Human Protein Atlas . . . . . . . . . . . . . . . . . . . . . . . 11 Assay Specificity and Sensitivity . . . . . . . . . . . . . . . . . . . 12 Antibody arrays on beads . . . . . . . . . . . . . . . . . . . . . . . 18 Mass spectrometry based proteomics . . . . . . . . . . . . . . . . 20 Bridging proteomic methods . . . . . . . . . . . . . . . . . . . . . . 20 Blood 23 Blood components . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Fighting infections with antibodies . . . . . . . . . . . . . . . . . . 25 Blood coagulation and plasma preparation. . . . . . . . . . . . . . 28 Biobanks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 The detectable plasma proteome . . . . . . . . . . . . . . . . . . . 31 Biomarker discovery in plasma 33 Sample selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Study size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Target selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Experimental design . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Statistical tools for data analysis . . . . . . . . . . . . . . . . . . . 37 Present Investigation 41 Pre-analytical sample preparation (I-III) . . . . . . . . . . . . . . . 42 Biomarker discovery and verification (I, III-V) . . . . . . . . . . . . 45 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . 50 51 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 vii ! viii

Description:
Abstract. This thesis is about protein profiling in serum and plasma using antibody . protein profiling of blood preparations using suspension bead arrays. sue preparation step for IHC can also be implemented for sample prepa-.
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