Antibody-basedsubcellularlocalizationofthehumanproteome

MarieSkogs

LicentiateThesisKTH-RoyalInstituteofTechnology

SchoolofBiotechnologyStockholm,Sweden2016

©MarieSkogs2016KTH–RoyalInstituteofTechnologySchoolofBiotechnologyDivisionofProteomicsandNanobiotechnologyScienceforLifeLaboratoryTomtebodavägen23ASolnaSwedenTRITA-BIOReport2016:13ISSN1654-2312ISBN978-91-7729-010-0 CoverillustrationbyInaSchuppeKoistinenwww.inasakvareller.seOtherillustrationsbyMarieSkogsunlessotherwisestatedAkademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan iStockholm framlägges till offentlig granskning för avläggande av teknologielicentiatexamen ibioteknologionsdagenden8 juniklockan14.00 iAlfa2,ScienceforLifeLaboratory,Tomtebodavägen23A,Solna.

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AbstractThis thesis describes the use of antibodies and immunofluorescence forsubcellularlocalizationofproteins.Thekeyobjectiveisthecreationofanopen-source atlas with information on the subcellular location of every humanprotein. Knowledge of the spatial distribution and the precise location of aprotein within a cell is important for its functional characterization, anddescribing the human proteome in terms of compartment proteomes isimportanttodeciphercellularorganizationandfunction.Immunofluorescence and confocalmicroscopy of cultured cellswere used forhigh-resolution detection of proteins on a high-throughput scale. Critical toimmunofluorescence results are sample preparation and specific antibodies.Antibodystainingofcellsrequiresfixationandpermeabilization,bothofwhichcanresultinlossorredistributionofproteinsandmaskingofepitopes.Ahigh-throughputapproachdemandsastandardizedprotocolsuitableforthemajorityof proteins across cellular compartments. Paper I presents an evaluation ofsample preparation techniques from which such a single fixation andpermeabilization protocolwas optimized.Paper II describes the results fromapplying this protocol to 4000 human proteins in three cell lines of differentorigin.Paper III presents a strategy for application-specific antibody validation.Antibodiesarethekeyreagentsinimmunofluorescence,butallantibodieshavepotential for off-target binding and should be validated thoroughly. Antibodyperformance varies across sample types and applications due to thecompetition present and the effect of the sample preparation on antigenaccessibility. In this paper application-specific validation forimmunofluorescence was conducted using colocalization with fluorescentlytaggedproteinintransgeniccelllines.Keywords:Human proteome, Subcellular localization, Organelles, Immunofluorescence,Fixation,Permeabilization,Antibodyvalidation

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SammanfattningDennaavhandlingberöranvändandetavantikropparochimmunofluorescenceför lokalisationsbestämning av proteiner. Målet med arbetet har varituppbyggnadenavenwebbaseradatlasöverdensubcelluläralokalisationenhosalla mänskliga proteiner. Kunskap om ett proteins lokalisation är en viktigaspektförattutrönaproteinetsfunktion.Därtillärkunskapomproteomethosenskilda organeller och subcellulära enheter viktigt för att förstå cellensorganisationochfunktion.Immunofluorescence och konfokalmikroskopi av odlade celler användes förstorskalig lokalisationsbestämning av proteiner med hög precision. För attsäkerställa bra resultat i immunofluorescence är provpreparering samtspecifika antikroppar av högsta betydelse. Infärgningmed antikroppar kräverfixering och permeabilisering av cellerna vilket kan resultera i förlust elleromfördelningavproteiner samtmaskeringav epitop. För en storskalig ansatstilllokalisationsbestämningavallamänskligaproteinerkrävsenstandardiseradprovförberedning lämplig för majoriteten av alla protein i alla subcellulärastrukturer. I artikel I utvärderades ett antal olika fixerings ochpermeabiliseringsalternativfrånvilkaettoptimaltprotokollvaldes.Iartikel IIanvändesdettaprotokollför4000mänskligaproteineritreolikacellinjer.Artikel III presenterar en strategi för applikationsspecifikantikroppsvalidering.Antikropparärnyckelreagensiimmunofluorescence,menallaantikropparharpotentialförkorsreaktivitetochmåstevaliderasnoggrant.Antikropparsprestationvarierarmellanprovtyperochapplikationerberoendepåförekomstenavkonkurrerandeproteinerochprovprepareringenspåverkanpåepitopet.Idennaartikelanvändesöverlappmedfluorescenttaggatproteinitransgena cellinjer för applikationsspecifik validering av antikroppar förimmunofluorescence.

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ListofpublicationsΙ StadlerC,SkogsM, BrismarH,UhlénM, LundbergE.A single fixation

protocol forproteome-wide immunofluorescence localizationstudies. JProteomics73(6),1067-78(2010)doi:10.1016/j.jprot.2009.10.012

ΙΙ Fagerberg L, Stadler C, Skogs M, Hjelmare M, Jonasson K, Wiking M,Abergh A, Uhlén M, Lundberg E. Mapping the subcellular proteindistribution in three human cell lines. J Proteome Res 10(8), 3766-77(2011)doi:10.1021/pr200379a

ΙΙΙ SkogsM, Stadler C, SchuttenR,HjelmareM,GnannC, Poser I,HymanAA, Uhlén,M Lundberg E. An antibody validation scheme forimmunofluorescenceusinggenetagging.Manuscript

RespondentscontributionstotheincludedpublicationsΙ Intellectualinputinstudydesignandmanuscriptwriting ΙΙ PerformedpartoftheIFexperimentsΙΙΙ Majorityofstudydesignandexperimentalplanning.Participatedinthe

laboratorywork.Allanalysis.Majorityofmanuscriptwriting.

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AdditionalpublicationsnotincludedinthisthesisFagerberg L, Hallström BM, Oksvold P, Kampf C, Djureinovic D, Odeberg J,Habuka M, Tahmasebpoor S, Danielsson A, Edlund K, Asplund A, Sjöstedt E,LundbergE, SzigyartoCA,SkogsM, Takanen JO,BerlingH,TegelH,Mulder J,Nilsson P, Schwenk JM, Lindskog C, Danielsson F,Mardinoglu A, SivertssonA,vonFeilitzenK,ForsbergM,ZwahlenM,Olsson I,NavaniS,HussM,Nielsen J,PontenF,UhlénM.Analysisofthehumantissue-specificexpressionbygenome-wide integration of transcriptomics and antibody-based proteomics.Mol CellProteomics13(2),397-406(2014)DanielssonF,SkogsM,HussM,RexhepajE,O'HurleyG,KlevebringD,PonténF,Gad AK, Uhlén M, Lundberg E. Majority of differentially expressed genes aredown-regulated during malignant transformation in a four-stage model. ProcNatlAcadSciUSA110(17),6853-8(2013)FagerbergL,OksvoldP,SkogsM,AlgenäsC,LundbergE,PonténF,SivertssonA,OdebergJ,KlevebringD,KampfC,AsplundA,SjöstedtE,Al-KhaliliSzigyartoC,Edqvist PH, Olsson I, Rydberg U, Hudson P, Ottosson Takanen J, Berling H,Björling L, Tegel H, Rockberg J, Nilsson P, Navani S, Jirström K, Mulder J,Schwenk JM, Zwahlen M, Hober S, Forsberg M, von Feilitzen K, Uhlén M.Contribution of antibody-based protein profiling to the human Chromosome-centricProteomeProject(C-HPP).JProteomeRes12(6),2439-48(2013)DanielssonF,WikingM,MahdessianD,SkogsM,AitBlalH,HjelmareM,StadlerC,UhlénM,LundbergE.RNAdeepsequencingasatoolforselectionofcelllinesfor systematic subcellular localization of all human proteins. J Proteome Res12(1),299-307(2013)JakobsenL,VanselowK,SkogsM,ToyodaY,LundbergE,PoserI,FalkenbyLG,BennetzenM,Westendorf J,NiggEA,UhlenM,HymanAA,Andersen JS.Novelasymmetrically localizing components of human centrosomes identified bycomplementaryproteomicsmethods.EMBOJ30(8),1520-35(2011)

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AbbreviationsBAC BacterialartificialchromosomeCDR ComplementaritydeterminingregionDAPI 4’,6-diamidino-2-phenylindoleDNA DeoxyribonucleicacidFab FragmentantigenbindingFc FragmentcrystallizableFPKM FragmentsperkilobaseoftranscriptpermillionmappedreadsGFP GreenfluorescentproteinHPA HumanproteinatlasIF ImmunofluorescenceIHC ImmunohistochemistryMS MassspectrometryPFA ParaformaldehydePTM PosttranslationalmodificationRNA RibonucleicacidWB Westernblot

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ContentsAbstract iListofpublications iiiAbbreviations vIntroduction 11.CellBiology 3

Thefirstdescriptionofthesmallroom 4Cellorganization 4Cellcultivation 5Visualizingcells 6

2.Genesandproteins 7Genes 7Proteins 8Howmanygenesandproteinsdowehave? 8Proteomics 9Proteomicsforsubcellularlocalization 10

3.Antibodies 13Antibodiesintheimmunesystem 13Antibodystructure 14Antibodydiversity 16Antibodiesasresearchtools 17Antibodybinding 18Antibodyapplications 20Antibodyvalidation 22Antibodygenerationprojects 23

4.Immunofluorescence 25Samplepreparation 25Fluorescencemicroscopy 27ValidationofIFresults 28

5.HumanProteinAtlas 29HPAantibodies 30SubcellularProteinAtlas 30ValidationstrategiesintheSubcellularProteinAtlas 32

6.Presentinvestigation 35PaperΙ 35PaperΙΙ 36PaperΙΙΙ 37Futureperspectives 39Acknowledgements 41

7.References 438.Appendix:Includedpublications 53

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IntroductionIn the quest to understand the human body we can choose to study it ondifferent levels;organismlevel,organlevel, tissuelevel,cell levelormolecularlevel.Thisthesisdescribesthestudyofindividualcellsinthelaboratory,whichare used as representatives of the cells in the body. Just like the tissues andorgans carry out specialized functions in the body, cells are made up oforganelles and specialized cellular compartments with distinct functions. Thepapers included in this thesiswere conductedwith the aim to determine theconstituentsofthesecompartments.Knowledgeofthebuildingblocksofthecellwillenableabetterunderstandingofthecellularmachineryandwillcontributetotheunderstandingofthemolecularbiologyofthehumanbody.Proteins are thebuildingblocks and themachineryof cells and constitute themajority of the cellular dry mass. To understand the cell it is thereforenecessarytostudyitsproteins.Proteinlocationisoneimportantaspectsinceitgivescluesonpossiblefunctionandinteractionpartners.The studies in this thesis have been performedwithin the framework of TheHumanProteinAtlasproject andhaveutilized the large amountof antibodiesproducedwithintheproject.Antibodiesareproteinsproducedbytheimmunesystem that protect against infections by recognizing and binding pathogens.Thenaturalbindingcapacityofantibodieshasmadethemanimportanttoolinboth research and therapeutics. Within The Human Protein Atlas projectantibodies towards all human proteins are produced and used to detect thetargetproteininamultitudeofassays.Localizationstudiesareperformedusingimmunofluorescence where antibodies coupled to fluorophores and used to

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staincells.Whenimagedwithconfocalmicroscopythisapproachenablesinsitulocalizationofproteinswithhighresolution.In this thesis I will first describe the background to the research area; cellbiology,genesandproteinsbeforemovingovertoadescriptionofantibodiesandtheiruseasresearchreagents.IwillthendescribeimmunofluorescenceandTheHuman Protein Atlas project and conclude with a description of the threepublicationsthatconstitutethisthesiswork.The threepapersdescribe someof the steps taken toproduce theSubcellularProtein Atlas. Localizing the entire human proteome is a huge effort and toachievethisgoalasinglestandardizedsamplepreparationprotocolsuitableforthe majority of proteins had to be chosen. The first paper describes theevaluation of different protocols for sample preparation forimmunofluorescence.Inthesecondpaperthisprotocolwasappliedto∼20%ofthehumanproteome.Thisshowedthattheproposedsetupforhigh-resolution,high-throughputlocalizationusingantibodiesandtheprotocolfrompaperonewasfeasibleandtheamountofdataproducedenabledinitialconclusionabouttheorganizationofthecellularproteomestobedrawn.The thirdpaperdealswithantibodyvalidation.Antibodiesarevery importantandcommonlyusedresearchreagentsthatcanbegeneratedtobindalmostanytarget protein of interest. However the binding between antibody and targetproteiniscomplexanddependentonassayconditionsandcompetingproteins,andthereforeantibodyperformancediffersmarkedlybetweenapplicationsandsamples.Inthispaperon-andoff-targetbindingandbatch-to-batchdifferencesin immunofluorescence were assessed for a large number of antibodies bycolocalizationwithfluorescentlytaggedprotein.

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1.CellBiologyThecellisthebasicunitoflifeandthebuildingblockofeveryorganism.Nooneknowsexactlyhowmanycellsthehumanbodyismadeupof,butanestimationfrom counting organ by organ states that there are over 1 000 billions!1Amongst thesecells thereareapproximately2300differentcell types thatareallspecializedfordifferentfunctionsandenvironment2.Thereareforexampleshort-livedepithelial cells in thecolon thatare shedafter5-7days3and therearelong-livedneuronsthatareformedinearlylifeandneverreplaced4.Thereare the small and motile sperm and the large egg that with its 0.1 mm canalmost be seen with the naked eye5. There are osteoblasts that form bone6thereby providing structural support to the body and there are leukocytes(white blood cells) that circulate through the body to detect and destroypathogens5.Severalcelltypescantogethermakeupatissueandseveraltissuestogethercanmakeanorgan.Thepancreas for example is anorganwith two functions; aiddigestion of food by secretion of digestive enzymes into the intestine andcommunicatethenutrientstatustothebodybysecretionofhormonesintothebloodstream. These two functions are performedby twodifferent tissues, theexocrine and the endocrine. The exocrine tissue consists of two cell types7;acinar cells that secrete digestive enzymes such as proteases, amylases andlipasesandductcellsthatsecretefluidthatmixwiththedigestiveenzymesandfood.Theendocrinetissuesconsistsoffivecelltypes8;themostabundantbeinginsulin-producing beta cells but also alpha, delta, epsilon and pancreaticpolypeptidecellsthatsecretethehormonesglucagon,somatostatin,ghrelinandpancreaticpolypeptiderespectively.

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ThefirstdescriptionofthesmallroomThe knowledge of our microscopic constituents would never have beenachievedwasitnotforthedevelopmentofmicroscopesandsamplepreparationtechniques. The first microscopes were developed simultaneously by severalscientistsstartingwithsimplelensesformagnificationandreading.In themiddle of the 17th centuryHooke used a compoundmicroscopewith adouble lenssystemtoprovidethe firstdetaileddescriptionof themicroscopicworldinhisMicrographiapublishedin1665.Hecoinedandusedthelatinwordcell,meaningasmallroom,todescribethestructureofdeadcork9butdidnotknowthathewasactuallyvisualizingtheelementaryunitoflife.Theself-taughtmicroscopistLeeuwenhoekusedasmallself-madehigh-magnifyingsingle lensmicroscope to study and first describe living cells of a number ofmicrorganisms9, 10. Many scientist worked with microscopic examinations ofcellsandin1838and1839SchleidenandSchwannformulatedwhatcametobeknown as the cell theory; that states that both plants and animal tissues aremadeupof elementaryunitswhichare cells9. Itwas still unknownwhere thecellscamefromandthetheoryofspontaneousgenerationstatedthatinanimatematterspontaneouslygenerateorganisms.ThankstomanyresearcherssuchasvonMohlwhodiscoveredcelldivisionbypartitioning,andRemakwho tracedindividual cells in frog embryos11, Virchow could in 1855 postulate that cellscome from cells. During the nineteenth century further improvements inmicroscopy made it possible to visualize smaller subcellular structures andmanyofthelargerorganellescouldbedescribed.

CellorganizationThe cell is highly organized into different compartments with differentfunctions(Figure1).Compartmentsthatareenclosedbyamembranearecalledorganelles. The cell is surrounded by the cell membrane, also called plasmamembrane, which shields the cell from the environment and preserves theintracellularmilieu.Itisalipidbilayerofphospholipidswithhydrophobiccoreand hydrophilic surfaces. The membrane contains proteins involved incommunicationandtransportinandoutfromthecell.Inthemiddleofthecellis thenucleussurroundedby thenuclearmembrane. Inside thenucleus is theDNA and all the regulatory proteins that affect which geneswill be active orsilent.Further,thenucleushasmanyspecializedsubdomains,thebiggestisthenucleolusinvolvedinsynthesisandprocessingofribosomalRNA12.Therestofthe cell outside the nucleus is the cytoplasm that contains the soluble cytosoland many organelles and compartments, such as the mitochondria thatproduces the majority of the energy needed by the cell, the membranous

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networkoftheendoplasmicreticulumwheremembraneandsecretedproteinsaresynthesizedandlipidsproduced,theGolgiapparatusthatmodifiesproteinsanddispatchthemforvesicletransporttotheirfinaldestination.Anetworkofthree different cytoskeletal filaments provides shape, robustness andtransportation routes5. Actin filaments support the plasma membrane, formsprotrusions and is involved inmovement. Intermediate filaments support thenuclear membrane, provides mechanical strength and is important for cellsunderhighmechanicalstresssuchasskin,hairandnails.Microtubulefilamentssupports the spatial organization of organelles and provides a transportnetworkthroughoutthecell.

Figure 1. Schematic representation of a cell with selected organelles andsubstructures.AdaptedfromanillustrationbyAnnicaÅberg.

CellcultivationNormal cells in the body reside in a tissue context and rely heavily onintercellular communication to coordinate building and maintenance of thattissue.Asafetysystemisinplacesothatthecellswillonlyproliferategiventheappropriatesignalandwillcommitapoptosisratherthanrisktissueintegrity.Ifcells like this are removed from their context and cultivated invitro,theywillnot proliferate. In tumors, genetic aberrations renders the cells insensitive to

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these controls and the cells proliferate uncontrollably and eventually ametastasis can form where the cell acquire the ability to grow in a differentplace than its intentional. These deregulated cells are easier to grow in thelaboratory as they are immortal and continue to proliferate and thereforecancer cells were the first human cells to be grown in the laboratory in the1950s13. Cells canalsobe immortalized invitro bymutationsor expressionofrelevantproteins14. Invitro cultivatedcell lineswillnotbe identical to their invivo counterpart. In general cells grown in the laboratory down regulateexpressionoftissuespecificgenesandgenesinvolvedinadhesionandsignalingwhilegenesinvolvedinproliferationandenergymetabolismareupregulated15,16.Cancercelllinesandinvitroimmortalizedcelllinesarestillimportantmodelsystemsbecausetheyare fromhumanoriginyetcanbemanipulatedandtheycanbeexpandedtolargenumbersandstoredinthefreezerforlongerperiods.

VisualizingcellsCellsarecolorlessandtranslucentandhardtoseeunderthemicroscopeduetolack of contrast. Therefore cells are normally visualized with phase-contrastmicroscopy or after staining with a contrast-enhancing dye. There are manydifferentdyesusedtostaincellssuchashematoxylinthatstainnucleiblueandeosin that stain cytoplasm and cell membrane pink. Fluorescent microscopyuses fluorescent molecules for detection. These can either be small-moleculedyes that stain different compartments and organelles such as DAPI (4’,6-diamidino-2-phenylindole) that binds to DNA17, fluorescent proteins such asgreenfluorescentprotein(GFP)orfluorescentprobesconnectedtoanaffinity-bindingreagentthatbindsthetargetprotein.Themostcommonlyusedaffinity-binding reagents are antibodies discussed in chapter 3. Several of the smallmoleculestainsaswellasantibodystainingis incompatiblewith livecellsandrequire sample preparation by fixation and permeabilization that will bediscussedinchapter4.

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2.GenesandproteinsProteins are the building blocks and the machinery of cells; they form theorganelles, they execute nearly all functions, they listen to stimuli from thesurroundingsandtheysendsignalstoothercells.Genesareblueprints fortheproductionofproteinsandeverycellinthehumanbodycarriesthesamesetofblueprintsintheirDNA(withafewexceptionslikeBcellsdescribedinchapter3andthegametes).Thedifferenceobservedbetweencelltypesarisesnotfromthegenes itself,but fromavaryingexpressionof thegenes.Thisresults intheproduction of a cell-type specific set and amount of proteins. The humangenomewas sequenced in200118,19 andknowledgeof the genetic codewas atremendously important step towards understanding of human biology.However thiswasonly a first step andweare still struggling to answerbasicquestions like which parts of the genome that are translated to proteins andexactlyhowmanyfunctionalgenesandproteinswehave20.

GenesDNA (deoxyribonucleic acid) is located inside the nucleus and carriesinformationfromonegenerationtothenext.Itconsistsoftwonucleotidechainsthattogetherformahelix.Eachnucleotideconsistsofthesugardeoxyribose,aphosphategroupandanitrogen-containingbase5.ThesugarandthephosphateformthebackboneoftheDNAchainandthebasesprotrudeandbase-pairwithbasesfromanotherchaincreatingadoublehelix.Therearefourdifferentbases;guanine(G),adenine(A), cytosine(C)and thymine(T)andA isalwayspairedwithTandGwithC.ThetotallengthofDNAinahumancellisabout2m,and

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this is divided into 46 chromosomes and tightly packed to fit in the nucleus5.ThegenomeistheentireDNAcontentinacellandageneisastretchofbasesthattogetherformablueprintfortheproductionofaprotein.Thefourdifferentbasesareread in tripletscalledacodon. In theblueprint thesecodonsdenotedifferent amino acids and the order of the codons in a gene determines theorder in which the amino acids shall be joined together to build a protein.Combining4differentbases into tripletsproduces64different combinations5,someofthesedenotethesameaminoacid,andsomearestartandstopcodons.TheprocessofproducingproteinsfromDNAstartswithDNAbeingtranscribedintomessengerRNA(ribonucleicacid).ThisisamoleculecloselyrelatedtoDNAbut it is single stranded and contains the sugar ribose instead of deoxyriboseandthebaseuracil(U)insteadofthymine.RNAistranscribedfromthegeneinthenucleusandthentransferredtothecytoplasmwhereitmeetsaribosome,acomplex of RNA and protein that is the factory building proteins from theinformationembeddedintheRNAsequences.

ProteinsProteinsarebuiltupofastretchofaminoacids.Eachaminoacidconsistsofacentralcarbonatom,acarboxylicacidgroup,anaminogroupandasidechain.Thecarboxylgroupbondswithanaminogroupofanotheraminoacidstoformthe backbone of the protein chain. There are 20 different side chains5 withdifferentchemicalproperties;hydrophobic,polarorcharged.Everyproteinhasa particular three-dimensional structure determined by the amino acidsequence5. They fold so that hydrophobic patches meet in the inside,hydrophilicresiduesareontheoutsideandsothatchargedresiduesofdifferentpolarity interact. The primary structure is the order of the amino acids, thesecondarystructurearethehelixesorβ-sheetsthattheaminoacidchainformsandthetertiarystructureisthefullthree-dimensionalfoldoftheprotein.Someproteins form complexes with other proteins thereby building a quartenarystructure.

Howmanygenesandproteinsdowehave?Only about 1% of the genome codes for proteins21. There are also regulatorysequencessurroundingthegenesthatregulategeneexpressionandnon-codinggenesthataretranscribedtoRNAbutnottoprotein.TheseRNAmoleculescanhave regulatory functions or carry out important tasks in the cell such astransferRNAthatfetchesaminoacidsinthecytoplasmandguidesthemintothe

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correctposition in the ribosome5.Ensembleversion83.38 (www.ensembl.org,October 2015) contains 20 313 protein coding genes and 25 180 non-coding.Theremainingsequenceisreferredtoas“junk”DNAsinceafunctionhasnotyetbeenfound.Atypicalgenecontainsseveralcodingregions,exons,withnon-codingregions,introns,inbetween.TheentirestretchofDNAisfirsttranscribedfromthestartcodon to the stop codon. The produced RNA is then processed to cut out theintrons. This can be done is several ways, using different numbers of exons,resulting in different length and properties of the produced proteins, calledsplicevariants.After theprotein isproduced it is subject topost-translationalmodifications (PTMs) where different functional groups such as sugar areattachedatspecificsites.BydifferentialsplicingandPTMsasinglegenecangiverisetoseveralproteinvariants.Thesevariantsmayhavecommontraitsbutthedifference can result in that they interact with different partners, performslightlydifferentfunctionsoraretransportedtodifferentlocations.DuetothemodificationsonRNAandprotein level thenumberofproteinsarefargreaterthanthenumberofprotein-codinggenesanditisdependentonthedefinitionofwhat constitutesadifferentprotein.Todetermine thenumberofprotein-coding genes predictions aremade from the gene sequence based onknowledgeofwhataprotein-codinggene should look likewith start andstopcodon,regulatorysequencesetc.Buttheonlywaytoknowhowmanygenesareprotein-codingistoobservetheproteinbyexperimentalmeans.Inthereleaseof neXtProt22 3.0.28 (www.nextprot.org, January 2016) 18% of the predictedprotein-codinggeneshadnoexperimentalevidenceonproteinlevel.

ProteomicsTo truly understand cells we must study their proteins. There are manydifferentaspectsof importancetostudy,suchasexpressionpatterns, location,interactions, pathways, splice variants, PTMs, structure and biochemicalfunctions23. Proteomics is the study of proteomes, a proteome being all theproteins inacomplexmixture,oftenusedtodenotetheproteomeofaspeciesbut also to denote a smaller mixture such as an organelle proteome or apathwayproteome.Duetothecomplexityofproteinisoforms,thecollectionofproteinsinacellislargeandcomplexandnoteasytodefine.Someproteinsarerequired for general cell maintenance and survival, called housekeepingproteins,andhaveasimilarexpressionlevelsinallcells.Butalargepartoftheproteomeisspecializedandisexpressedincertaincelltypesoratacertaintimepointsinacellslife.Thereforetheproteomeisalsotime-dependent.

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ProteomicsforsubcellularlocalizationOne of the protein attributes that should be characterized in order tounderstandcellbiologyisproteinlocation.Forproteinswherethereis limitedor no information available, knowledge of its location gives clues on possiblefunctions and interaction partners and constitutes a starting point for furtherstudies.Definingtheproteomesofsubcellularstructuresandorganelles isoneimportant step towards understanding cellular anatomy. Protein localizationcan be donewithmass spectrometry (MS) or by the imaging basedmethodsimmunofluorescence(IF)andfluorescentproteintagging.WhenusingMSforsubcellularlocalizationcellsaretraditionallyfractionatedbycentrifugation, and the different fractions analyzed. Advantages of MS is thesimultaneousanalysisofthousandsofproteinsandtherelativeeasewithwhichproteinscanbequantifiedanddynamicchangesfolloweduponperturbation24-26.Traditionalfractionationbycentrifugationislimitedinwhichcompartmentscan be separated, and the level of purity. But with newer methods such asProtein Correlation Profiling (PCP)27, 28, LOPIT29 and proximity biotinylation(BioID)30 even some compartments that cannot easily be fractionated can beanalyzed. With imaging based methods only one or a few proteins can beanalyzed simultaneously, but a very high resolution can be achieved indecipheringorganellesandsub-compartments.WhilstMSapplicationsanalyzesanaverageacrossabulkofcells, imagingbasedmethodsdetect theprotein insitu with single-cell resolution and can detect cell-to-cell variability, such asproteinsthatchangelocationorexpressionlevelduringthecellcycle.HowevertherearestilllimitationsinforgenomewidelocalizationsstudiesMShas not been the method of choice due to the limitations in deciphering allorganellesandsub-compartments.Inthisregardimagingbasedtechniquesarebettersuited.Imagingbasedmethodsdetectstheproteininsituwithsingle-cellresolution and can reach high spatial resolution. Whilst MS applicationsanalyzesanaverageacrossabulkofcells, imagingbasedmethodshavesingle-cell resolution and can detect cell-to-cell variability, such as proteins thatchangelocationorexpressionlevelduringthecellcycleTaggingproteinsofinterestwithfluorescentproteinssuchasGFPisacommonstrategy for studying protein function and location. This has been donesuccessfully in genome-wide studies in yeast31,32 and for parts of the humanproteome33. An advantage with this method is the possibility to follow theprotein over timewith live cell imaging to detect protein dynamics. Potentialproblemswith taggedproteins in localization studies aremislocalizationsdueto the tag or due to overexpression34 and the laborious task of creating cellsexpressingthetaggedprotein.

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IFuses fluorescently labeledantibodies forsubcellular localizationandwillbediscussed in chapter 4. IF is a powerful tool for localization studies as everyantibodycanbeusedtoevaluatetheendogenousproteinlocationacrossmanydifferentcellandtissuesamples.

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3.AntibodiesAntibodiesareapartofthe immunesystem.Theyareproteinsmadebywhiteblood cells called B cells and protect against infections by recognizing andbinding pathogens. Every B cellmakes a unique antibody,with affinity for adifferent target, and this large variety provides protection against a widerepertoireofpathogens.Scientistshavetakenadvantageofthenaturalbindingcapacityof antibodies anduse themas tools for research anddiagnostics andfortreatmentofdisease.Wecannowproduceantibodiestargetingbasicallyanyproteinwewanttostudyandwecanmodifytheantibodyproteinintobindingreagentswithnewproperties.

AntibodiesintheimmunesystemThehistoryofourknowledgeonantibodiesstartswithobservationofimmunityto the smallpox, one of the world’s most devastating illnesses that has onseveral occasions wiped out large parts of the population35. The observationthatsurvivorsacquiredimmunitytothediseaseleadtothewidespreadpracticeofvariolation,wherematerialfrominfectedindividualswereintroducedtonon-immune individuals forprotection36.Thiswas a ratherunsafeprocedurewithside effects in the formof acquired smallpox or other transmitteddiseases. Itwasalsowidelyknownfolkwisdomthatmilkmaidsafteracquiringthecowpoxwere immune to small pox and this lead to the procedure of vaccination thatwasdescribedbyJennerin177636.

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Itwasat this timeunknownhow immunitywasconferredbutduring the late19th centurywhenBehring andKitasato showed that transfer of sera fromanimmunized animal could cure an infected animal37 it was apparent thatimmunityresidedinsera.Ehrlichhypothesizedthatbloodcellsproducedside-chainswithbindingsitesbindingtotoxinsandpathogens38andwasthefirsttousethetermantibody39.Inthe1920sand30sAvery,HeidelbergerandMarrackshowedthatantibodieswereproteins40,41andFagraeusshowedthattheywereproducedbyBcells42.Bcellsareproducedinthebonemarrowfrompluripotenthematopoieticstemcellsthatgiverisetoallbloodcells43.InthebonemarroweachBcellundergoesgene rearrangements that determines the type of antibody that the cell willproduce.When theB cells leave thebonemarrow they express antibodies ontheoutsideof thecellmembrane,Bcellreceptors,andcirculatethebloodandlymphsystemtodetectintrudingpathogens.Uponbindingofatargetthecellisstimulated to proliferate and start secreting soluble antibodies to fight theinfection.Antibodiescaninactivateapathogenbybindingtoitandcoveringitbut binding also attracts other immune system cells such as phagocyticmacrophages that destroy the target44. The initial pool of antibodies have aflexible binding site with capacity to adapt to a range of similar targetsenhancing the immune systems potential for detection of pathogens45. Uponinfection several different antibodieswill bind to the intruding pathogen andthecellscarryingantibodieswiththestrongestbindingwillmultiplytoproduceasmuchantibodiesasareneeded5.TheBcellwillalsomakesmalladjustmentsofthegenomecodingforthebindingsitetoallowevenbetterbinding.SomeoftheseBcellswithatailoredbindingsitewillpersistandcanquicklybeactivatedagain upon subsequent encounter of the same pathogen. This provides long-termimmunityandisthephenomenausedinvaccination.

AntibodystructureAll antibodies have the same basic Y shaped structure (Figure 2) with twoidenticalarmseachharboringabindingsiteanda long leg that interactswithother parts of the immune system. It is a protein complex consisting of fourproteinchains,twoidenticalheavychainsandtwoidenticallightchains44.Boththe light chains and the heavy chains consist of one variable region and oneconstantregion.Thevariableregionhashighvariabilitybetweenantibodiesandencompasses the binding site whereas the constant region is more similarbetweeneachantibody46.ThetwoarmswiththeantigenbindingsitesarecalledFabfragmentsforfragmentantigenbindingandthestemiscalledFcfragmentforfragmentcrystallizablefromtheearlystudieswithenzymesthatcleavedtheantibody producing three fragments44. There are three distinct regions in the

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variable chains that have the highest variability, denoted hypervariableregions44. The hypervariable regions are close together on the surface of thethree-dimensional protein structure and form the complementarity-determining regions, CDRs46,which are responsible for binding.One light andoneheavychainpairtocreateabindingsurfacewheretheCDRsmeet,andtwoof these dimers then join together through the heavy chains to form the Y-shapedantibody.

Figure 2. Model of an antibody based on X-ray diffraction and schematicrepresentation of an antibody and the nomenclature to describe its parts.Adaptedfrom1IGT47usingSwiss-PdbViewer

Antibodiesarealsoreferredtoas immunoglobulins(Ig)andhumanshave fiveimmunoglobulin classes, IgM, IgD, IgG, IgA and IgE divided on basis of theproperties of the heavy chain constant part44 (the Fc fragment). This partdetermineswhichFcreceptors,andtherebywhichcelltypes,theantibodywillinteract with. IgG is the most prevalent antibody in blood and recruitsphagocyticcells.IgAismorecommoninsecretionsandtheirspecialFcportionisrequiredfortransportationtothesesites.IgMisthefirstantibodytoappearin an immune response, typically has low affinity but is a potent complementactivator.IgDisrareinserumandmainlyboundtothesurfaceofBcellsanditsfunctionis largelyunknown48, IgEprotectsagainstparasitesandis involvedinallergies.Allclassescanbemadeasamembrane-boundandasasecretedform.After leaving the bone marrow B cells make membrane-bound IgM and IgD.Thesefirstantibodieshaveapromiscuousbindingandcanbindseveralsimilarpatterns allowing the immune system to recognizing a larger number ofpathogens. Upon activation cells switch to secretion and later in the immuneresponsesomecellsswitchantibodyclassandstartsproducingIgG,IgAorIgE5,49 thathasastrongerbindingduetotheaffinitymaturationprocessdescribedbelow.

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AntibodydiversityFormostproteinstheaminoacidsequenceisdeterminedbythegenomeandisidentical in every cell. This can however not be true for antibodies since thehumanbodycontainsbillionsofBcellseachproducingantibodieswithauniquebindingsite.InsteadthediversitystemsfromrearrangementsofDNAduringBcell development in the bone marrow complemented by fine-tuning of thebindingbypointmutationsthatoccurafterbindingofantigen.Thisisachievedthroughfourdifferentprocesses5:combinationsofdifferentgenesegmentsintoa heavy or light chain, junctional diversity from joining these segments in animpreciseway,theparingofheavyandlightchainandaffinitymaturationafterbindingofantigen.Theconstantparts,C,consistofonegenesegmentandthevariableregion,V,isacombinationofgenesegments:2forlightchainsand3forheavychains.Thereare several different versions of the segments and already combinations oftheseleadtoalargediversity.Thejoiningofthesesegmentsintoasinglegeneisdonebyhomologousrecombinationusingrepetitiveflankingsequences.Thisisdone in an imprecise way with a random number of bases being added orremoved which adds variations by changing the structure of the antibodyproteinandhowtheCDRswillbepositioned.Thelightandtheheavychainsarerearrangedirrespectiveandthecombinationofthesetwoisonemoresourceofvariation. These three processes that happen in the bone marrow under theearly B cell development often result in failure to produce an antibody; forexample by gene rearrangement that disrupts the regulatory sequences or bychangingtheopenreadingframe.Alsonotallheavyandlightchainsareabletobind each other in the correct way to form a functional entity. It is evenestimated that thisprocedure failsmoreoften than it succeeds5,50.Bcells thatfailtoproduceanantibodyarenotgivensignalstoproliferateanddiethroughapoptosis43. It is also possible that the produced binding site binds to a self-protein, a part of the organism. This is called autoimmunity and these cellswouldinadiseasefreestatereceivesignalstoactivateapoptosis43,50.Thefourthprocessofdiversity,affinitymaturation,differsfromtheothersandtakesplace after theB cell receptorhasbounda target51. Pointmutationsareintroducedinthevariableregionoftheantibody.Thesepointmutationsoccurmuch more frequently52 than spontaneous mutations and are called somatichypermutations.Upon infectionseveralBcellswillhaveantibodiesbinding tothepathogen,butwithdifferentaffinitiesandthecellswithhighestaffinitywillpreferentially be selected for proliferation by stimulation from other immunesystemcellsandoutcompeteweakerbinders5.Pointmutationsthatimprovetheaffinity will thus be multiplied. This allows the immune system to tailor theoriginalantibodyrepertoiretomatchtheintrudingpathogen.

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AntibodiesasresearchtoolsDuetotheirabilitytospecificallybindthetarget,antibodiesweresoonusedbyresearchersasatooltostaintissuesandcells.Todayantibodiesareextensivelyusedinresearchforavarietyofapplications.Apartfrombeingusedasreagentsin imaging procedures they are used to detect proteins in plasma and otherfluidsandtocapturethetargetfromacomplexsolutionforsubsequentanalysis.Therearealso47antibody-basedbiopharmaceuticalsusedtotreatavarietyofdiseases53andmanymoreareusedasdiagnostictools54.E.g.stainingoftumorbiopsies with an antibody towards Ki-67 is a clinical routine to assessproliferationstatusofdifferenttumorsasaprognosticmarker55.The first antibodies used for research were obtained by immunizing animalswiththeproteinofinterest,orasmallerpartoftheprotein.TheanimalsBcellsproduce antibodies and after some time the serum can be collected. Thisprocedure produces polyclonal antibodies, a mixture of antibodies withdifferentbindingsitesbindingtodifferentpartsofthetarget. Immunizationofthesameantigeninasecondanimalwillresultinadifferentbatchofantibodieswith potential differences in the binding sites and especially in the relativeamountsofantibodiestowardseachbindingsite56.Thismeansthatifavaluablepolyclonalantibodyrunsout it isnotalwayseasytoobtainanotheroneofthesamequality.NormalantibodyproducingBcellscannotbemaintainedinthelaboratorybutin 1975 Köhler and Milstein fused B cells with myeloma cells, a cancer celloriginatingfromwhitebloodcells,creatingthehybridomacell linethatcanbepropagatedinthelaboratoryandthatsecreteantibodiesofthespecificityoftheoriginal B cell57. These antibodies with identical binding sites are calledmonoclonalantibodies.Themonoclonalantibodywasamilestoneforthefieldsof therapeutics and diagnostics since it ensured unlimited supply ofantibodies54.AsamplecontainingalargenumberofBcellsfromanimmunizedanimalareusedasstartingmaterialandalargepartoftheworkisscreeningtheobtainedhybridomaclones forantigenbindingsinceonlyasmall fractionwillbind the protein of interest. Today there are also recombinant monoclonalantibodies58 where the gene sequence can be manipulated by geneticengineering to combine different gene fragments, remove gene fragments orchange amino acids. This enables creation of custom made antibodies, forexample by combining any antibody class with any binding site, making anantibody with two different binding sites or attaching a toxin on antibodiestargeting tumors59.Theproducedgenesequences can thenbe introduced toaproductionsystem,suchascelllines,yeastorbacteria60.It is also possible to select binders in vitro thereby circumventing theimmunizationstep.Inphagedisplay59,60afabfragmentlibraryisexpressedon

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thesurfaceofbacteriophagesandthetargetproteinisthenusedtofishoutanyphagethatbindstoit.ThelibraryiscreatedbyamplifyinggenesegmentscodingforthevariableregionoftheheavyandlightchainsbyPCRfromalargenumberofB cells andassemble into fab fragments61.Theproduced fab fragmentgenesequencesareintroducedintothegenomeofbacteriophagesfusedtothegeneforacoatprotein.Afterfishingoutthegoodbindersthegenesequencescanberetrievedbysequencingtheboundphages.There are several different binding reagents developed from antibodies59, 60.These are often smaller parts of the antibody since the smaller size permitsbetter tissuepenetration,enablesproduction insimplerorganismsand lossoftheFcpartlowersimmunogenicity.ExamplesaretheFabfragmentthatcanbeused alone or as a F(ab)2 with two joined fragments and the single chainfragment variable (scFvs), thatusesonly thevariablepartsof light andheavychains linked together62. There are also binding reagents based on otherscaffolds than antibodies. Examples are Affibodies63 that are based on theantibody-binding domain of bacterial Protein A and designed ankyrin repeatproteins (DARPins)64 based on the ankyrin protein used for protein-proteininteractioninmanyorganisms.

AntibodybindingThe natural binding capacity of antibodies is what makes them outstandingresearchreagents.Butthenaturalpromiscuityofbindingandthecomplexityofperformance across samples and applications is a challenge to researchers.Proper use of antibodies as research reagents must always include suitablevalidationoftheantibodiesandtheobtainedresults.EpitopesandparatopesThemoleculethatisrecognizedbytheantibodyiscalledantigenandthepreciseamino acids that is bound by the antibody is the antigenic determinant orepitope44. Antibodies can bind either linear or conformational epitopes, thelinearbeingaconsecutivestretchofaminoacidsdirectlyfollowingeachotherintheproteinsequenceandtheconformationalepitopebeingaminoacidsthataredistantintheprimaryaminoacidsequencebutbroughttogetherbythefoldofthe protein44. A linear epitope is 5-9 amino acids65, 66. These short epitopeswouldtheoreticallygiverisetomuchcross-reactivitytootherproteinsbutitisalso a conformational component involved, and the linear epitope must bepositioned correctly for recognition65. Which type of epitope is generated isinfluencedbythestateoftheantigenusedtoimmunizetheanimal.Full-lengthproteins and longer antigens are more likely to fold and give rise to

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conformationalepitopeswhilstsmallpeptidesgiverise to linearepitopes.Theepitope type have big implications for antibody performance, an antibodyrecognizingaconformationalepitopecannotdetectthetargetinaWesternblotwithdenaturedproteins67andviceversa.Some protein sequences seem to trigger the immune system and areimmunogenichotspots,andantibodiestowardsthesewillbeobservedineverybatch of polyclonal antibodies. It is wise to avoid very short peptides forimmunization since a short sequence may not include more than one highlyimmunogenic epitope. Polyclonal antibodies consist of a pool of antibodiesbinding different epitopes. Different applications and sample preparationtechniques affectswhichepitopes are available andpolyclonalsworkacross abroader rangeof applicationsdue to their ability tobinddifferent epitopes. Ifpolyclonalantibodiesareseparated intodifferentbatcheswithsingleepitopesthey are less likely to function across applications68. The binding site of theantibody contains about 50 amino acids but only about 15 are involved inbindingtoanantigen,thesearecalledtheparatope49.AffinityAffinityisameasureofthebindingstrength.Thebindingbetweenantigenandantibody is achieved by noncovalent interactions, so calledweak interactions,whichincludehydrophobicforces,electrostaticforces,vanderWaalsforcesandhydrogenbonds44,69.Toachievea strongbindingwithonlyweak interactions,many interactionsneed tobepresent69and thebestaffinitybetweenabinderandatargetisachievedwhenthereisperfectshapecomplementaritybetweenthebindingsurfacesandperfectlymatchedchargedistribution70.Thestrengthof the interactions is affected by ionic strength, pH and detergents44 andwilltherefore vary with sample type and experimental conditions. Other factorssuch as protein interactions, PTMs, and fixation affects which epitopes areexposedandavailableforinteractions.On-andoff-targetbindingAntibodies can have on-target and off-target binding (Figure 3), on-targetdenoting binding to the intended antigen and off-target binding beinginteractions with other proteins. The off-target binding can either behydrophobic stickiness or electrostatic interactionswith the Fc part or cross-reactivity involving the CDRs in the antigen binding site49, 71. Cross-reactivitycanbeduetoasimilarepitopeonanotherproteinor theparatopebindinganunrelated epitope through conformational flexibility.Due to that the antibodybindingsiteconsistsofapproximately50aminoacidsandatypicalparatopeofonly 15, the antibody binding site can contains several paratopes capable ofbindingdifferenttargets49.

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Figure3.On-andoff-targetbindingofantibodies.

SpecificitySpecificity of an antibody is the ability to discriminate between severalmolecules competing for the binding site49. Specificity for a monoclonalantibodydependson theaffinitiesof competingmolecules relative to target70.For a polyclonal antibody the specificity also depends on the fraction ofantibodies that interact with the competing molecule72. When only a smallsubpopulation interacts with the cross-reactive molecule the polyclonalantibodymixture can still be considered specific. The promiscuity of the low-affinity IgM/IgD antibodies is due to a high conformational flexibility of thebinding site45. A flexible binding surface that can interact with a variety oftargetscannotreachhighaffinitiesdueto theenergycostof inducedfit70.Theaffinity maturation process that produces high affinity antibodies leads tohigherrigidityofthebindingsiteandtailoringofthesurfaceanditsfunctionalgroups to the target49. High affinity can be related to high specificity in thathigher affinity involves a more rigid geometrical binding area that excludesmostcompetition70.Antibodyselectivityistheabilitytodetectselectivelyonlythetargetproteininanassay73.Selectivityisdependentonthedetectionlimitoftheassayaswellasthe affinity and concentration of competing molecules relative to the target.Almostallantibodieshavethecapacitytointeractwithmoleculesotherthantheintended target, albeit hopefully with much lower affinities. Low affinityinteractionsmay fallunder thedetection limitof thesystembuta lowaffinitycompetitorinhighabundancemaybedetected.

AntibodyapplicationsProtein research and proteomics need binders against all proteins and allpossible protein variants and today polyclonals, monoclonals and different

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fragmentsareallextensivelyused.Polyclonalshavethebenefitofquickerandcheapergenerationandthepotential toworkacrossmanyapplicationsduetothe different pools of included antibodies performing in different settings74.Monoclonalswill never run out and can be tailored to the specific needs, buttakeslongertimetodevelopandhaveahighercostofgeneration.Researchers typically use several different techniques to study a protein andcan benefit from the broader performance of polyclonals. In therapeutics anddiagnostics antibodies are typically used for one application only, requires astable source of reproducible binders and cannot use polyclonals due topotentialimmunogenicity53.Antibodies and other binders are used in a large number of differentapplications todetect, capture,measureor visualizeproteins. InWesternblot(WB)antibodiesareusedtodetectproteinsinamembraneafterseparationbysizethroughelectrophoresis75.Inenzyme-linkedimmunosorbentassay(ELISA)antibodiesareusedtodetect targetproteins insamplessuchasplasma,bloodand urine by either attaching the sample or antibodies to a solid support,applying the other reagent (antibody or sample), anddetecting binding by anenzymereaction76,77.Thecommonpregnancytestsdetectthehormonehumanchorionic gonadotropin in urine by a sandwich ELISA using two antibodiestargeting different parts of the protein78. In immunoprecipitation79 (IP),antibodies are used to capture proteins from a solution, and the proteins canthenbeanalyzedbyWBorMS80.Inflowcytometry,antibodieslabelproteinsonthe cell surface and labeled cells can be distinguished from the rest of thepopulation81.Inimmunohistochemistry82(IHC)antibodiesareusedtostainthintissuesectionstoexamineproteinexpressionacross tissuesandcell types. InIF,describedinthenextchapter,antibodieslabeledwithfluorophoresareusedtostaincellsortissuestodetectlocationofproteins.Severaloftheapplicationsdescribedaboverequiredetectionoftheantibodiesafter binding of the target. Visualizing binding can be done in several ways.Either the antibody itself is labeled or more commonly the target-bindingantibodyisdetectedbyasecondarylabeledantibody.Inthesecondaryantibodyapproachtheprimaryantibodytargetingtheproteinofinterestisfirstallowedtobind.Thenafterwashingawayunboundantibodythesecondaryantibodyisapplied. Secondary antibodies bind the constant part of the primary antibodyandwilldetectanyantibodyfromthatspecies.Theuseofsecondaryantibodiesisconvenientsinceitcircumventslabelingofeverysingleantibody.Antibodiesaretypically labeledwithenzymesproducingacoloredproductuponadditionof substrate or fluorophores but radioactive isotopes and gold are also useddepending on application. It is often possible to use multiple antibodiessimultaneously,labeledwithdifferentfluorophoresorenzymes.

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AntibodyvalidationThe promiscuity of antibody binding combined with the large number ofcompeting epitopes available in a sample makes validation of antibodyspecificity crucial toavoid false results.This involvesa combinationof carefulantigen selection, antibody purification, validation of produced binders andapplication-specificvalidationofresults83.Antigensmustbe selected tominimizepotential cross-reactivityderived fromsimilaritytootherproteinsandoneshouldconsiderwhethertheantibodywillbeusedinapplicationswithfoldedordenaturedprotein.Shortantigensof10-20aminoacidswillbe less likely to foldandwill result in linearepitopesandperform well mainly in applications with denatured protein while full-lengthproteinswillleadtomoreconformationalepitopes.Designingantigenbasedonlow sequence similarity to other proteins avoids cross-reactivity of linearepitopes but not potentially similar conformational epitopes. Differentstrategies forantibodypurificationcanbeused.ThereareaffinitypurificationmatricesthatbindallIgGsandaffinitypurificationusingtheantigenasligand.Itis a common strategy to validate all produced antibodies with a standardmethod,oftenWB84.Howeverthespecificityoftheantibodywilldependontheconcentration of off-targets relative to target85 and the effect of samplepreparationonavailabilityofepitopes.Thereforeitisimportanttovalidatetheantibodiesintherelevantapplicationandsampletype86.Thiscanbeincludedaspart of the experimental design and the strategies will vary for everyapplication,butstrategiesthatareoftenaverygoodchoiceistheuseofdoublebinders83 or knockouts84. Non-specific off target effects are avoided by usingblockingagentsinthebufferthatblocknon-specificbindingsitesorcontrolledfor by performing parallel control experimentswith a similar antibody of thesameclassandorigin.Most researcherswithin biological fieldswill use antibodies at some point intheir career, and often they are chosen from a commercial supplier of whichtherearenumerous.Oneofthewebsitesthatlistandcompareantibodiesholdsmore than 2 million products from 71 providers (www.antibodypedia.com,2016-04-11). During the last few years there have been several storieshighlighting problems with specificity and reproducibility of antibodies86, 87leading to a discussion and increased awareness of the complexity of usingantibodies. Studies have shown that many antibodies bind more than oneprotein88,89andthatabouthalfof5000commerciallyavailableantibodiesfailastandardized IHC analysis90. There are efforts to standardize validation ofantibodies91 but a consensus has not been reached and may likely never bebecause of the different demands by different applications. However for theindividual researcher in the quest for an antibody there are guidelines andsuggestion to follow for selection and validation84,92. Researchers need to be

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criticaltotheresultsobtainedbyantibodiesandrealizethatantibodiesareonlyvaluableresearchtoolsifproperlyused.

AntibodygenerationprojectsThefieldofaffinityproteomicsdependonavailabilityofbinderstowardseveryproteinandeveryproteinmodificationofinterest83,93.Thereforeseveralprojectonlarge-scalegenerationofantibodiesorotherbindershaveemerged.Thevalue and importanceof generating antibodies towards the entirehumanproteomewasdiscussedalreadyinthe1960sintheMolecularAnatomy(MAN)program94, 95. The program wanted to provide the first detailed analysis ofhumancells,anddidamongstotherthingsdevelopcentrifugationtechnologiesfor cell fractionation. In the 1980s when 2-dimensional gel electrophoresisemerged,theideasfromtheMANprojectwasrecoveredintheHumanProteinIndex96 program that was based on 2D gel electrophoresis and wanted tocatalog all human proteins by collecting information on genetic position,position in 2D gel, amino acid composition, disease relation and subcellularlocation95. The Human Protein Index project aimed at producing antibodiestowardthetissuespecificproteinsobservedin2Dgelelectrophoresis97.Three European programs, ProteomeBinder, AffinityProteome and Affinomicshave developed or coordinated development and validation of binders ofdifferenttypestowardsaselectionoftargets98.AprogramrunbytheNationalInstituteofHealth(NIH)producesdifferentantibodytypesagainsttranscriptionfactor (proteincapture.org). A program at National Cancer Institute (NCI)produces monoclonal antibodies and focuses on targets related to cancer(antibodies.cancer.gov). The Human Proteome Organization (HUPO) wasstarted in 2001 to coordinate researchers in proteomics and promoteproteomics technology development93. Already from the start the need forantibodieswasrealizedandin2010theHumanProteomeProjectwasinitiatedas an international collaborative effort to map the human proteome bycoordinatingtheworkalreadyongoingwiththecomplementarytechniquesMSandaffinity-basedproteomics99.OneofthemembersofthiscollaborationisTheHumanProteinAtlas(HPA)projectthatgeneratesantibodiesandusestheminIHC to detect protein expression in tissues and in IF to detect proteinsubcellularlocationincells.TheHPAprojectisdescribedindetailinchapter5.

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4.ImmunofluorescenceImmunofluorescence(IF)istheuseoffluorescentlylabeledantibodiesandwasfirst described by Coons et al in 1941100. IF is used to detect the endogenousprotein in cell or tissue samples and when combined with a high-resolutionimaging technique such as confocalmicroscopyhigh spatial resolution can beobtained.Critical to reliable results in IFarebothantibodyspecificityand thesamplepreparationprocedure.Thedescriptionprovidedheredealsmainlywithcell samplesandnot tissues since cell linesare themodel systemused in thisthesiswork.

SamplepreparationCell samples for IF need to be fixed andpermeabilized. The cellmembrane isimpermeable toproteinsas largeas antibodies andhencepermeabilizationofthemembraneisrequiredtoallowantibodyaccesstothecellsinterior.Fixationis required to retain proteins in their native location during permeabilizationandsubsequentsamplepreparationandanalysis.Twomajorstrategiesarecommonlyusedforsamplepreparationfor IF,eithersimultaneous fixation and permeabilization by organic solvents or fixation bychemical cross-linking followedbypermeabilizationbydetergent101.Themostcommonlyusedsolventsaremethanol,ethanoloracetone.Theyalldehydratethe cell causing protein precipitation102. Membrane lipids are solubilized andextracted permitting antibody access but soluble and low-molecular weightantigensmayalsobeextracted.

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Cross-linking for IF is done with formaldehyde that forms bridges betweenproteins. Formaldehyde is prepared fromparaformaldehyde (PFA) and reactsmainlywithnitrogensofproteinsandcreatesamatrixofcross-linksthattrapsalsonucleicacidsandcarbohydrates103.Aftercross-linkingthemembraneisstillintact and must be permeabilized. Permeabilization is commonly done withsaponins104 or non-ionic detergents. Saponins remove cholesterol from themembranescreatingsmallholes105.Theeffect is transientandonlypresentaslong as saponin is present in the buffer104 and membranes with little or nocholesterol such as the inner mitochondrial membrane will not bepermeabilized.NonionicdetergentsuchasTritonX-100,Tween20andNonidetP-40solubilizemembranelipidscausingdisruptionofmembrane,aprocessthatmayalsosolubilizeandwashawaymembraneproteins106.All fixation and permeabilization alternatives will lead to distortion of theoriginal cell architecture107,108. Therefore the choice of protocol is a balancebetween acquiring sufficient fixation to not lose the protein of interest andenoughpermeabilization to allowentranceof the antibodywhilst keeping theprotocolasmildaspossibletoretainoriginalcellstate.Thedifferentmodesofaction of the two sample preparation strategies, precipitation versus cross-linking, infers differences in epitope accessibility. Solvents cause shrinkage,denaturationandlossofsomesolubleproteinsbutisquickandthereforebetterfor proteins that would dissociate during the slower PFA fixation109. Cross-linking preserves the secondary structure110 but epitopes may be masked bycross-linksandharshpermeabilizationallowsaccesstomorecompartmentsbutmayriskthelossofmoremembraneproteins.Forsomeepitopesacombinationof theprotocolsworksbest, for examplePFA inmethanol111, PFA followedbypermeabilization with methanol112 or methanol followed by PFA113. Whenanalyzingawell-knownproteinthechoiceofprotocolcanbebaseduponpriorknowledge of that protein, such as solubility or expected location, but whenanalyzing a protein with unknown location an unbiased approach should betakenandpreferablyseveralprotocolsevaluated.After fixation and permeabilization the sample is incubatedwith an antibody.Either the antibody detecting the target is fluorescently labeled or a labeledsecondary antibody is used.Markers are often used to control for cell qualityand to relate the protein of interest to a reference. Several of the largerorganelles such as endoplasmic reticulum and mitochondria can easily bedistinguished due to their distinct structure and position whilst smallerstructuressuchasdifferenttypesofvesiclesordifferentregionsinthenucleusare dependent on markers for identification. Markers used are antibodiestowards well-known proteins for different compartments or small moleculedyes such as DAPI. When several antibodies are used simultaneously in asampletheymusteitherbeprimarylabeledwithdifferentfluorophoresorthey

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must be raised in different hosts and detected by secondary antibodies withdifferentfluorophores.

FluorescencemicroscopyAfter staining, the bound antibodies are detected by fluorescencemicroscopy.The basis for fluorescence microscopy is excitation light being absorbed byfluorophoresthatwillemitlightofalongerwavelengthwhenreturnedtotheirnormal energy state114. Different fluorophores have different absorption andemission spectra. The basic systems and basic sets of fluorophores typicallyallowsimultaneoususeoffourfluorophorespersamplelimitingthenumberofmarkers in a single experiment. Typically the different fluorophores aredetectedsequentiallytoavoidcrosstalk.Thebasic fluorescentmicroscopesarewide-fieldepifluorescencemicroscopes.In epi-illumination the objective is used both to focus excitation light on thesample and to collect the emitted light. Light from the light source passesthrougha filter that selects thedesiredexcitationwavelength.Emitted light isdirected towards the eyepiece or a camera after passing a filter that removeslightofundesiredwavelengths, forexamplereflectedexcitation light. Inwide-field microscopy the entire field-of-view is illuminated and light is collectedfromtheentiredepthofthesample.Thisproducesanimagethatisblurredbyout-of-focuslight.Confocalmicroscopy115usesopticalsectioningtoanalyzeonlyathinfocalplane,ahorizontalsliceofthesample,obtainingasharperimagebyremoving thehaze fromout-of-focus light.The sample is illuminatedpointbypointandapinholeinsertedattheimageplaneofthelensblockslightfromanyother Z-level than the focal plane from reaching the detector. The two maintypes of confocal systems are the laser scanning confocalmicroscope and thespinningdisc.The resolution of conventional light microscopy is limited by the physicalproperties of light. Resolution is the possibility to resolve two objects and isdefineastheminimaldistanceatwhichtwopointsaredissolvedasseparate114.Lightrays fromapointonthespecimenshould ideallytravel throughthe lenssystem and produce a single point in the image plane, however due to thediffraction of light the rays will be affected by the lenses encountered andproduceablurredspotwithbrightdisksurroundedbyfainterdisksalternatingwithdarkdisks,calledAirydisks.Theresolutionobtainedinasystemdependson the wavelength of light and the numerical aperture of the objective. Thenumerical aperture is a measure of the light gathering properties of theobjective. With a high numerical aperture objective and visible light themaximum theoretical resolution is ∼200 nm116. Resolutions higher than the

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diffraction limit can be reached with super-resolution microscopy116, acollection of techniques that use different strategies to enhance resolution.Stimulated emission depletion (STED) uses spatially patterned excitation toanalyze the fluorescence from only a small region at a time. Single-moleculeimaging techniques such as stochastic optical reconstruction microscopy(STORM)andphotoactivatedlocalizationmicroscopy(PALM)imageasubsetofphotoswitchable fluorophores at a time, register their position and create theresultfromasuperpositionofimages.

ValidationofIFresultsFor IF there are a few types of application-specific validations available.Observedreductionordisappearanceofstaininguponknockdownorknockoutofthegeneofinterestaregoodnegativecontrols84thatvalidatesbindingoftherighttargetandcanalsodistinguishbetweentheintendedtargetandoff-targetbinding if they occur in non-overlapping compartments. A similar but lessprecise strategy is analysis in samples with and without expression of theprotein, estimated fromRNA-seqdata. Application-specific validation can alsobe achieved by two antibodies targeting different parts of the protein. In asimplesetuptheywouldvalidateeachotherbystainingthesamelocation.Foramore precise validation proximity ligation assay117 can be used where afluorescentsignalisobtainedonlywhenthetwoantibodiesarebindinginverycloseproximity.Hereitisassumedthattheprobabilityislowthatacompetingbindingsiteshouldbeavailableforbothantibodiesclosetogether.Detectionoffluorescentlytaggedproteincanbeusedtovalidatethattheantibodyiscapableof detecting the target proteinusing the current samplepreparationprotocol.Cells expressing taggedprotein are also useful as a validation of the obtainedlocations. If the tagged protein is notmislocalized and the antibody does notshow cross-reactivity than the tagged protein and the endogenous protein incontrol cells should show the same result. Tagged protein for antibodyvalidationisevaluatedinPaperΙΙΙ.

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5.HumanProteinAtlasTheHPAprojectstartedin2003attheRoyalInstituteofTechnology(KTH)118.Theaimwastoexpandtheknowledgeofthehumanproteomeusingantibody-basedmethods.Thisgene-centricprojecthasnowgeneratedrabbitpolyclonalantibodies targeting the majority of all human protein-coding genes. TheantibodiesareusedinIHCfordetectionofproteinsacrossamultitudeofhumantissuesandinIFforsubcellularlocalizationofproteinsincelllines.Theresultsare publically available in the online Human Protein Atlas database(www.proteinatlas.org). The tissue section of the atlas provides detailedinformationonprotein expression acrosshealthy andmalignant tissueswhilethe subcellular atlas provides high-resolution subcellular details of proteinlocation in cell lines. All information on antigen sequence, validation and allimagesareavailableinanopen-sourcedatabase.AmilestoneintheprojectwasachievedinNovember2014withthepublicationofthefirstdraftofthehumantissuespecificproteomeintheformofatissue-based map of the human proteome integrating data from transcriptomicsanalysisand IHCaffinityproteomics119.Thedifferent tissueproteomesaswellas genes with elevated expression in each tissue were presented along withtissueprofiles foreachgene. InDecember2016asimilargoalwillbereachedforthesubcellularatlasdescribingtheorganelleandcompartmentproteomes.

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HPAantibodiesBioinformatics selection of a suitable antigen is of highest importance togenerate antibodies with minimized cross-reactivity83. HPA antibodies areproduced towards antigens of 16-202 amino acids, called Protein EpitopeSignature Tags (PrESTs) with low similarity to other proteins120.Transmembraneregionsareavoidedandwhenpossibletheantigenischosentocoverallisoforms.EpitopemappingofHPAantibodieshaveshownthatPrESTsproduceonaveragethreelinearepitopes121of5-7aminoacids65andasmallerportionofconformationepitopes(lessthan25%)67.PrEST sequences are cloned into a bacterial expression vector together withalbuminbindingprotein and ahexahistidine tag and sequence verifiedbeforeproduction in E.Coli. The produced protein fragments are purified using thehexahistidinetagandanalyzedbyelectrosprayMSforcorrectmolecularweightbeforeimmunization.Antibodiesarepurifiedfromserumbyatwo-stepaffinitypurification process122. First the sample is depleted from antibodies targetingthe tag, and then the antigen is used as affinity ligand to purifymonospecificpolyclonal antibodies. The purified antibodies are quality controlled using aproteinarrayofPrESTs,whereaselectivebindingofthetargetisrequiredoveralargenumberofotherPrESTs122.Antibodies approved in PrEST array are used for WB, IHC and IF. WB isperformed using a standardized high-throughput setup with five differentsamples. IHCisdoneontissuemicroarrayswith48normaltissuescomprisingthemajororgansfromthreeindividualsand20cancersindifferentstagesfrom216tumors123.

SubcellularProteinAtlasThe subcellular atlas124 aims to provide detailed spatial information on everyproteinssubcellular locationanddefinetheproteomesofthemajororganellesandcompartments.ThetechniquesusedareIFandconfocalmicroscopyofcelllines. Confocal microscopy provides high-resolution images and the samplepreparationaswellasimageacquisitionisautomationcompatible.Celllinesarea good model system as they are easy to proliferate and can generate anunlimitednumberofcells.Thereleaseofversion14inOctober2015containedsubcellularlocationdatafor10003genes.Everyantibodyisanalyzedinthreedifferentcelllineschosenfromapanelof18cell linesofdifferent tissueorigin.Allcell lineshavebeenRNAsequencedandthesevaluesareusedasaguidetochoosecelllineswheretheproteinislikely

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tobeexpressed.To reacha complete coverageof theproteomenewcell linesarecontinuouslyaddedtothepanel.Samplepreparationisdonein96-wellglassbottomplates inapipettingrobottoaid throughputand reproducibility.Cells are fixedandpermeabilizedusingPFAandTritonX-100basedonaprotocol-evaluationprojectdiscussedinPaperΙ.ThreemarkersareusedinadditiontotheHPAantibodyineverysample;DAPIstainingofnucleus,anantibody towardscalreticulinorKDEL forendoplasmicreticulumandanantibody towardsα-tubulin formicrotubules(Figure4).Theprimary antibodies are detected with fluorescently labeled secondaryantibodies. Any off-target binding from the secondary antibody is monitoredthrough a control well in every sample plate, where the primary antibody isomitted.

Figure 4. Typical IF staining from the subcellular atlas showing the fourindividual channels and the merged image. Antibody HPA005785 targetingCD44antigeningreenshowstainingoftheplasmamembrane,DAPIstainingofDNA inblue, endoplasmic reticulum (calreticulin) in yellow,microtubules(α-tubulin)inred.

Imageacquisitioniscurrentlydonemanuallytoensurethebestpossibleimagequality and representative images. Aminimum of two images is acquired foreach sample,more if avariedpatternof expression isobserved.Theobtainedimagesaremanuallyannotatedbyanoperatorwithnoinformationonproteinidentity.Inthefollowingstepasecondoperatorcuratestheresultsbasedonallantibodies for a gene in all stained cell lines taking into account all validationdataandpublishedliterature.Today19locationsareannotated(Table1).Thestandardsetofreferencemarkers limitswhich locationscanbedifferentiated,

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resulting in grouping of locations, for example all vesicle-like patterns areannotated as vesicles although they are comprised of endosomes, lysosomes,peroxisomesetc.Table1. Annotatedlocations Nucleus Nucleuswithoutnucleoli Nucleoli Nuclearmembrane Cytoplasm Mitochondria Endoplasmicreticulum TheGolgiapparatus Vesicles Centrosome Microtubuleorganizingcenter Cytoskeleton(Microtubules) Cytoskeleton(Actinfilaments) Cytoskeleton(Intermediatefilaments) Cytoskeleton(Cytokineticbridge) Plasmamembrane Focaladhesions Celljunctions Aggresome

ValidationstrategiesintheSubcellularProteinAtlasSeveraldifferentstrategiesareusedtovalidatetheresultsobtained,asdifferentstrategiesaresuitablefordifferentgenes(Table2).Iftheintendedtargetisnotpresent a low-affinity off-target binding can result in a false localization andusingRNA-seqvaluesasaguidetoselectcelllinesforeachgeneminimizessuchfalsepositives.Forwell-knownandextensivelystudiedgenescompliancewithpublishedliteraturemaybeenough,andallresultsarecomparedtoinformationinUniProtonsubcellularlocationbasedonexperimentaldata.Forgeneswherethere is no available data or contradictory information on location in theliterature additional validation is needed. HPA aims to use paired antibodies,where two ormore antibodies generated towards different parts of the sameproteinshouldgive thesameresult123. In thecase that twoantibodiesarenotavailable, other application-specific approaches such as siRNA andcolocalizationwithtaggedproteinarealsoused.

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siRNAknockdownvalidationhas beenperformed for a subset of the genes intheatlas125andthecurrentversioncontain250siRNAvalidatedgenes.WhennodownregulationisobserveduponsiRNAtransfectionitisunclearwhetherthisreflect a non-specific staining, or an inefficient knockdown. For stainings ofmultiple locations itmay be difficult to decipher if all locations have reducedintensity. Staining in a cell line expressing the tagged target protein is apromising strategy for antibody validation and this strategywas evaluated inPaper ΙΙΙ and the current version of the atlas contain 94 genes with thisvalidationtype.Thisstrategyisdependentontheavailabilityoftransgeniccelllinesexpressingthetaggedtargetprotein.Aftercompletedanalysistheoperatormakingthefinalcurationtakesall thesedata into consideration and a validation score is given to each antibodyreflectingtheamountofvalidationdataavailabletosupporttheresultfromthatantibody.Table2. Validationstrategiesinthesubcellularproteinatlas 1.RNA-seqdata 2.Comparewithliterature 3.Multipleantibodies 4.Knockdownoftargetprotein 5.Co-localizationwithfluorescentlytaggedprotein

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6.PresentinvestigationThischapterdescribesthepapersincludedinthisthesis.Thefull-lengtharticleswithalldetailsareincludedasappendices.TheworkpresentedisapartoftheSubcellular Protein Atlas project and describes some of the steps towards asubcellular atlas based on IF and confocal microscopy. The projects involvesample preparation optimization, a description of the human subcellularproteome for the first fifth of the analyzed protein-coding genes and a newvalidationstrategy.TheprojectsallinvolvetheuseofHPAantibodiesandwouldnothavebeenpossiblewithoutthecollectiveworkoftheHPAteamthatdesignandproducetheantigensandpurifyandvalidatetheantibodies.

PaperΙ In order to create a subcellular atlas featuring the location of every singleprotein a high throughput pipeline for sample preparation and subsequentimmunofluorescence microscopy was needed. Since proteome wide studiescannotaffordtailoringtheassaytoeveryindividualprotein,compromiseshavetobemadeandthebestsuitableprotocolforamajorityoftheproteinsselected.Different proteins will have different optimal fixation and permeabilizationprotocols, but in order to localize proteins in a high throughput fashion wewantedtochooseonesingleprotocolsuitableforthemajorityofallproteinsinallthemajorcompartmentsinthecell.Sixdifferentprotocolsweretested,threebasedondehydrationbyalcoholsandthreebasedoncross-linkingbyPFAfollowedbypermeabilizationbydetergent.

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Thecross-linkingprotocolswere4%PFAwith0.1%saponinfor15minutesor4%PFAfor15minutesfollowedby0.1%TritonX-100for3x5minor0.05%for2x3. The protocols based on dehydration were methanol, ethanol or iso-propanolfor5min.Theseprotocolswereevaluatedinthreedifferentcelllinesusing 18 antibodies targeting 18 proteins localizing to 11 cellularcompartments;nucleus,nucleolus,nuclearmembrane,cytoplasm,mitochondria(inner and outer membrane), the Golgi apparatus, endoplasmic reticulum(lumen and membrane), vesicles, cell membrane and cytoskeleton (actinfilamentsandmicrotubules).Over300sampleswerestainedandimaged.Thereweremarkeddifferencesintheoutcomeofthedifferentprotocolsacrosscellularlocations.Cytoskeleton,endoplasmicreticulumandtheGolgiapparatuswere well preserved by all protocols and all protocols allowed access tonucleoli.Dehydrationprotocolsresultedinmoreshrinkingofthecellsandfailedtodetectsolublecytoplasmicproteins,mitochondriaandmostoftheproteinsinthecellmembranebutshowedlessbackgroundmakingcytoskeletalfibersmorepronounced. Cross-linkingworkedwell for themajority of compartments andpreservedsolubleproteinsbetterbutshowedahigherbackgroundstaining.Thetwo concentrations of Triton-X-100 showed similar results but as previouslyknown saponin could not permeabilize the inner mitochondrial membranemakingTritonX-100bettersuitedforgenome-widelocalization.The results showed that cross-linking is essential to maximize coverage ofproteinsinallcompartments.PFAfollowedbythelowerconcentrationofTritonX-100waschosenasthestandardprotocolforthesubcellularatlas.

PaperΙΙ This paper presents the results obtained in the subcellular atlas project afterapplyingtheprotocolfromPaperΙto19%ofthehumanprotein-codinggenes.Thisfirstcompilationofresultsdemonstratedthefeasibilityandpotentialoftheestablished pipeline for high-throughput localization of proteins using IF andconfocalmicroscopyandencompassedenoughdatatodrawconclusionsabouttheorganizationoftheproteome.Atotalof4005antibodiestargetingproteinsfrom 4005 geneswere analyzed in three cell lines. The cell lineswere A-431(epidermoidcarcinoma),U-2OS(osteosarcoma)andU-251MG(glioblastoma),chosentobefunctionallydifferentwithdifferentorigintherebymaximizingthetotalnumberofexpressedgenes.Theproducedimageswereannotatedtooneorseveralof16compartments:cytoplasm,nucleus,nuclearmembrane,nucleoli,mitochondria, endoplasmic reticulum, theGolgi apparatus, plasmamembrane,focal adhesions, cell junctions, centrosome, aggresome, microtubules, actinfilaments,intermediatefilamentsandvesicles.

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89% of the proteins were detected in at least one cell line, corresponding tomore than 3500 proteins. The majority of proteins showed ubiquitousexpression and were detected in all three cell lines. A hierarchical clusteringshowed that the organelle proteomesof the three cell lineswere very similarbecause they cluster together for every organelle. In addition organelleswithsimilarfunctionclustertogether,suchastheGolgiapparatusandvesicles.EnrichmentanalysisofGeneOntologytermsforthegenesthatwereexpressedin one cell line only showed enrichment of biological processes related tocentralnervoussystemdevelopmentforU-251MGinaccordancewithitsbrainorigin. For A-431 and U-2 OS terms such as receptor activity and signaltransduction related to the plasma membrane were enriched. This complieswith the hypothesis that states that ubiquitously expressed genes are oftenintracellularandcelltypeortissuespecificgenesareoftensecretedorpresentonthecellmembrane126.The most commonly observed compartments were cytoplasm and nucleusfollowed bymitochondria, plasmamembrane, vesicles, nucleoli and the Golgiapparatus.Themajorityofproteinslocalizedtoonecompartment(55%),butalargefractionshowedtwo(38%)ormorethantwo(7%)locations.Thefractionof proteins withmultiple locations was surprisingly high. Thismay partly beexplainedbythatpreviousdefinitionsoforganelleproteomesbasedonMSandfractionated samples may not have had the resolution or the experimentaldesign to detect multiple locations due to focusing on a single compartment.Thisnumbermayalsobeinfluencedbynon-specificbindingorcross-reactivitybytheantibodiesandmorevalidationoftheresultsisneeded.

PaperΙΙΙ Currently,thereisnoconsensusonthesinglebestwaytovalidateantibodiesforIF. An appealing approach that was assessed in this study is overlap withfluorescently tagged target protein in transgenic cell lines. Antibodies thatcolocalizewiththetaggedproteinarevalidatedascapableofbindingthetargetprotein using the current fixation and permeabilization protocol and sampletype.Additionalbenefitsofthisvalidationapproachisthatwhenthelocationofthe taggedprotein and that of endogenousprotein stainedby the antibody incontrolcellsareidenticalthelocationofthatproteinhasbeenvalidatedbytwodifferentmethods.Inthisstudy108transgeniccelllinesexpressingdifferentGFPtaggedproteinswere stained by 1-3 antibodies, in total 197 antibodies. All cell lines weregenerated by the group of Anthony Hyman atMax Planck Institute andwere

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HeLa cells transfected by bacterial artificial chromosomes (BAC). The use ofBAC allows large transgenic inserts containing the gene in its native contextwith native promotor and regulatory elements. This leads to close toendogenous expression levels and avoids overexpression artifacts commonlyobserved with standard methods based on cDNA and standard promotors.Howeveralsoatendogenouslevelsmislocalizationsmaybeobservedduetothetag,andinthisstudyonlycell linesexpressingthetaggedproteininalocationsupportedbyexperimentalsubcellularlocationdatafromUniProtwereused.136of197testedantibodiesweresuccessfullyvalidatedascapableofbindingthe tagged protein. These antibodies were further used to stain endogenousprotein in untransfected cells. For these antibodies the locations obtained fortagged protein and endogenous protein in control cells where furthercompared.For39%oftheantibodiestheproteinlocationwasidenticalandforanadditional43%therewereatleastonecommonlocation.Fortheremainingantibodieseither theendogenousproteincouldnotbedetectedor therewerenolocationsincommon.Differencesinobtainedlocationscaneitherbeduetointerference of the tag or cross-reactivity of the antibody. The only way toassess this is toperformadditionalexperiments, forexamplebysiRNA,whichwasusedinthisstudyfortwointerestinggenes.TheantibodieshavealsobeenevaluatedbyWBasapartof theHPApipeline.There was no strong correlation between the performances in the twoapplications showing that an application-specific validation is important. Webelieve that thenotionof goodversusbad antibodies aswell as thenotionofvalidated antibodies shouldbe abandoned in favorof validationof the resultsobtained in the specific assay. An antibody with off-target effects in oneapplicationmightperformwell inanother.Likewiseanantibodyaccompaniedby extensive validation data may give false positives in a sample lacking thetarget.As a result the next version of the subcellular atlas will give scores for eachlocationinsteadofaspreviouslyforeachantibodyoreachgene.Thevalidationscore will reflects the amount of data available to support that location. Toobtain a high validation score a location must either be detected by twoindependent antibodies, by one antibody and a corresponding taggedprotein,byoneantibodyandvalidatedbysiRNAorbyoneantibodywherethelocationissupportedbypublishedliteraturefromexperimentaldata.

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FutureperspectivesThe subcellular atlas has been released yearly since 2007 and now containslocation data for 10 003 genes. However only half of these genes (48%) areaccompanied by sufficient validation and the work continue to increase thisnumber. Since no validation strategy is perfect for all genes a combination ofmultiple antibodies, siRNA and gene tagging is currently used and moretechniquessuchCRISPRknockoutcelllinesmightbeadoptedinthefuture.WeshowedthattheprotocolfromPaperΙcouldbeusedsuccessfullyinahigh-throughputsetuptolocalizeproteinswithIF.Howeverwealsoshowedthatforsomeantibodiesotherprotocolsmightperformbetter.Thereforewewillnowinclude a second protocol in the subcellular atlas, using dehydration bymethanol. Antibodies that have failed the cross-linking protocol but whereadditional data is needed for validationof locationwill nowbe analyzedwiththisprotocoltoincreasecoverageandimprovevalidationintheatlas.The majority of the confocal images in the atlas have so far been acquiredmanuallybutanautomatedacquisitioniscurrentlyunderpilottesting.Likewiseautomated image annotation using pattern recognition is desired, both toincreasethroughput,improveaccuracyofannotationsandtoaddmoredetailedannotations by subdividing categories. Training of classifiers and hierarchicalclusteringhasbeenusedto identifymisannotated images fromthesubcellularatlas127andworkinthisareacontinues.In Paper ΙΙ we compiled the subcellular location data for 19% of the humanproteome.Since thenmoredatahasbeengeneratedand inDecemberof2016approximately80%ofthenon-secretedhumanproteincodinggeneswillhavebeenanalyzedandannotatedforsubcellularlocation.Thislargeamountofdatawillenabledetaileddescriptionofcompartmentproteomesforalltheannotatedcompartments,aswellasathoroughdescriptionofthefractionofproteinswithmultiple locations and differences in location across cell lines. It will also bevaluabletocomparethedatatoothertechniquesusedforlocalization,suchasMSandBioID.Inlifescienceresearchthereisnowanincreasingawarenessofthecomplexityofantibodyperformance.Several journalshave takenactionandnowdemandmore information and validation before publication128, 129. Since this is nowgaining more attention it will hopefully lead to that every researcher usingantibodiesnoticetheseissuesandtakeappropriateaction,whichwill improveboth research quality and reproducibility. If researchers demand moreinformation from the companies before purchase then companies will be

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“forced” toproducemorevalidationdataand todisclosemore informationonthevalidationexperiments.Researchersalsoneedtohavesensibleexpectationson antibodies as reagents and realize that while companies can validate theantibodies, the validation of obtained results lies with the end user. There isalready a vast resource of antibodies available and the most reasonable wayforwardistominethisresourcefortheonesthatarebestsuitedforthespecificapplicationandsample.

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AcknowledgementsThis thesis work has been going on for many years and there are countlesspersons thatdeserve a thankyou.This ismyopportunity to acknowledge theoneswhowere invaluable inmaking this happen. I alsowant to extend a bigthankyoutoallofyouthatcouldnotbementionedinperson,toeveryonewhometmeinthelaborlunchroomandhelpedmeorinspiredme.Thebiggest contributor to this coming through isof courseEmma –myboss,supervisorandfriendwhopushedmeintostartingmystudies, inspiredmetopushmylimitsandwhomIadmiregreatly.Thankyouforguidanceandsupportineverything.My cosupervisorMathias, thank you for initiating the HPA and transferringyourinspirationandenthusiasmtoallofus.ThankstoEmma,Frida,PeterTandLars forproofreadingthisthesis,andtoPeterNforthesisqualityreview.ThankstoKnutandAliceWallenbergFoundationforfinancialsupport.TheCellProfilinggroup,thepeoplewhoI’vespentsomuchtimewiththroughthese years. You have all contributed to my projects but our great group ofdifferentpersonalitiesandexperienceshasgivenmesomuchmoreandgettingtoknowyouhasbeenthebestpartofthisjob.Thankyou!EspeciallyIwanttothankMartinwhohasworkedwithmesinceforeverandbeenaperfectdesk-neighbor. Charlotte for improving the work environment and letting sunthrough thewindows, for the trips toHeidelberg and Crete and for extensivehelp with many things, especially the BAC manuscript.Mikaela for sharingeverything from career discussion to costumes and fictional writing, for thetrips toNorwayandSanDiegoand forbringing lotsof laughter into theofficespace.FridaforthenicetimewespentinCreteandVancouverandforbringabroad set of topics into the lunch room and updating me on anything fromculture,fashionandweavingtoFPKM,RutgerandChristianformassiveworkin the laboratory with BAC cell lines andHammou and Diana for help withsiRNA. I want to thank the CellPro PhD students for lunches, support, textmessages and craziness. Lovisa, Anna, Jenny, Peter, Devin and Lars, thisgroupwouldnothavebeenthesamewithoutyou. ThankstoUllaandAnnicaforteachingmethingsoftheoutsideworldwhilstlockedintothepsychoplasm.IwanttothankeveryoneworkingonSciLifeAlfa2.ThankyouallmembersofBiobankprofiling,Tissueprofiling,ClinicallyappliedproteomicsandCellscreening for the good work environment and for answering questions,contributingtotheProteomicsclubandtheSjövikenretreats.Specialthanksto

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JanandTonyfordiscussionsaboutmouseversushumanandforhelpwithWB.ThankstoClaudiaforhelpwithdiscussionsandlabworkonCo-IP.ThankstoeveryoneinTheHumanProteinAtlasprojectformakingtheatlaspossible and for sharing your knowledge when needed. Especially Kalle,Fredric, Linn and Åsa for providing the IT and bioinformatics necessary forsuccessfulprojects.ToallformercoworkersinAlbanovaFloor3thatmademymasterthesisandfirst jobs inKTHBiotechnologyaniceexperience.Special thanks toTorbjörn,ImmunotechandtheProteinfactoryforeverythingIlearnedfromyou.Thanks to the administrative staff for help with everything necessary, fromparentalleavetoChristmasparties,especiallyLottaandMartina.ThankstoAnthonyHymanandInaPoseratMaxPlanckInstitute,Dresden,forcollaborationsusingBACcelllines.ThankstoInaSchuppeKoistinenforallowingmetousethebeautifulpaintingofacelltoillustratethecoverofthisbook.Thanks to my friends from KTH, Beata, Maria and Karin, for being greatfriendsandsharingstudies,jobsandfamilylife.ThankstoBeataforguidingmetomyfirstjobintheHPA!ThankstoUmgängetandallfriendsoutsideresearchforsupportingmeandmyfamilywhenneeded.Ifeelblessedtohaveyouinmylife.ThankstomyparentsEvaandKjell,mybrotherTomasandmynewerfamilymembers Janne and Annika for being my family and helping with anythingneeded. To StorFamiljen Skogs – it is great to be a part of this large andeasygoing family where something fun is always happening. To mygrandparentsKarin and Gunnar,whom Imiss dearly andwhomade a largeimpactonmylifegrowingup.Tomysmall familyJohan,Nils andTom- I loveyoumorethananythingelse.Thank youNils andTom for bringing sunshine intomy life every day and toJohan foragreeingtosomeofmynon-sensiblecraziness,forsayingnothingofthelateeveningsatworkduringthesiswritingandforyourunconditionallove.CellProfiling:keepupthegoodwork!Iwanttoseeafantasticcellatlasinthe

futureandIwillfeelproudoverwhatIwasapartof.Onecellisnocell–overandout.

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8.Appendix:Includedpublications