Proteomic Profiling of Vesicular Organelles

INOM EXAMENSARBETE BIOTEKNIK,AVANCERAD NIVÅ, 30 HP

, STOCKHOLM SVERIGE 2017

Proteomic Profiling of Vesicular Organelles

HANNA HASSAN

KTHSKOLAN FÖR BIOTEKNOLOGI

KTH Biotechnology

Proteomic Profiling of Vesicular OrganellesHanna HassanSupervisor: Peter ThulExaminer: Emma Lundberg

2016

AbstractThe Human Protein Atlas (HPA) is a scientific research project aiming to map the entire human proteome using anantibody-based approach. The Subcellular Atlas that is a part of the HPA provides spatial information about thesubcellular location of proteins. Currently, the Subcellular Atlas does not go beyond the annotation “vesicles” forproteins showing a dot-like staining. In this study a proteomic profiling of three vesicular-like organelles, peroxisomes,endosomes and lysosomes was conducted to expand the annotations in the HPA’s subcellular atlas and to identifynew members of the respective proteomes. The proteomic profiling was done by indirect immunofluorescenceand colocalization studies with organelle specific markers. By this method, the location of proteins that werepreviously described to target one of the studied organelles could be verified, while the subcellular location ofso far uncharacterized proteins could be identified. In total, 91 proteins were identified to localize to the threestudied vesicular organelles. This includes proteins whose subcellular location has previously been described suchas PEX14 (peroxisome), LAMTOR4 (lysosome) and Rabankyrin-5 (endosome), but also proteins like HEATR4(peroxisome) and PIK3R1 (lysosome) that were not associated with any organelle before. The observations in thisstudy serve as a validation that colocalization studies are useful when verifying a previously described subcellularlocation and identifying unknown locations of proteins. Moreover, the high-resolution immunofluorescent imagesallowed single-cell analyses showing proteins that are only expressed in a subpopulation of cells, proteins thatare only localized to a subpopulation of organelles or proteins that are only present in a subcompartment of theorganelle. Altogether, this study has verified known locations as well as identified the location of proteins, which willresult in more precise annotations for vesicles in the HPA’s Subcellular Atlas.

SammanfattningHuman Protein Atlas (HPA) ar ett vetenskapligt forskningsprojekt vars mal ar att kartlagga det manskliga proteometmed hjalp av antikroppar. HPAs subcellulara atlas ger information om ett proteins subcellulara plats. For tillfalletbenamns bilder i denna atlas som har en prickliknande infargning endast som lokaliserade till ”vesiklar”. I dennastudie forekommer en protein-profilering av de tre vesikel-liknande organellerna peroxisomer, endosomer ochlysosomer for att utoka HPAs subcellulara atlas och identifiera nya medlemmar av respekive proteom. Denna protein-profilering gjordes genom indirekt immunofluorescensteknik och co-lokaliseringsstudier med organell specifikamarkorer. Tidigare kanda platser for protein blev validerade och okanda platser for protein blev identifierade. Totaltblev 91 protein lokaliserade till de tre studerade organellerna. Detta inkluderar protein vars subcellulara plats tidigarebeskrivits sa som PEX14 (peroxisom), LAMTOR4 (lysosom) och Rabankyrin-5 (endosom), men aven protein somHEATR4 (peroxisom) och PIK3R1 (lysosom) som inte varit associerade med nagon organell. Resultaten i dennastudie validerar co-lokaliseringsstudier som anvandbara nar verifiering av ett tidigare kant subcellulart omradeoch identifiering av okanda omraden protein tillhor studeras. De hogupplosta immunofluorescerande bilderna tillatenkelcell-analyser som visade protein som endast uttrycktes i en underpopulation av celler, protein som endastlokaliserade till en underpopulation av organeller eller protein som endast finns i ett delomrade av en organell.Denna studie har verifierat kanda platser och identifierat platser protein befinner sig i vilket kommer resultera i enmer detaljerad annotering for vesiklar i HPAs subcellulara atlas.

KeywordsProteomic profiling — Colocalization — Immunostaining — Peroxisome — Endosome — Lysosome

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Proteomic Profiling of Vesicular Organelles — 2/22

Contents

Introduction 3Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Proteomic Profiling of Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Materials and Methods 3Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Cell Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Immunostaining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Image Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Image Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Results 4Immunostaining of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Members of the Peroxisomal Proteome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Members of the Endolysosomal Proteome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Candidate Proteins with an Unknown Location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Discussion 9Immunostaining of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Colocalization with Organelle Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Candidate Proteins with an Unknown Location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Proteomic Profiling of Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Conclusion 10

Future Perspectives 10

Acknowledgments 10

References 10

Appendix 12


Introduction

THE Human Protein Atlas (HPA) is a project aimingto map the entire human proteome through antibody-based proteomics and transcriptomics. The first ver-

sion of the HPA was released in 2005 and as of April 2016,the database has been updated with its fifteenth release. [1] [2]The atlas is divided into four subparts: normal tissue, cancertissue, subcellular and cell line. Today, the database containsprotein data covering 86% of the predictive human genesand more than 11 million images with primary data from im-munofluorescence (IF) and immunohistochemistry. [3] TheHPA’s Subcellular Atlas provides spatial information aboutthe subcellular location of proteins. Identifying the locationsof proteins is important for a better understanding of the in-teraction and cellular functions of proteins. Proteins rarelyact alone and if two proteins are located to the same location,it is more likely that they interact with each other or have aneffect on the function. [4] This information can in turn alsobe valuable to develop more effective treatments for differentdiseases by e.g. simplifying the target identification processfor drug discovery. Currently, the HPA does not go beyondthe annotation category “vesicles” for proteins with a dot-likestaining. However, there are different types of vesicles andvesicle-like organelles in the cell. Therefore a more detailedannotation that shows what type of organelle this protein islocated in is needed. The aim of this study is to expand theannotation category ”vesicles” in to a more specific one byusing resources from the HPA’s subcellular atlas. This wasachieved by conducting proteomic profiling of three vesicular-like organelles through antibody-based techniques.

VesiclesVesicles, a category comprised of many different organelles,are small structures enclosed by a membrane and are involvedin a variety of functions such as transportation, sorting, stor-ing and metabolism. The vesicular-like organelles that willbe studied in this project are peroxisomes, endosomes andlysosomes. Peroxisomes are multifunctional organelles thatharbour a variety of enzymes and are involved in several an-abolic and catabolic cellular pathways. The main functionof peroxisomes is β -oxidation of long- and very long-chainfatty acids. In addition, peroxisomes carry out importantreactions such as phospholipid biosynthesis, chemical detox-ification, oxidation of purines, polyamines and some aminoacids. Many of these reactions involve hydrogen peroxide,which is why they were named peroxisomes. [5] Endosomesare formed via endocytosis and their main function is to sortthe material that has been taken up by the cell. There are threemain types: early, late and recycling. Early endosomes matureinto late endosomes that fuse with lysosomes for degradationof material. Recycling endosomes are sub-compartments ofthe early endosome that recycle material to the plasma mem-brane. [6] [7] Lysosomes are organelles that contain severaldifferent enzymes and are responsible for the degradation ofmolecules internalized within the cell. [8] [9]

Proteomic Profiling of OrganellesIdentifying proteins that are located in a certain organellecan be approached in different ways. The general way is theisolation of the organelle of interest followed by the identi-fication of the residing proteins mass by mass spectrometry.The results are then analyzed by a protein database to deter-mine which proteins were found in the sample. [10] Anotherapproach is to use immunofluorescence or green fluorescentprotein (GFP). These approaches use fluorescence to visual-ize e.g. molecules and organelles. Locations of subcellularproteins can be determined by labeling proteins of interestwith fluorescent dyes, followed by image acquisition usingfluorescence microscopy. There are two main techniques thatare used for labeling proteins, namely gene tagging with a flu-orescent protein and immunolabeling with fluorophores (alsoknown as immunofluorescence). This study will focus onusing indirect immunofluorescence to detect proteins. Indirectimmunofluorescence uses a primary and secondary antibodyto target and detect the protein of interest. The primary anti-body targets the protein of interest and is indirectly coupledto a fluorophore by the use of a secondary antibody. The fluo-rophore enables the detection of the targeted protein throughvisualization. [11] Colocalization is the comparison of thespatial distribution of two fluorescently labeled molecules andcan be used to determine if two molecules are associated tothe same organelle. In this method, the protein of interest istargeted with a fluorophore and the organelle of interest withanother fluorophore. If the signal of the fluorophores overlap,the protein localizes to the organelle. [12]

AimThe main aim of this project is to expand the annotations forproteins with a vesicle-like pattern in the HPA by identifyingproteins located in peroxisomes, endosomes and lysosomes.Using indirect immunofluorescence, colocalization with or-ganelle specific markers and confocal microscopy, the proteinslocalized to these organelles can be determined. Another aimof this project is to validate HPA-antibodies by using themto identify the subcellular location of proteins with a knownlocation.

Materials and MethodsData AnalysisUsing the UniProtKB database, lists of proteins with the sub-cellular location peroxisome, endosome or lysosome, thatwere asserted experimentally were generated. The gener-ated lists contained proteins with known subcellular locations.These lists were ran against a compiled table of HPA-antibodyannotations. The compiled HPA-antibody table consisted ofantibodies annotated to be targeting proteins with a positivevesicular staining in U-2 OS cells. The HPA-antibody tableentries also had a FPKM value over 0.5, ensuring that thegene of interest was expressed in the cell line. The proteinsfrom the UniProtKB lists that were found in the compiledHPA-table were chosen as candidates to be tested. A model


for the identification of novel peroxisomal, lysosomal andendosomal proteins was built in KNIME (v. 3.1.2) [13] usingthe image segmentation data that is generated for all images inthe Subcellular Atlas. [14] Images of identified members wereused to train the model. The number of extracted featureswas reduced by removing features that showed only a lowvariance for vesicular staining. The features of the identifiedmembers were used to define the center of cluster by k-means.A list of vesicular proteins that were closest to the clustercenter was generated. The generated list was compared toentries in UniProtKB and the proteins that had no or a curatedsubcellular location were chosen as candidates for testing.

Cell CultivationU-2 OS cells, a cell line derived from osteosarcoma, grewin McCoy’s 5A medium (Sigma-Aldrich and Lonza) supple-mented with 10% Fetal Bovine Serum (FBS, Sigma-Aldrich)and 1% L-glutamine in a 37◦C and 5.2% CO2 environment.10 000 cells/well were seeded onto a 96-well glass bottomplate (Greiner) coated with 40 µl 12.5 µg/ml fibronectin andincubated for 24h before immunostaining, in the same envi-ronment and temperature.

ImmunostainingThe growth medium was removed from the 96-well glassbottom plate and the cells were washed with PBS. Thecells were then fixed by incubation with 40 µl ice cold 4%paraformaldehyde (PFA, VWR) diluted in PBS for 15 min-utes. After removal of PFA, the cells were permeabilized byincubating with 40 µl PBS containing 0.1% Triton X-100(Sigma-Aldrich) for 3x5 minutes followed by a washing stepwith PBS. Primary rabbit HPA-antibodies were diluted to2-4 µg/ml in blocking buffer (PBS + 4% FBS) containing anorganelle specific marker. Organelle markers used: mono-clonal mouse anti-ABCD3 diluted 1:1000 (Atlas Antibodies)for the peroxisome, monoclonal mouse anti-VPS26A diluted1:800 (Atlas Antibodies) and a monoclonal mouse anti-EEA1diluted 1:250 (BD Biosciences) for the endosome and a poly-clonal sheep anti-LAMP-1 diluted 1:80 (R&D systems) forthe lysosome. Endosome and lysosome organelle markerswere used together. After incubation overnight in 4◦C the cellswere washed with 40 µl PBS 4x10 minutes. 40 µl blockingbuffer containing 1 µg/ml of the secondary antibodies goatanti-rabbit Alexa 488, goat anti-mouse Alexa 555 and donkeyanti-sheep Alexa 633 (Life Technologies) was added andincubated in room temperature for 90 minutes. Subsequently,the blocking buffer containing antibodies was removed andthe nuclei of the cells were stained with the addition of 50µl DAPI (Invitrogen) diluted 1:500 in PBS. After 10 minutesDAPI was removed and the cells were washed with 40 µlPBS 4x10 minutes. The plate was then mounted with 85%glycerol in 10xPBS and sealed with foil cover.

Image AcquisitionImages of the immunofluorescently stained U-2 OS cells wereacquired manually in sequential steps (one for each dye) tominimize spectral bleed-through. This was done in room tem-perature using Leica SP5 DM6000 CS confocal microscope,a 63X/1.4 numerical aperture oil immersion objective andthe software LAS AF (Leica Microsystems). The confocalsettings were as follows: 16 bit acquisition, 600 Hz, line aver-age 2, pixel size 80 nm. The detector gain was adjusted foreach sample to obtain an image with a high signal to noiseratio and the maximum allowed gain was set to 800 for theHPA-antibody channel.

Image AnalysisThe acquired images were visually inspected for a colocaliza-tion between the protein of interest and the organelle marker.The images were analyzed and merged by using ImageJ.

ResultsImmunostaining of ProteinsThe aim of this study was to identify proteins localized toperoxisomes, endosomes and lysosomes. This was done byusing organelle markers for these three organelles togetherwith HPA-antibodies targeting previously selected protein can-didates to immunostain U-2 OS cells. The manually acquiredimages of cells were visually inspected for colocalizationbetween proteins of interest and organelle markers. The HPA-antibody was labeled green, the nuclei was stained blue withDAPI and the organelle markers for peroxisomes, endosomesand lysosomes were labeled with different fluorophores. In thefigures shown in this result section all the organelle markersare shown in red to enable easier detection of colocalizationwith the green HPA-antibody.The candidates were divided into one out of four different cat-egories depending on the type of overlap the images showed(Fig.1). The categories were:

i Complete overlap: vesicles detected by the HPA-antibody (green channel) and organelle marker (redchannel) colocalize.

ii Partial overlap: not all vesicles detected by the organellemarker colocalize with the HPA-antibody staining

iii Fractional overlap: a fraction of the vesicle detected bythe organelle marker colocalizes with the HPA-antibodystaining. A fractional overlap was only seen when usingendosomal organelle markers.

iv No overlap: vesicles detected by the HPA-antibody andorganelle marker do not colocalize.

Members of the Peroxisomal ProteomeOut of 48 proteins with a known location that were tested,19 showed a complete overlap, 7 a partial overlap and 22


Figure 1. Three categories of overlap patterns. First row represents a complete overlap, second row a partial overlap and thethird row a fractional overlap. The HPA (Green) column shows the HPA-antibody and the marker (Red) column an organellemarker.

of them had no signal overlapping with the peroxisomal or-ganelle marker. One of the tested proteins SCP2 (non-specificlipid-transfer protein) showed a distinct staining in only somecells. (see Fig. 2 and Appendix 2.) 57 proteins with an un-known location were tested with the peroxisomal organellemarker. These proteines were predicted by a trained modelin KNIME for having a similar staining pattern with proteinsknown to localize to the peroxisome and lysosome. 5 out of57 candidates showed a complete overlap and 52 showed nooverlapping signal with the peroxisomal marker.

Members of the Endolysosomal Proteome

The endosomal and lysosomal candidates with a known loca-tion were tested together as the organelles have no clear-cutboundary between them. Both organelles had 55 proteinsselected as candidates to be tested each and 20 of these candi-dates were described to be located in both locations. There-fore 90 candidates in total were tested for colocalization withthe LAMP-1 antibody as a marker for lysosomes and theVPS26A antibody for endosomes. Out of these 90 candidates12 showed a complete overlap, 17 a partial overlap and 61


Figure 2. Immunostaining of three different proteins. HPA-antibody is shown in green, the peroxisomal marker ABCD3 in redand nuclei stained with DAPI in blue. A) IF-staining of a known peroxisome protein, peroxisomal membrane protein PEX14.B) IF-staining of an unknown peroxisomal protein, HEAT repeat-containing protein 4, showing a complete overlap with theperoxisomal organelle marker. C) IF-staining of a known peroxisomal protein SCP2, non-specific lipid-transfer protein,showing a complete overlap with one cell. This is an example of a subpopulation of peroxisomes between cells.

no overlap with the lysosomal organelle marker. For the en-dosomal marker VPS26A, 3 showed a complete overlap, 6 apartial overlap, 8 a fractional overlap and 73 no overlap. Thethree proteins showing a complete overlap are all associatedwith the retromer, a complex of proteins that is involved inrecycling transmembrane receptors from endosomes to thetrans-Golgi network, according to the UniProtKB database.The VPS26A organelle marker is therefore thought to be an an-tibody more suited for targeting the retromer complex ratherthan the entire endosome. The early endosomal organellemarker EEA1 was thereafter used. 60 candidates were testedin total with this marker, 55 candidates from the first experi-ment described above and 5 additional candidates (lysosomalcandidates that showed a colocalization with the former endo-somal marker VPS26A). Out of these 60 candidates 2 showed

a complete overlap, 1 a partial overlap, 10 a fractional overlapand 47 no overlapping signal with the endosomal marker.

57 proteins with an unknown location were tested with thelysosomal organelle marker. These proteins were predictedby a trained model in KNIME for having a similar stainingpattern with proteins known to localize to the peroxisome andlysosome. Out of the 57 candidates, 1 showed a completeoverlap and 4 a partial overlap with the lysosomal marker. 52showed no overlapping signal with the lysosomal organellemarker. For examples of acquired images see Fig. 3.

In total, 91 proteins (97 antibodies) out of 179 proteins(195 antibodies) were identified to localize to the three studiedvesicular organelles. Table 1 summarizes the amount of anti-bodies tested for each vesicle-like organelle and what overlapcategory they fall under. The number seen in parenthesis de-


Figure 3. Immunostaining of four different proteins. HPA-antibody is shown in green, the organelle marker in red and nucleistained with DAPI in blue. A) IF-staining of a known lysosomal protein, HLA class II histocompatibility antigen DP alpha 1chain. B) Staining of a known endosomal protein, Rabankyrin-5. C) IF-staining of a protein known to be a part of the retromercomplex, vacuolar protein sorting-associated protein 35. D) IF-staining of an unknown lysosomal protein, phosphatidylinositol3-kinase regulatory subunit alpha, showing a complete overlap with the lysosomal organelle marker

scribes the amount of proteins targeted as the same proteincould be targeted by several antibodies. If available, more thanone antibody per protein was used in order to validate HPA-antibody stainings. However, in Table 2 only the amount ofantibodies used is shown due to only one antibody being usedper protein. This is because identifying previously unknownmembers of the organelles’ proteomes does not validate the

HPA-antibodies. In Table 3 the amount of vesicular organellesseen in the cytoplasm are presented. The number for eachorganelle is an average count of vesicles when comparingthree cells from the same image. One acquired image waschosen for each organelle. Tables containing the antibodiestested for each organelle marker can be found in Appendix 1.


Table 1. Results after testing protein candidates with a known subcellular location. The amount of antibodies tested followedby the number of proteins tested in parenthesis are shown.

Category Peroxisome Endosome Lysosome Retromer

Complete overlap 19(16) 2 12 3

Partial overlap 7(6) 1 17 6

Fractional overlap 0 10 0 8

No overlap 22(16) 47(44) 61(55) 73(68)

Total 48(38) 60(57) 90(84) 90(84)

Table 2. Results after testing protein candidates with an unknown subcellular location. The amount of antibodies tested areshown.

Category Peroxisome Lysosome

Complete overlap 5 1

Partial overlap 0 4

Fractional overlap 0 0

No overlap 52 52

Total 57 57

Table 3. The amount of vesicular organelles visualized by the organelle markers. The number shown below is the averagevesicle count for each organelle when comparing three cells in the same acquired image.

Organelle Amount of vesicles

Peroxisome 140

Endosome 118

Lysosome 64

Table 4. Proteins with a previously unknown location colocalizing with one of the organelle markers.

Protein Gene name Subcellular location

HEAT repeat-containing protein 4 HEATR4 Peroxisome

Annexin A10 ANXA10 Peroxisome

Transmembrane protein 41B TMEM41B Peroxisome

Proline-rich transmembrane protein 4 PRRT4 Peroxisome

Zinc finger protein 709 ZNF709 Peroxisome

Phosphatidylinositol 3-kinase regulatory subunit alpha PIK3R1 Lysosome


Candidate Proteins with an Unknown LocationAcquired images of proteins that colocalize with the used or-ganelle markers were used to train a model for the predictionof proteins that have similar features. The predicted proteincandidates were tested if they had a previously unknown lo-cation. Out of the 57 tested candidates, 10 colocalized eitherpartially or completely with the organelle markers for the per-oxisome and lysosome. The tested candidates that showed acomplete overlap with an organelle marker signal are shownin Table 4.

DiscussionImmunostaining of ProteinsThe aim of this study was to identify proteins localized to per-oxisomes, endosomes and lysosomes by immunostaining withspecific organelle markers. The immunostaining in this studyshowed four different colocalization patterns: a complete,partial, fractional and no overlap. A complete overlap withthe specific organelle marker signal shows that the targetedprotein is located in all organelles detected by the markerwhereas a partial overlap shows that the targeted protein isonly located in some organelles. A fractional overlap with theorganelle marker signal, which was only seen with the endo-somal organelle markers, shows that only a part of the labeledorganelle colocalizes with the HPA-antibody. This type ofoverlap could be due to optical or chromatographical aberra-tions. However, this was not deemed to be the case as thisoverlap pattern was only seen with the endosomal candidates.This fractional overlap is thought to be due to the domain thetargeted protein is localized to inside the endosome. If theorganelle marker and the targeted protein have a fractionaloverlap, they could be localized to different domains in theendosome. [15] [16]

Colocalization with Organelle MarkersOut of the 91 proteins seen to colocalize with the used or-ganelle markers, 40 candidate proteins showed a completeoverlap with the organelle marker signal. These includeknown members of the three studied organelles such as PEX14(peroxisome), LAMTOR4 (lysosome) and Rabankyrin-5 (en-dosome). This supports that the observed colocalization withthe chosen markers did show if the protein was a member ofone of the organelles or not. The 88 candidates that showed nocolocalization with the organelle markers could be due to theused antibody being unspecific and therefore targeting some-thing else. Another reason is that the previously determinedsubcellular location of a protein could have been detected in aspecific cell line or in a specific condition different from whatwas used in this study.

Candidate Proteins with an Unknown LocationOut of the 57 protein candidates with previously unknownlocations that were tested, 10 showed either a partial or a com-plete overlap with the used organelle markers. These resultsneed to be verified to be able to draw the conclusion that these

proteins belong to the organelle they were observed to localizeto. This is important since unspecific binding is a major issuewhen drawing conclusions from studies based on a singleantibody. Preferably, the location should be verified by anantibody-independent method. In this study certain antibodiestargeted the same protein and as the results showed that theoutcome was the same for both antibodies this could serve asanother type of validation. However, to raise the validity ofthe results, verifying antibody specificity would be necessary.Antibody specificity in IF can be validated by a reduction ofthe protein expression, which would correlate with a decreaseof the antibody signal in IF. Methods to reduce the proteinexpression are for example the use of CRISPR for a geneknockout, which completely removes the protein, or a knock-down through siRNAs. The 10 HPA-antibodies that showedan overlap have not been validated through siRNA knockdownor a gene knockout. However they have all been validatedthrough Western blot. E.g. the used HPA-antibody targetingHEAT repeat-containing protein 4 was seen to completelyoverlap with the signal from the peroxisome organelle marker.The Western blot results showed a single band correspondingto the predicted size in kDa for this antibody. This resultmakes it reasonable to concludes that the antibody used is spe-cific. Another example is the phosphatidylinositol 3-kinaseregulatory subunit alpha protein that was seen to overlap withthe lysosomal organelle marker. The antibody targeting thisprotein showed a band of predicted size in kDa (+/-20%) withadditional bands present in the Western blot. To ensure thatthis antibody is specific, a better Western blot result would beoptimal.

Proteomic Profiling of OrganellesThere are different approaches to study the proteomes of or-ganelles. The vast majority of proteomic studies are basedon mass spectrometry (MS) studies whereas this study usedimmunofluorescent labeling. Immunofluorescent labelingon a proteome- and genome-wide level is only done by theHPA. Both of these approaches have limitations when identi-fying what proteins are members of the organelles of interest.To study the peroxisome, endosome and lysosome, the MS-based approach would first need to isolate and purify theseorganelles from a sample and reduce the complexity. A disad-vantage with these steps is that the isolation can be challengingto succeed with and during the complexity reduction, impor-tant proteins can be lost and will therefore not be able tobe identified. [17] The immunofluorescent labeling approachdoes not need to isolate, purify or reduce the complexity ofthe sample and does therefore not lose any proteomic infor-mation. However, this approach can cause artifacts and doesrequire cell fixation as well as permeabilization, which canbe challenging when dealing with different cell lines. [11] Itis also limited to the antibodies that are available. The IF-approach, if colocalization is used, is not reliant on databasesto identify whether a protein is located in a certain organellebut it is reliant on antibody specificity. The accuracy of the


location visualized of native proteins in the microscope can beaffected by antibody specificity which can result in mislocal-ization. However, it has been shown that immunofluorescenttagging is a reliable technique. [18] Both of these approacheshas validation of protein identification as an obligatory stepbut this is not always provided in literature, making this ashared limitation between the approaches. Which one is betterthan the other depends on what questions you want to answeras both approaches have their advantages and disadvantages.For example the MS-based approach can identify many pro-teins at once for an isolated organelle whereas the IF-approachfocuses on the colocalization of one protein at a time (when vi-sualizing the results through microscopy). The MS-approachis good for quantification, whereas the IF-approach is better atdetecting finer sub-structures or overlaps such as the fractionaloverlap observed in this study. The MS-approach would bemore useful if proteomes were to be compared at differentconditions or time points since the IF-approach would needthe entire IF antibody repertoire, consequently taking moretime and resources. The IF-approach can however show if thetargeted protein is also located in another organelle, a separatecompartment, if it is cell cycle dependent or expressed in onlya subpopulation of the cells, which the MS-approach cannot.An example of this is the protein SCP2 (see Fig.2C) whichshowed an expression in only some cells where it also over-lapped with the peroxisome organelle marker. This proteinin particular has an isoform which is located in peroxisomeswhich could explain why its expression was only seen in somecells and not all. It could also be due to cell cycle dependencyor that some cells have a subpopulation of peroxisomes withdifferent content. [19]

ConclusionIn this study 91 proteins localized to the studied vesicular or-ganelles could be determined and the acquired images will bepublicly available on the Human Protein Atlas. Many of thetested proteins were also found to not colocalize with an or-ganelle marker. However, the obtained results do suggest thatcolocalization studies could be a good approach to validate theknown subcellular location of proteins with organelle markersor map the proteome of vesicular organelles. Mapping theseproteomes would further expand the annotations for proteinslocated in vesicular organelles in the Human Protein Atlas.This will enable users of the atlas to get a more specific subcel-lular location, giving them valuable information for medicaland biological research.

Future PerspectivesThe main aim of this study was to identify the location ofknown and unknown peroxisomal, endosomal and lysoso-mal proteins. In retrospect, many antibodies were not testeddue to time constraint. Therefore a future step for this studywould be to test more antibodies to detect more proteins thatare members of these vesicular organelles’ proteomes. Fur-

ther validation of the results through antibody validation andsuper-resolution microscopy to analyze the fractional overlappattern would also be encouraged as well as analyzing moreorganelles through colocalization in the long run.

AcknowledgmentsI would like to express my deepest gratitude to Emma Lund-berg who gave me the golden opportunity to work with thisproject, for the encouraging comments and support through-out this process. Furthermore, I would like to sincerely thankmy supervisor Peter Thul for being so understanding and al-ways answering my questions, for the useful remarks and forhelping me through the ups and downs of this project. I wouldalso like to thank the Cell Profiling group for creating such alovely working environment, helping me when in need andalways making my day brighter with laughter!

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[5] Vasily D Antonenkov, Silke Grunau, Steffen Ohlmeier,and J Kalervo Hiltunen. Peroxisomes are oxidative or-ganelles. Antioxidants & redox signaling, 13(4):525–537,2010.

[6] Nature home page [Internet], 2016 [Cited May 20]. Avail-able from: http://www.nature.com/subjects/endosomes.

[7] I. Dikic. Endosomes. Molecular Biology IntelligenceUnit. Springer New York, 2008.

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[9] Nature home page [Internet], 2016 [Cited May 20]. Avail-able from: http://www.nature.com/subjects/lysosomes.

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[12] Kenneth W Dunn, Malgorzata M Kamocka, and John HMcDonald. A practical guide to evaluating colocaliza-tion in biological microscopy. American Journal ofPhysiology-Cell Physiology, 300(4):C723–C742, 2011.

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[16] Birte Sonnichsen, Stefano De Renzis, Erik Nielsen, JensRietdorf, and Marino Zerial. Distinct membrane domains

on endosomes in the recycling pathway visualized bymulticolor imaging of rab4, rab5, and rab11. The Journalof cell biology, 149(4):901–914, 2000.

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AppendixAppendix 1. Tables containing the antibodies tested for each organelle marker. Information such as what type of overlap wasobserved and if the location of the targeted protein was previously known or unknown can be seen below.

Table 5. Peroxisome Organelle Marker ABCD3Antibody Ensembl id Gene name Overlap Location

HPA000262 ENSG00000109436 TBC1D9 None UnknownHPA001242 ENSG00000243978 RGAG1 None UnknownHPA001493 ENSG00000125846 ZNF133 None UnknownHPA001569 ENSG00000145675 PIK3R1 None UnknownHPA002114 ENSG00000115233 PSMD14 None UnknownHPA003251 ENSG00000251369 ZNF550 None UnknownHPA003300 ENSG00000183475 ASB7 None UnknownHPA003642 ENSG00000187105 HEATR4 Complete UnknownHPA003720 ENSG00000181704 YIPF6 None UnknownHPA004909 ENSG00000115592 PRKAG3 None UnknownHPA005469 ENSG00000109511 ANXA10 Complete UnknownHPA005552 ENSG00000068366 ACSL4 None KnownHPA005821 ENSG00000085982 USP40 None UnknownHPA006764 ENSG00000060971 ACAA1 None KnownHPA007244 ENSG00000060971 ACAA1 Complete KnownHPA008862 ENSG00000102910 LONP2 None KnownHPA011861 ENSG00000107897 ACBD5 Complete KnownHPA012145 ENSG00000107897 ACBD5 Complete KnownHPA012571 ENSG00000095970 TREM2 None UnknownHPA014946 ENSG00000166471 TMEM41B Complete UnknownHPA015049 ENSG00000160055 TMEM234 None UnknownHPA016495 ENSG00000106609 TMEM248 None UnknownHPA017322 ENSG00000197601 FAR1 Partial KnownHPA017992 ENSG00000166783 KIAA0430 None KnownHPA019365 ENSG00000005469 CROT None KnownHPA019527 ENSG00000242110 AMACR Partial KnownHPA019556 ENSG00000143921 ABCG8 None UnknownHPA019827 ENSG00000165609 NUDT5 None UnknownHPA020099 ENSG00000197943 PLCG2 None UnknownHPA020235 ENSG00000127980 PEX1 Partial KnownHPA020260 ENSG00000095321 CRAT None KnownHPA020912 ENSG00000242110 AMACR None KnownHPA021192 ENSG00000161533 ACOX1 None KnownHPA021195 ENSG00000161533 ACOX1 Partial KnownHPA021302 ENSG00000133835 HSD17B4 Partial KnownHPA021311 ENSG00000133835 HSD17B4 Complete KnownHPA021479 ENSG00000133835 HSD17B4 None KnownHPA021575 ENSG00000141569 TRIM65 None UnknownHPA021593 ENSG00000115425 PECR Complete KnownHPA022130 ENSG00000198721 ECI2 None KnownHPA022904 ENSG00000197448 GSTK1 Complete KnownHPA022991 ENSG00000167536 DHRS13 None UnknownHPA023157 ENSG00000197566 ZNF624 None UnknownHPA023454 ENSG00000166329 CCDC182 None UnknownHPA023561 ENSG00000171206 TRIM8 None UnknownHPA024785 ENSG00000147647 DPYS None Unknown

Continued on next page


Table 5 – continued from previous pageAntibody Ensembl id Gene name Overlap Location

HPA027135 ENSG00000116171 SCP2 None KnownHPA027317 ENSG00000116171 SCP2 Complete (not all cells) KnownHPA029012 ENSG00000198198 SZT2 None KnownHPA029065 ENSG00000011485 PPP5C Complete KnownHPA030209 ENSG00000018510 AGPS Complete KnownHPA030211 ENSG00000018510 AGPS Complete KnownHPA030226 ENSG00000140548 ZNF710 None UnknownHPA031323 ENSG00000197568 HHLA3 None UnknownHPA031626 ENSG00000198721 ECI2 Partial KnownHPA031630 ENSG00000143157 POGK None UnknownHPA031838 ENSG00000115956 PLEK None UnknownHPA032026 ENSG00000117528 ABCD3 None KnownHPA032027 ENSG00000117528 ABCD3 Complete KnownHPA032141 ENSG00000162928 PEX13 Complete KnownHPA035840 ENSG00000087008 ACOX3 Complete KnownHPA035870 ENSG00000055147 FAM114A2 None UnknownHPA037000 ENSG00000033178 UBA6 None UnknownHPA038052 ENSG00000110013 SIAE None UnknownHPA039324 ENSG00000087470 DNM1L None KnownHPA039584 ENSG00000131379 C3orf20 None UnknownHPA040626 ENSG00000092470 WDR76 None UnknownHPA042830 ENSG00000034693 PEX3 Complete KnownHPA044157 ENSG00000212123 PRR22 None UnknownHPA044175 ENSG00000258436 RNASE12 None UnknownHPA044732 ENSG00000183292 TISP43 None UnknownHPA045018 ENSG00000163807 KIAA1143 None UnknownHPA046104 ENSG00000142655 PEX14 Complete KnownHPA046373 ENSG00000224940 PRRT4 Complete UnknownHPA047631 ENSG00000242612 DECR2 Partial KnownHPA047844 ENSG00000172661 FAM21C None UnknownHPA048668 ENSG00000274349 ZNF658 None UnknownHPA049231 ENSG00000142655 PEX14 Complete KnownHPA050036 ENSG00000176896 TCEANC None UnknownHPA050077 ENSG00000101417 PXMP4 Complete KnownHPA051282 ENSG00000121691 CAT None KnownHPA051966 ENSG00000162735 PEX19 Complete KnownHPA052708 ENSG00000100372 SLC25A17 None KnownHPA053153 ENSG00000242852 ZNF709 Complete UnknownHPA053646 ENSG00000180011 ZADH2 None KnownHPA054039 ENSG00000187498 COL4A1 None UnknownHPA055664 ENSG00000131944 C19orf40 None UnknownHPA055805 ENSG00000188707 ZBED6CL None UnknownHPA055838 ENSG00000121691 CAT None KnownHPA056933 ENSG00000011485 PPP5C None KnownHPA057168 ENSG00000139697 SBNO1 None UnknownHPA057603 ENSG00000133805 AMPD3 None UnknownHPA057637 ENSG00000148832 PAOX None KnownHPA058296 ENSG00000143502 SUSD4 None UnknownHPA058466 ENSG00000101417 PXMP4 None KnownHPA059737 ENSG00000134594 RAB33A None Unknown




HPA060972 ENSG00000100372 SLC25A17 Complete KnownHPA061006 ENSG00000152954 NRSN1 None UnknownHPA061550 ENSG00000075643 MOCOS None UnknownHPA061693 ENSG00000163125 RPRD2 None UnknownHPA066512 ENSG00000169957 ZNF768 None UnknownHPA071502 ENSG00000177990 DPY19L2 None UnknownHPA072710 ENSG00000152049 KCNE4 None UnknownHPA073653 ENSG00000172239 PAIP1 None UnknownHPA073760 ENSG00000131373 HACL1 None Known

Table 6. Endosome Organelle Marker EEA1Antibody Ensembl id Gene name Overlap Location

HPA001467 ENSG00000079950 STX7 None KnownHPA003524 ENSG00000103811 CTSH None KnownHPA004167 ENSG00000108774 RAB5C Partial KnownHPA004426 ENSG00000197746 PSAP None KnownHPA006615 ENSG00000075785 RAB7A None KnownHPA006964 ENSG00000075785 RAB7A None KnownHPA007728 ENSG00000185359 HGS Fractional KnownHPA014717 ENSG00000170088 TMEM192 Fractional KnownHPA017672 ENSG00000213088 ACKR1 None KnownHPA017910 ENSG00000092871 RFFL None KnownHPA017967 ENSG00000231389 HLA-DPA1 None KnownHPA018156 ENSG00000164733 CTSB None KnownHPA019053 ENSG00000073921 PICALM None KnownHPA019204 ENSG00000010270 STARD3NL None KnownHPA019513 ENSG00000082805 ERC1 None KnownHPA020998 ENSG00000188186 LAMTOR4 None KnownHPA023920 ENSG00000136933 RABEPK None KnownHPA024235 ENSG00000029725 RABEP1 None KnownHPA024705 ENSG00000155975 VPS37A None KnownHPA024781 ENSG00000155975 VPS37A None KnownHPA024817 ENSG00000104497 SNX16 None KnownHPA025960 ENSG00000156675 RAB11FIP1 None KnownHPA026531 ENSG00000136643 RPS6KC1 None KnownHPA028162 ENSG00000134262 AP4B1 None KnownHPA028598 ENSG00000072274 TFRC Complete KnownHPA028747 ENSG00000030582 GRN None KnownHPA031470 ENSG00000160179 ABCG1 None KnownHPA031565 ENSG00000136643 RPS6KC1 None KnownHPA034808 ENSG00000114331 ACAP2 Fractional KnownHPA035584 ENSG00000068650 ATP11A None KnownHPA036033 ENSG00000196455 PIK3R4 None KnownHPA036162 ENSG00000158411 MITD1 None KnownHPA036163 ENSG00000158411 MITD1 None KnownHPA037400 ENSG00000205302 SNX2 None KnownHPA037612 ENSG00000214357 NEURL1B None KnownHPA037726 ENSG00000107560 RAB11FIP2 None KnownHPA039734 ENSG00000136100 VPS36 None KnownHPA040727 ENSG00000206418 RAB12 None Known




HPA040802 ENSG00000069329 VPS35 Fractional KnownHPA040978 ENSG00000176428 VPS37D None KnownHPA041019 ENSG00000124222 STX16 None KnownHPA041138 ENSG00000124067 SLC12A4 None KnownHPA042231 ENSG00000141971 MVB12A None KnownHPA042629 ENSG00000123154 WDR83 None KnownHPA043348 ENSG00000167987 VPS37C None KnownHPA047373 ENSG00000028528 SNX1 Fractional KnownHPA049374 ENSG00000129515 SNX6 Fractional KnownHPA050351 ENSG00000095066 HOOK2 None KnownHPA050520 ENSG00000134070 IRAK2 None KnownHPA050918 ENSG00000122705 CLTA Fractional KnownHPA052900 ENSG00000197746 PSAP None KnownHPA057498 ENSG00000122958 VPS26A Fractional KnownHPA058342 ENSG00000106460 TMEM106B Fractional KnownHPA059143 ENSG00000141367 CLTC Fractional KnownHPA059590 ENSG00000198689 SLC9A6 None KnownHPA061447 ENSG00000149823 VPS51 None KnownHPA063748 ENSG00000198720 ANKRD13B None KnownHPA065849 ENSG00000185722 ANKFY1 Complete KnownHPA066538 ENSG00000105355 PLIN3 None KnownHPA070346 ENSG00000164342 TLR3 None Known

Table 7. Endosome (Retromer) Organelle Marker VPS26AAntibody Ensembl id Gene name Overlap Location

HPA001467 ENSG00000079950 STX7 None KnownHPA003524 ENSG00000103811 CTSH None KnownHPA004167 ENSG00000108774 RAB5C Fractional KnownHPA004426 ENSG00000197746 PSAP Fractional KnownHPA006615 ENSG00000075785 RAB7A Partial KnownHPA006964 ENSG00000075785 RAB7A None KnownHPA007728 ENSG00000185359 HGS Fractional KnownHPA014717 ENSG00000170088 TMEM192 Partial KnownHPA017672 ENSG00000213088 ACKR1 None KnownHPA017910 ENSG00000092871 RFFL None KnownHPA017967 ENSG00000231389 HLA-DPA1 None KnownHPA018156 ENSG00000164733 CTSB None KnownHPA019053 ENSG00000073921 PICALM None KnownHPA019204 ENSG00000010270 STARD3NL None KnownHPA019513 ENSG00000082805 ERC1 None KnownHPA020998 ENSG00000188186 LAMTOR4 None KnownHPA023920 ENSG00000136933 RABEPK None KnownHPA024235 ENSG00000029725 RABEP1 None KnownHPA024705 ENSG00000155975 VPS37A None KnownHPA024781 ENSG00000155975 VPS37A None KnownHPA024817 ENSG00000104497 SNX16 None KnownHPA025960 ENSG00000156675 RAB11FIP1 None KnownHPA026531 ENSG00000136643 RPS6KC1 None KnownHPA028162 ENSG00000134262 AP4B1 None KnownHPA028598 ENSG00000072274 TFRC None Known




HPA028747 ENSG00000030582 GRN Fractional KnownHPA031470 ENSG00000160179 ABCG1 None KnownHPA031565 ENSG00000136643 RPS6KC1 Fractional KnownHPA034808 ENSG00000114331 ACAP2 Partial KnownHPA035584 ENSG00000068650 ATP11A None KnownHPA036033 ENSG00000196455 PIK3R4 None KnownHPA036162 ENSG00000158411 MITD1 None KnownHPA036163 ENSG00000158411 MITD1 None KnownHPA037400 ENSG00000205302 SNX2 Partial KnownHPA037612 ENSG00000214357 NEURL1B None KnownHPA037726 ENSG00000107560 RAB11FIP2 None KnownHPA039734 ENSG00000136100 VPS36 None KnownHPA040727 ENSG00000206418 RAB12 None KnownHPA040802 ENSG00000069329 VPS35 Complete KnownHPA040978 ENSG00000176428 VPS37D None KnownHPA041019 ENSG00000124222 STX16 None KnownHPA041138 ENSG00000124067 SLC12A4 Partial KnownHPA042231 ENSG00000141971 MVB12A None KnownHPA042629 ENSG00000123154 WDR83 None KnownHPA043348 ENSG00000167987 VPS37C None KnownHPA047373 ENSG00000028528 SNX1 Partial KnownHPA049374 ENSG00000129515 SNX6 Complete KnownHPA050351 ENSG00000095066 HOOK2 None KnownHPA050520 ENSG00000134070 IRAK2 None KnownHPA050918 ENSG00000122705 CLTA None KnownHPA052900 ENSG00000197746 PSAP Fractional KnownHPA057498 ENSG00000122958 VPS26A Complete KnownHPA058342 ENSG00000106460 TMEM106B Fractional KnownHPA059143 ENSG00000141367 CLTC None KnownHPA059590 ENSG00000198689 SLC9A6 None KnownHPA061447 ENSG00000149823 VPS51 None KnownHPA063748 ENSG00000198720 ANKRD13B None KnownHPA065849 ENSG00000185722 ANKFY1 Fractional KnownHPA066538 ENSG00000105355 PLIN3 None KnownHPA070346 ENSG00000164342 TLR3 None Known

Table 8. Lysosome Organelle Marker LAMP-1Antibody Ensembl id Gene name Overlap Location

HPA000262 ENSG00000109436 TBC1D9 None UnknownHPA001242 ENSG00000243978 RGAG1 Partial UnknownHPA001467 ENSG00000079950 STX7 Partial KnownHPA001493 ENSG00000125846 ZNF133 None UnknownHPA001569 ENSG00000145675 PIK3R1 Complete UnknownHPA002114 ENSG00000115233 PSMD14 None UnknownHPA003251 ENSG00000251369 ZNF550 None UnknownHPA003300 ENSG00000183475 ASB7 None UnknownHPA003524 ENSG00000103811 CTSH None KnownHPA003642 ENSG00000187105 HEATR4 None UnknownHPA003720 ENSG00000181704 YIPF6 None UnknownHPA004167 ENSG00000108774 RAB5C None Known




HPA004426 ENSG00000197746 PSAP Complete KnownHPA004909 ENSG00000115592 PRKAG3 None UnknownHPA005469 ENSG00000109511 ANXA10 None UnknownHPA005821 ENSG00000085982 USP40 None UnknownHPA006615 ENSG00000075785 RAB7A Complete KnownHPA006964 ENSG00000075785 RAB7A Partial KnownHPA007728 ENSG00000185359 HGS Partial KnownHPA008763 ENSG00000030582 GRN Partial KnownHPA012571 ENSG00000095970 TREM2 None UnknownHPA014717 ENSG00000170088 TMEM192 Complete KnownHPA014946 ENSG00000166471 TMEM41B None UnknownHPA015049 ENSG00000160055 TMEM234 None UnknownHPA016495 ENSG00000106609 TMEM248 None UnknownHPA017655 ENSG00000197217 ENTPD4 None KnownHPA017967 ENSG00000231389 HLA-DPA1 Complete KnownHPA018156 ENSG00000164733 CTSB None KnownHPA019204 ENSG00000010270 STARD3NL Complete KnownHPA019556 ENSG00000143921 ABCG8 None UnknownHPA019827 ENSG00000165609 NUDT5 Partial UnknownHPA020099 ENSG00000197943 PLCG2 None UnknownHPA020998 ENSG00000188186 LAMTOR4 Complete KnownHPA021506 ENSG00000204386 NEU1 None KnownHPA021575 ENSG00000141569 TRIM65 None UnknownHPA022991 ENSG00000167536 DHRS13 None UnknownHPA023157 ENSG00000197566 ZNF624 None UnknownHPA023235 ENSG00000104324 CPQ None KnownHPA023454 ENSG00000166329 CCDC182 None UnknownHPA023561 ENSG00000171206 TRIM8 None UnknownHPA024522 ENSG00000185722 ANKFY1 None KnownHPA024785 ENSG00000147647 DPYS None UnknownHPA024817 ENSG00000104497 SNX16 None KnownHPA025960 ENSG00000156675 RAB11FIP1 None KnownHPA026419 ENSG00000119396 RAB14 None KnownHPA028162 ENSG00000134262 AP4B1 None KnownHPA028598 ENSG00000072274 TFRC Partial KnownHPA028747 ENSG00000030582 GRN Partial KnownHPA030226 ENSG00000140548 ZNF710 None UnknownHPA031068 ENSG00000064601 CTSA Partial KnownHPA031323 ENSG00000197568 HHLA3 Partial UnknownHPA031470 ENSG00000160179 ABCG1 None KnownHPA031565 ENSG00000136643 RPS6KC1 Partial KnownHPA031630 ENSG00000143157 POGK None UnknownHPA031838 ENSG00000115956 PLEK None UnknownHPA034597 ENSG00000136710 CCDC115 Partial KnownHPA034808 ENSG00000114331 ACAP2 None KnownHPA035526 ENSG00000163820 FYCO1 None KnownHPA035584 ENSG00000068650 ATP11A None KnownHPA035870 ENSG00000055147 FAM114A2 None UnknownHPA036033 ENSG00000196455 PIK3R4 None KnownHPA036733 ENSG00000124333 VAMP7 None Known




HPA036923 ENSG00000138246 DNAJC13 None KnownHPA037000 ENSG00000033178 UBA6 None UnknownHPA037400 ENSG00000205302 SNX2 Partial KnownHPA037612 ENSG00000214357 NEURL1B Complete KnownHPA037726 ENSG00000107560 RAB11FIP2 None KnownHPA038052 ENSG00000110013 SIAE None KnownHPA038052 ENSG00000110013 SIAE None UnknownHPA038053 ENSG00000110013 SIAE None KnownHPA038421 ENSG00000114742 WDR48 None KnownHPA039584 ENSG00000131379 C3orf20 None UnknownHPA039734 ENSG00000136100 VPS36 Partial KnownHPA040626 ENSG00000092470 WDR76 Partial UnknownHPA040727 ENSG00000206418 RAB12 Partial KnownHPA040802 ENSG00000069329 VPS35 Complete KnownHPA041138 ENSG00000124067 SLC12A4 None KnownHPA041995 ENSG00000169682 SPNS1 Complete KnownHPA042988 ENSG00000169682 SPNS1 None KnownHPA044157 ENSG00000212123 PRR22 None UnknownHPA044175 ENSG00000258436 RNASE12 None UnknownHPA044732 ENSG00000183292 TISP43 None UnknownHPA045018 ENSG00000163807 KIAA1143 None UnknownHPA046373 ENSG00000224940 PRRT4 None UnknownHPA047373 ENSG00000028528 SNX1 Partial KnownHPA047844 ENSG00000172661 FAM21C None UnknownHPA048668 ENSG00000274349 ZNF658 None UnknownHPA049374 ENSG00000129515 SNX6 Partial KnownHPA049876 ENSG00000101160 CTSZ None KnownHPA050036 ENSG00000176896 TCEANC None UnknownHPA050520 ENSG00000134070 IRAK2 None KnownHPA050918 ENSG00000122705 CLTA Complete KnownHPA052900 ENSG00000197746 PSAP None KnownHPA053153 ENSG00000242852 ZNF709 None UnknownHPA053478 ENSG00000109323 MANBA None KnownHPA054039 ENSG00000187498 COL4A1 None UnknownHPA055489 ENSG00000116954 RRAGC Complete KnownHPA055664 ENSG00000131944 C19orf40 None UnknownHPA055805 ENSG00000188707 ZBED6CL None UnknownHPA055838 ENSG00000121691 CAT None KnownHPA057168 ENSG00000139697 SBNO1 None UnknownHPA057498 ENSG00000122958 VPS26A Complete KnownHPA057603 ENSG00000133805 AMPD3 None UnknownHPA057966 ENSG00000163820 FYCO1 None KnownHPA058296 ENSG00000143502 SUSD4 None UnknownHPA058342 ENSG00000106460 TMEM106B Partial KnownHPA059143 ENSG00000141367 CLTC Partial KnownHPA059381 ENSG00000176978 DPP7 None KnownHPA059737 ENSG00000134594 RAB33A None UnknownHPA061006 ENSG00000152954 NRSN1 None UnknownHPA061385 ENSG00000038532 CLEC16A None KnownHPA061550 ENSG00000075643 MOCOS None Unknown




HPA061693 ENSG00000163125 RPRD2 None UnknownHPA061701 ENSG00000156171 DRAM2 None KnownHPA063891 ENSG00000164073 MFSD8 None KnownHPA064306 ENSG00000141564 RPTOR Complete KnownHPA065849 ENSG00000185722 ANKFY1 None KnownHPA066285 ENSG00000073060 SCARB1 Partial KnownHPA066512 ENSG00000169957 ZNF768 None UnknownHPA067637 ENSG00000115194 SLC30A3 None KnownHPA070346 ENSG00000164342 TLR3 None KnownHPA070642 ENSG00000162736 NCSTN None KnownHPA070738 ENSG00000103111 MON1B Partial KnownHPA071502 ENSG00000177990 DPY19L2 None UnknownHPA072710 ENSG00000152049 KCNE4 None UnknownHPA073653 ENSG00000172239 PAIP1 None Unknown

Appendix 2. Extra images of SCP2 only overlapping with the peroxisome marker in some cells. Images in the order:Merge, HPA-antibody targeting SCP2, nucleus and peroxisomal organelle marker (ABCD3).

Merge


HPA-antibody


DAPI


Peroxisomal organelle marker (ABCD3)

Proteomic Profiling of Vesicular Organelles

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