Attack by Peptide-Specific CTL Protect Vascular Endothelial Cells from An

Two Preferentially Expressed Proteins Protect Vascular Endothelial Cells from an Attack by Peptide-Specific CTL

This information is current as Daniela S. Thommen, Heiko Schuster, Mario Keller, Sarika of September 24, 2021. Kapoor, Andreas O. Weinzierl, Cuddapah S. Chennakesava, Xueya Wang, Lucia Rohrer, Arnold von Eckardstein, Stefan Stevanovic and Barbara C. Biedermann J Immunol published online 27 April 2012

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 27, 2012, doi:10.4049/jimmunol.1101506 The Journal of Immunology

Two Preferentially Expressed Proteins Protect Vascular Endothelial Cells from an Attack by Peptide-Speciﬁc CTL

Daniela S. Thommen,* Heiko Schuster,† Mario Keller,* Sarika Kapoor,‡ Andreas O. Weinzierl,† Cuddapah S. Chennakesava,* Xueya Wang,* Lucia Rohrer,‡ Arnold von Eckardstein,‡ Stefan Stevanovic,† and Barbara C. Biedermann*,x

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-specific CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide–HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by Downloaded from

small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF56–64 and CD59106–114, were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-speciﬁc source protein expression that compensated for poor HLA-A*02 binding afﬁnity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expres-

sion of SMCY311–319, an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY311–319- speciﬁc CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary http://www.jimmunol.org/ high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides. The Journal of Immunology, 2012, 188: 000–000.

uman vascular endothelial cells (EC) form the inner sional APC, they might by this mechanism escape CTL-mediated lining of blood vessels and maintain organ homeostasis injury and death. A cell-speciﬁc peptide repertoire presented by H by several key functions; that is, undisturbed healthy EC endothelial MHC class I molecules would explain tissue or organ prevent blood coagulation (1, 2), regulate vasomotion (3, 4). and predilection of immune-mediated injury such as seen during

actively participate in leukocyte trafficking (5, 6). EC are an ex- graft–versus-host disease (10, 11). The hypothesis that EC present by guest on September 24, 2021 posed tissue that is in intimate contact with transmigrating ef- a different repertoire of MHC class I-restricted peptides was sup- fector lymphocytes in the course of immune responses (7). EC ported in the past by the following in vitro observations. First, the express histocompatibility Ags, that is, the molecular motifs rec- activation of human CD8+ T lymphocytes by professional APC led ognized by the TCR (8), and therefore may serve as targets for to a subset of effector CTL that recognized and killed preferen- Ag-specific effector lymphocytes. CD8+ MHC class I-restricted tially leukocyte-derived target cells but ignored EC from the same CTL are activated by professional APC (e.g., dendritic cells) donor (12). Second, EC were poor targets for peptide-specific CTL and differentiate to become effector CTL (9). In the course of allo- due to an impaired capacity to present certain immunodominant or autoimmune disorders, effector cells could rapidly eliminate Ags such as SMCY311–319, a male-specific, HLA-A*02–restricted EC when they shared the MHC class I-restricted peptide profile minor histocompatibility Ag, as efficiently as other target cell with dendritic cells. In contrast, when EC would present a com- types (13). This cellular difference was not due to a general re- pletely different peptide profile than leukocyte-derived profes- sistance of EC to CTL-mediated target cell lysis, to lack of co- stimulation, or to low MHC class I expression (13). Taken to- *Department of Biomedicine, University of Basel, CH-4031 Basel, Switzerland; gether, these observations are explained by a different repertoire †Department of Immunology, Institute for Cell Biology, University of Tu¨bingen, of MHC class I ligands presented by EC. To prove that EC indeed D-72076 Tu¨bingen, Germany; ‡Institute for Clinical Chemistry, University Hospital Zurich, CH-8091 Zurich, Switzerland; and xDepartment of Clinical Medicine, Uni- present a different peptide repertoire compared with syngeneic versity of Basel, CH-8345 Adetswil, Switzerland leukocyte-derived B lymphoblastoid cells (BLC) serving in pre- Received for publication May 23, 2011. Accepted for publication March 27, 2012. vious experiments as professional APC, we grew up sufficient This work was supported in part by Swiss National Science Foundation Grants 3100- numbers of EC and BLC from the same donor, isolated HLA- 118096/1 (to B.C.B.) and 313530-119219 (to D.S.T.). A*02–bound peptides from the two cell types, and characterized Address correspondence and reprint requests to Prof. Barbara C. Biedermann, Inter- the resulting cell type-specific peptide profiles by mass spec- nal Medicine, University of Basel, Stapfetenstrasse 18, CH-8345 Adetswil, Switzer- trometry. By using this approach we identified HLA-A*02 ligands land. E-mail address: [email protected] prevailing on EC, on BLC, or on both cells. Some endothelial The online version of this article contains supplemental material. peptides seemed to be specific for EC, that is, they were never Abbreviations used in this article: AUC, area under the curve; BLC, EBV- immortalized B lymphoblastoid cell; EC, vascular endothelial cell; FDR, false dis- identified so far on BLC. However, we found no novel or unique covery rate; LC-MS, liquid chromatography/mass spectrometry; DMFI, change in endothelial peptide species, but all were previously detected on mean fluorescence intensity; MFI, mean fluorescence intensity; MS, mass spectrom- other epithelial cell lines or in tumor tissues. Two peptides were etry; siRNA, small interfering RNA; TFA, trifluoroacetic acid. present at dominant levels on EC. We further investigated mech- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 anisms for this preferential peptide presentation on EC such as 1)

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101506 2 PROTEIN EXPRESSION PROTECTS ENDOTHELIAL CELLS FROM CTL expression levels of source proteins, 2) binding affinity to HLA- solid tissue analysis (15). In brief, 1 vol lysis buffer containing PBS, 0.6% A*02, and 3) half-life of peptide/HLA-A*02 complexes. Finally, CHAPS, and complete protease inhibitor (Roche) was added to snap-frozen we used RNA interference to knock down the source proteins of cell pellets and the samples were homogenized by intense shaking for 1 h at 4˚C. Afterward, samples were sonicated and debris was removed by cen- the most abundant endothelial HLA-A*02–restricted peptides and trifugation and additional passing through a 0.2-mm filter (Sartorius, Go¨t- tested the impact of this treatment on peptide presentation and on tingen, Germany). Immune precipitation of HLA-A*02 molecules was the susceptibility of EC and BLC to CTL-mediated killing. performed using the HLA-A*02–specific Ab BB7.2 covalently coupled to cyanogen bromide-activated Sepharose 4B (GE Healthcare, Freiburg, Germany; 40 mg Sepharose/mg Ab). MHC molecules and peptides were Materials and Methods eluted in 0.1% trifluoroacetic acid (TFA) and peptides were isolated by Cell lines ultrafiltration through a centricon 10 kDa cut-off membrane (Millipore, Schwalbach, Germany). For liquid chromatography/mass spectrometry All studies involving primary human cells were approved by the Ethical (LC-MS) analysis, 20% of each sample was desalted and concentrated Review Board (Ethikkommission beider Basel). HUVEC (EC) were iso- using C18 Zip Tips (Millipore) according to the manufacturer’s instruc- lated from umbilical cords by enzymatic digestion as described (13). EC tions. Peptides were eluted in 50 ml 80% acetonitrile/0.1% TFA evaporated were cultured in complete medium 199 containing 20% FCS, 2 mM to nearly complete dryness by vacuum centrifugation and finally taken up m L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin (all from in 5 ml loading solvent (2% acetonitrile, 98% H2O, 0.1% TFA). Invitrogen Life Technologies, Carlsbad, CA), supplemented with fibroblast growth factors (20 ng/ml human acidic fibroblast growth factor and 20 ng/ Nicotinylation of peptides ml human basic fibroblast growth factor; both from PeproTech, London, Chemical modification of synthetic peptides with deuterated nicotinic acid U.K.) and heparin (0.2 mg/ml; Sigma-Aldrich, Saint Louis, MO). EBV- and peptide mixtures eluted from MHC precipitation with nicotinic acid immortalized syngeneic BLC were grown from cord blood mononuclear were performed as previously described (16). In brief, peptides were first cells (14). BLC were cultured in complete RPMI 1640 (Invitrogen Life Downloaded from guanidinated in freshly prepared 2.5 M O-methylisourea hemisulfate at Technologies) containing 10% FCS, 2 mM L-glutamine, 100 U/ml peni- basic pH (.10.5) for 10 min at 65˚C to protect lysine side chains. Reaction cillin, and 100 mg/ml streptomycin. The EBV-transformed B cell line JY, was terminated by addition of formic acid. Guanidinated peptides were the TAP-deficient T2 cell line (both gifts from A. Cerny, Lugano, Swit- then loaded on C18 Micro spin columns (Thermo Fisher Scientific) and zerland), and the colon carcinoma cell line LS174 (a gift from G. Spagnoli, modified by slowly passing 1 ml of a 2.2 mg/ml solution of either 1H -or Basel, Switzerland) were cultured in complete RPMI 1640. For some 4 2D -nicotinoyloxysuccinimide (diluted in 50 mM phosphate buffer [pH experiments, Flu - and SMCY -specific, HLA-A*02–restricted 4 58–66 311–319 8.5]) over the column. After three washing steps with double distilled H O, CTL clones were used and kept in culture as previously described (13). 2

potential tyrosine modifications were removed by passing 1 ml 50% hy- http://www.jimmunol.org/ Quantification of HLA surface expression droxylamine solution over the column. Following another three washing steps with double distilled H2O, peptides were finally eluted in 80% EC were detached from cell culture flasks using Accutase (PAA Labora- acetonitrile/0.1% TFA and volumes were adjusted by vacuum centrifuga- tories, Pasching, Austria) treatment for 3 min at 37˚C and subsequently tion. Concentration of nicotinylated peptides was assessed using UV ab- transferred to centrifugation tubes. BLC were resuspended by repeatedly sorbance at 261 nm on a NanoDrop UV/Vis Spectrophotometer (Peqlab, pipetting and transferred to centrifugation tubes. Cells were filtered Erlangen, Germany) against a standard curve generated from different con- through a 70-mm nylon mesh to avoid clumping, washed twice with PBS, centrations of nicotinoyloxy-succinimide. For synthetic peptides, purity and counted. For each donor 5 3 105 EC or BLC were transferred to after deuterated nicotinylation was shown to be .80% using HPLC microtiter plates and washed twice with ice-cold FACS buffer (PBS, 2% (Waters), and identity of peptides was confirmed using MS. FCS, 2 mM EDTA). Unless noted otherwise, all further steps were performed at 4˚C. Cells were stained with HLA-A*02–specific BB7.2, HLA- Nanoflow LC-MS/MS by guest on September 24, 2021 A/B/C–specific W6/32, or respective isotype controls (BioLegend, San MS was performed on an LTQ Orbitrap XL mass spectrometer (Thermo m Diego, CA) at saturating conditions (10 g/ml Ab diluted in FACS buffer). Fisher Scientific, Bremen, Germany) equipped with a nanoelectron spray After two washing steps with FACS buffer, cells were stained with sec- ion source coupled to an UltiMate 3000 RSLCnano UHPLC system 9 ondary FITC-conjugated anti mouse F(ab )2 fragments (Dako) diluted (Dionex, Sunnyvale, CA). After injection samples were loaded on a 2 cm 1:100 in FACS buffer. Quantification beads (Quifikit; Dako) were washed PepMap100 C18 Nano-Trap column (Dionex) within 10 min at a flow rate of twice with FACS buffer and afterward stained with the same concentration 4 ml/min and 3% solvent B. Peptides were then separated on a 50 cm of secondary Ab. Both cells and quantification beads were finally washed PepMap C18 column with a particle size of 2 mm (Dionex) running at 45˚C m twice with FACS buffer and after addition of 5 l 7-aminoactinomycin D with a flow rate of 300 nl/min and a gradient ranging from 3 to 30% solvent analyzed on a FACSCanto II analyzer (BD Biosciences). For each sample B within 140 min. For absolute quantification a different gradient was used 250,000 events were recorded and each experiment was performed in trip- ranging from 10 to 60% solvent B within 240 min (solvent A, H2O, 0.02% licate. Cells were gated on single cells based on FSC-A/FSC-H parameters formic acid; solvent B, 20% H2O 80% acetonitrile, 0.04% formic acid). and viable 7-aminoactinomycin D-negative cells using FlowJo FACS data MS analysis was run in data-dependent acquisition using a top five analysis software (Tree Star). Generation of standard curve for quantification method (e.g., the five most intense ions with a positive charge between 2 and and subsequent calculation of surface expression were done according to 4 analyzed during survey scan were selected for fragmentation during each manufacturer’s instructions (Quifikit; Dako). scan cycle). Survey scans were performed in the Orbitrap at a resolution Isolation and sequence analysis of MHC class I-presented of 60,000 with a scan range of 450–650 m/z (450–750 m/z for absolute quantification). Peptides were fragmented using collision-induced disso- peptides ciation (normalized collision energy, 35%; activation time, 30 ms; isolation EC and syngeneic BLC from three different HLA-A*02–positive male width, 1.3 m/z) with resulting fragment ions (MS/MS scans) analyzed in donors were grown to large cell numbers and three independent peptide the linear ion trap. Dynamic exclusion was enabled for all runs (maximal isolation experiments were performed. Per experiment, on average 5.2 6 number of masses excluded at each time point [exclusion list size] 500; 0.9 3 108 EC were grown on 11,000 cm2 (Falcon/BD Biosciences, San duration of exclusion for each mass: 40s). Jose, CA) over 45 d to confluence. For nine repetitive passages, cells Data analysis were harvested by trypsin-EDTA (Invitrogen Life Technologies) digestion and reseeded finally on 64 gelatin-coated plates (Falcon/BD Biosciences, Data analysis was performed with Proteome Discoverer 1.3. Peak lists were product no. 353025). At the final cell harvest, trypsinized cells were searched against Swissprot (date, November 2010; taxonomy filter, Homo washed in complete medium 199, spun down, and 1.5 ml pelleted cells sapiens) using Mascot software version 2.2.04 with no cleavage specificity were snap frozen in liquid nitrogen. BLC were expanded as suspension selected. Precursor ion tolerance was set to 10 ppm and product ion tol- cultures at an average density of 0.3 3 106 cells/ml to a final cell number erance to 0.6 Da. Filters used for postprocessing analysis included 5 ppm of 1.9 6 0.5 3 109 cells per donor. BLC were also collected by centri- precursor ion tolerance, ionscore .20, and a maximum identification rank fugation and briefly treated with ice-cold trypsin-EDTA to mimic the of 3. Peptides showing no HLA-A*02 motif were also excluded from treatment of EC. Trypsin was neutralized with complete, ice-cold complete further analysis. Percolator tool was used to evaluate peptide confidence medium 199, the cells were spun down, and 1.5 ml pelleted cells were snap based on q-value with a strict target false discovery rate (FDR) of 0.05 frozen in liquid nitrogen until the isolation and identification of MHC (high confidence) and a relaxed target FDR of 0.15 (medium confidence). class I-bound peptides. HLA-presented peptides were obtained by immune Fragment spectra collected from peptides used for ranking were confirmed precipitation of HLA molecules using an adapted protocol developed for using synthetic peptides to ensure proper identification, in particular of The Journal of Immunology 3 peptides showing low confidence (FDR , 0.15). Ordering of peptides concentrations of the peptide under investigation, target cell killing by according to their abundance was based on automatically calculated area Flu58–66-specific CTL was incrementally inhibited. under the curve (AUC) with a 2 ppm precursor ion window relying solely on identification rank 1 peptides. For absolute quantification, AUC of re- T2 peptide binding assay spective modified peptides was determined using Xcalibur Qual Browser TAP-deficient T2 cells express largely unloaded MHC class I molecules with a mass accuracy of 2 ppm. that are unstable, and therefore HLA-A*02 surface levels are low. When T2 cells were incubated overnight at 37˚C with 1 3 1025 M peptide in mRNA isolation and RT-PCR complete RPMI 1640 (supplemented with only 2.5 instead of 10% FCS), Total RNA was isolated from 5 3 106 cells (EC, BLC, JY, T2, or LS174) HLA-A*02 surface levels did increase due to stabilization by specific using TRIzol reagent (Invitrogen Life Technologies) according to the peptide binding. Cells were washed and surface expression of HLA-A*02 manufacturer’s protocol. After reverse transcription (SuperScript; Invitrogen was determined by FACS using BB7.2 mAb (1:100; gift from Peter Life Technologies), cDNA coding for the genes of interest was amplified Cresswell, Yale Medical School, New Haven CT) or an isotype control Ab using the following primers (all from Microsynth, Balgach, Switzerland): (1:100; product no. mca928; Serotec), respectively. After washing, cells were incubated with FITC-labeled goat anti-mouse IgG secondary Ab 59-ATGGGGAAGGTGAAGGTCGG-39 and 59-AGGGATGATGTTCTG- (1:200, product no. 115-095-003; Jackson ImmunoResearch Laboratories). GAGAG-39 for GAPDH, 59-CTCTCCGCTCTCGCCCGCTA-39 and 59-A- GGAATGGGGTGGGTGGCAG-39 for PTRF, 59-TGGACAATCACAATG- All incubations were strictly performed on ice. Data acquisition was performed on a Cyan ADP FACS instrument using Summit Software (Dako). GGAAT-39 and 59-CAAGAGCAAAGGAGGAAGC A-39 for CD59, and 59- 5 9 9 Additionally, 1 3 10 cells per sample were collected in the lymphocyte CAAGAGGTGGAAACATACAG-3 and 5 -AGCACCACCGTAGATACA- Δ G-39 for DDX5. PCR for CD59 and GAPDH cDNA was performed with an gate and analyzed. The change in mean fluorescence intensity ( MFI) was calculated by subtracting the MFI with isotype control Ab from the MFI initial denaturation step at 96˚C for 5 min, then 35 cycles with 30 s dena- with BB7.2 mAb. The fluorescence index was calculated as ΔMFI with turation at 96˚C, 30 s annealing at 58˚C, 1 min elongation at 72˚C, followed peptide/ΔMFI without peptide (17). by the final extension for 10 min at 72˚C. Amplification of PTRF and DDX5 Downloaded from cDNA was done using annealing temperatures of 60 and 53˚C, respectively. Decay and half-life of the peptide/MHC class I complex Western blot For the determination of half-life of the HLA-A*02/peptide complexes, 6 T2 cells were loaded with peptide overnight at 37˚C in complete RPMI Cells (5 3 10 ; EC, BLC, JY, T2, or LS174) were solubilized in 200 ml lysis buffer (20 mM Tris HCl [pH 7.5], 150 mM NaCl, 1 mM DTT (Roche), 100 1640 (2.5% FCS). Peptide concentrations used for pulsing were adjusted to equalize the fluorescence index of T2 cells. After washing extensively, mMNa3VO4, 1 mM NaF, and 1% Triton-X in distilled H2O containing the m m cells were again incubated at 37˚C. At indicated time points (0, 2, 6, 24 h)

following protease inhibitors: 10 g/ml aprotinin, 10 g/ml leupeptin, 100 http://www.jimmunol.org/ mM PMSF (all from Sigma-Aldrich), and 5 mg/ml pepstatin (AppliChem, aliquots were stained for HLA-A*02 as described above and staining intensity assessed by specific DMFI was determined by flow cytometry (17). Darmstadt, Germany). The protein concentration was measured by a Bio- Rad protein assay (Bio-Rad, Hercules, CA) according to the manufacturer’s Target cell treatment by small interfering RNA instructions. Per sample, 30 mg protein was loaded onto the gel. Nonre- ducing conditions were used for CD59; all other proteins were separated The following small interfering RNAs (40 mM stock concentrations) from under reducing conditions. PTRF (43.5 kDa) and DDX5 (68 kDa) were Microsynth were used to knock down precursor proteins of prevalent separated on a 9% SDS-polyacrylamide gel, and CD59 (19 kDa) was peptides: CD59 small interfering RNA (siRNA), 59-GAAGUCUAAGAG- separated on a 15% SDS-polyacrylamide gel using a Bio-Rad Mini-Protean UGAAGUATT-39; PTRF siRNA, 59-GAGGAAAGAUUGAAUCCUAT-39; 3 Cell. The proteins were transferred onto cellulose nitrate (Protran BA83; DDX5 siRNA, 59-GCAGAUAGAAUGCUUGAUATT-39, and mock siRNA, Whatman Schleicher & Schu¨ll, Dassel, Germany) in TBS (25 mM Tris, 192 59-AGGUAGUGUAAUCGCCUUGTT-39. Early passage EC or exponen- 5 mM glycine, 20% methanol) and blocked in blocking solution (43.5 mM tially growing JY cells were seeded 1 d before transfection at 10 cells per by guest on September 24, 2021 Tris-HCl, 6.5 mM Tris-base, 150 mM NaCl, 0.02% Tween 20) containing well in complete medium 199 without penicillin/streptomycin in a gelatin- 5% fat-free milk. Proteins were detected with mAbs in saturating final coated six-well plate. The next day, the medium was replaced with 2.5 ml concentrations specific for PTRF (1:250, P87520; BD Biosciences), DDX5 prewarmed complete medium 199 without penicillin/streptomycin. RNA (1:750, Ab10261; Abcam, Cambridge, UK), and CD59 (1:200, MCA1054; complexes were prepared as follows (volumes for one well of a six-well Serotec, Oxford, U.K.). b-actin (1:80,000, AC-15; Sigma-Aldrich) was used plate): 10 nM siRNA was slowly mixed with Lipofectamine RNAiMAX as a loading control. All Ab incubation steps were performed for 60 min at transfection reagent (Invitrogen) in Opti-MEM I GlutaMAX medium (Life room temperature in blocking solution. After washing three times 10 min Technologies) according to the manufacturers’ instructions. Then, the with washing solution (43.5 mM Tris-HCl, 6.5 mM Tris-base, 150 mM complexes were added dropwise and gently to the cells. The medium was NaCl, 0.02% Tween 20), the peroxidase-conjugated Abs (peroxidase- changed after 16 h and the cells incubated at 37˚C for indicated times. Cells conjugated goat anti-mouse Ab [1:4000, 115-035-071; Jackson Immu- were either analyzed by Western blot for detecting protein levels or used for noResearch Laboratories, Westgrove, PA], peroxidase-conjugated rabbit cytotoxicity assays (13) or flow cytometry. anti-goat Ab [1:2000, P0449; Dako, Glostrup, Denmark]) were added and detected by the chemiluminescence substrate (SuperSignal West Pico; Calcein-release–based cytotoxicity assay Pierce Chemical/Perbio Science, Helsingborg, Sweden). Emitted light was CTL-mediated target cell lysis was measured by a calcein-release assay as collected on a photographic film (Biomax; Eastman Kodak, Rochester, NY) described (13). Adherent EC grown to confluency in 96-well flat-bottom and automatically developed (Curix 60; Agfa-Gevaert, Mortsel, Belgium). plates (Falcon) or JY cells were loaded for 30 min at 37˚C with 20 mM Peptides synthesis calcein-AM (Molecular Probes/ Invitrogen) in serum-free medium 199. Cells were then washed and bleached for 2 h at 37˚C in complete medium 199. The HLA-A*02–restricted peptides GILGFVFTL (Flu58–66), FIDSYICQV After washing twice with complete medium 199 and then assay medium (SMCY311–319), SLLDKIIGA (PTRF56–64), SLSEKTVLL (CD59106–114), (medium 199, 2% FCS, 5 mM HEPES, 2 mM L-glutamine, 100 U/ml peni- and YLLPAIVHI (DDX5168–176) were synthesized on an automated pep- cillin, and 100 mg/ml streptomycin), 50 ml assay medium was added to 10,000 tide synthesizer 433A (Applied Biosystems, Foster City, CA) using stan- target cells per well. CTL were washed, counted, and added to the calcein- dard 9-fluorenylmethyloxycarbonyl/tert-butyl strategy. Peptides used for loaded target cells at an E:T ratio of 10:1 (final volume, 200 ml/well) and spun absolute quantification were further purified using preparative HPLC to down. Spontaneous release was determined by adding assay medium to the ensure purities .95%. target cells; maximum release was obtained by adding lysis buffer (50 mM sodium borate in 0.1% Triton X-100 [pH 9.0; both from Sigma-Aldrich]). Competitive binding assay After incubation for 2 h at 37˚C, 75 ml supernatant was carefully removed and Peptide affinity for HLA-A*02 was quantified by a competitive binding transferred to another 96-well, flat-bottom plate. Released calcein was mea- 28 sured in a fluorescence multiwell plate reader (SPECTRAmax GEMINI-XS; assay. T2 cells were loaded with 10 M Flu58–66 in the simultaneous presence of different concentrations of either SMCY , DDX5 , Molecular Devices; excitation wavelength, 485 nm; emission wavelength, 311–319 168–176 538 nm). Percentage specific lysis was calculated as [(sample release 2 PTRF56–64, or CD59106–114. The competing peptides were present at in- 2 3 creasing concentrations ranging from 10212 Mto1024 M. Cells were spontaneous release)/(maximum release spontaneous release)] 100%. incubated overnight at 37˚C in complete RPMI 1640 (2.5% FCS). The next Statistical methods day, a calcein-release cytotoxicity assay was performed as described previously using a CTL line specific for Flu58–66-HLA-A*02 (13). In the Mean values between groups were compared using the Student t test. Un- 28 simultaneous presence of 10 M Flu58–66 and increasingly higher less indicated otherwise, means 6 SD of triplicates are shown. 4 PROTEIN EXPRESSION PROTECTS ENDOTHELIAL CELLS FROM CTL

Results flow cytometry using either HLA-A*02–specific mAb BB7.2 or HLA-A*02–restricted peptides presented by EC pan-HLA class I-specific mAb W6/32 (Fig. 1A). As expressed in median values and interquartile range (shown in parentheses), EC We characterized the endothelial and leukocyte-derived HLA- 3 3 A*02–restricted peptide profile in three independent experiments lines had on average 22 (16–29) 10 HLA-A*02 molecules or 3 3 analyzing cells from three healthy HLA-A*02–positive males. EC 88 (86–137) 10 HLA class I molecules per cell. Corresponding 3 and BLC were grown side-by-side under virtually identical con- BLC expressed on average 142 (129–151) 3 10 HLA-A*02 or ditions and harvested using the same procedure to avoid meth- 482 (436–515) 3 103 HLA class I molecules per cell. Therefore, odological bias. HLA surface expression was considerably higher the total number of HLA ligands was expected to be at least 5-fold in BLC compared with EC from all three donors, as determined by lower in EC compared with BLC. Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. (A) Quantification of surface HLA-A*02 molecules and total HLA class I molecules on EC and BLC. HLA-A*02 molecules represent ∼25% of all HLA class I molecules, both on EC and on BLC from positive donors. EC have ∼6-fold less HLA-A*02 or HLA class I molecules, respectively, than do BLC. (B) Quantification of HLA-A*02 ligands SLSEKTVLL (upper panel) and YLLPAIVHI (lower panel) from BLC (left) and EC (right). Synthetic peptides labeled with deuterated nicotinic acid (dNIC) were used for calibration (arrow). Ten picomoles of each spiked peptide was injected together with nicotinylated (NIC) peptide mixtures derived from HLA-A*02 ligand isolation and analyzed by LC-MS. The nicotinylated native peptide is marked by an arrow. Absolute quantification of SLSEKTVLL, the most prevailing ligand found on ECs, reveals comparable quantities (∼3 pmol) presented on EC as well as on BLC. Allotypic HLA-A*02 ligand YLLPAIVHI, which is present on EC at negligible concentrations (arrow in the lower right panel), confirms the relative predominance of SLSEKTVLL on EC. Mass spectra show the relative intensity averaged over the retention time of respective precursor ion peaks within the ion chromatogram. Shown is the result obtained from donor 1. Graphics were generated with Xcalibur 2.07 QualBrowser. The Journal of Immunology 5

The entire pool of peptides bound to endothelial or leukocyte was detected with highest abundance in EC lines closely followed HLA-A*02 molecules was isolated, fractionated, and the indi- by SLLDKIIGA, whereas both peptides were not among the top vidual components were characterized by tandem mass spec- rankings with respect to AUC values in BLC (Table I). trometry. As expected by the use of the A*02-specific Ab BB7.2, To achieve an exact quantitative determination of peptide most peptides encompassed the allele-specific peptide motif of amounts, stable isotope labeling experiments were performed. HLA-A*02 characterized by aliphatic anchor amino acids in po- Natural HLA-A*02 ligands from either source were labeled with sition 2 and at the C terminus. A total of 56 HLA-A*02 ligands nicotinic acid, whereas synthetic calibrating peptides were labeled were identified from three EC lines whereas ˃1400 HLA-A*02 with deuterated nicotinic acid (16). Spiking experiments using ligands were characterized in BLC extraction experiments (Sup- defined amounts of calibrating peptides led to a precise quanti- plemental Tables I, II). Note that SMCY311–319, a male-specific, fication of HLA-A*02 ligands (Fig. 1B, Supplemental Table immunodominant minor histocompatibility Ag (18), was never III) and confirmed the estimations from AUC values. Both identified in these isolates, neither in EC nor in BLC. The amino SLSEKTVLL (CD59) and SLLDKIIGA (PTRF) represented by acid sequences of the isolated HLA-A*02–restricted peptides far the most abundant peptides of EC, dominating the HLA- were used for sequence homology searches, and the source pro- A*02–mediated peptide repertoire (Table II). Although also oc- teins from which these peptides derive were identified. A com- curring in HLA-A*02 ligand pools of BLC, they play a minor prehensive listing of HLA-A*02 ligands, their source proteins, and quantitative role there and are concealed by vast copy numbers MS-relevant parameters are given in Supplemental Tables I and II. of other HLA-A*02 ligands such as YLLPAIVHI (DDX5) and EC and BLC shared 25 of the 56 identified peptide species, among AIVDKVPSV (COPG) (Table II). In particular, the PTRF-derived them processing products of RNA helicase DDX5 (YLLPAIVHI) peptide SLLDKIIGA was hardly detectable on BLC (Table I), Downloaded from and coatomer subunit gamma, COPG (AIVDKVPSV); both pep- highlighting the observation that in no other tissues or cell lines tides have been repeatedly found in HLA-A*02–extracted peptide analyzed so far, SLLDKIIGA and SLSEKTVLL were as dominant pools obtained from human tissues. Moreover, YLLPAIVHI has as in EC. These two peptides were repetitively identified as the been classified as an allotypic peptide presented on most HLA- most prevalent HLA-A*02 ligands in these cells. Knowing the A*02 expressing cells and characteristic for this HLA allotype total EC end BLC number per donor that entered these experi-

(19). Thirty-five peptides were found exclusively on EC but not on ments, and knowing the number of HLA-A*02 molecules per cell, http://www.jimmunol.org/ autologous BLC. we were able to calculate that SLSEKTVLL and SLLDKIIGA Two peptides, SLLDKIIGA and SLSEKTVLL, yielded extra- together represent .50% of all HLA-A*02–restricted peptides per ordinarily high signals in EC compared with other cells and were single EC but ,5% of the peptides presented by BLC. consistently identified in all three EC donors analyzed in this study, suggesting that these peptides are constitutively processed and The molecular basis for abundant and endothelial presented by the MHC class I pathway of EC (Table I). The proteins cell-selective presentation of HLA-A*02–bound peptides from which these two peptides arise are polymerase I and tran- The most obvious explanation and logical prerequisite for the script release factor (PTRF) (20, 21) and the complement inhibitor preferential presentation of MHC class I bound PTRF56–64 and by guest on September 24, 2021 CD59 (22). Both HLA-A*02–restricted peptides have been iso- CD59106–114 on EC would be the strong and cell type-specific lated previously from other tissues. However, the relative amounts expression of PTRF and CD59 genes. Therefore, we compared of PTRF- and CD59-derived peptides appeared higher in EC the mRNA and protein levels of DDX5, PTRF, and CD59 in EC compared with any other cell line and tissue from which these and syngeneic EBV-immortalized BLC from the same individual peptides have been previously isolated. Comparing the AUC val- as the EC, in the HLA-A*02–positive BLC line JY, and in TAP- ues of the different peptides in LC-MS experiments, SLSEKTVLL deficient T2 cells and LS174 colon cancer cells (Fig. 2). PTRF

Table I. Peptides identiﬁed in EC (top) and BLC (bottom) together with their ranking based on the relative abundance estimated by the AUC of respective precursor ions

EC Donor 1 EC Donor 2 EC Donor 3

Sequence AUC Rank AUC Rank AUC Rank SLLDKIIGA 1.57 3 107 2 3.53 3 107 3 4.82 3 107 3 SLSEKTVLL 5.48 3 107 1 9.46 3 107 1 9.29 3 107 1 YLLPAIVHI ND ND ND AIVDKVPSV ND 4.43 3 106 5 4.19 3 106 9 ILMEHIHKL ND ND 4.00 3 105 26 VLIPKLPQL ND ND ND YLPEDFIRV ND ND ND FIDSYICQV ND ND ND ILDQKINEV 3.48 3 106 6 3.66 3 106 9 5.60 3 106 8 BLC Donor 1 BLC Donor 2 BLC Donor 3

AUC Rank AUC Rank AUC Rank SLLDKIIGA ND ND ND SLSEKTVLL 2.87 3 107 226 6.95 3 106 80 ND YLLPAIVHI 2.25 3 108 37 ND ND AIVDKVPSV 1.30 3 109 3 1.98 3 108 2 1.54 3 105 55 ILMEHIHKL 2.43 3 108 31 5.65 3 106 108 ND VLIPKLPQL 2.83 3 109 1 7.72 3 105 400 ND YLPEDFIRV 2.88 3 108 24 9.50 3 106 56 ND FIDSYICQV ND ND ND ILDQKINEV 6.99 3 108 6 1.56 3 108 4ND 6 PROTEIN EXPRESSION PROTECTS ENDOTHELIAL CELLS FROM CTL

Table II. Absolute quantification of selected HLA ligands in two abundant structure protein b-actin was equally expressed in all consecutive runs, each containing 20% of complete isolation per cell line cell lines at the protein level (Fig. 2B). According to the SYF- ∼ 8 ∼ 3 8 of EC ( 10 cells) and BLC ( 4 10 cells) from donor 1 with 0.3 (run PEITHI algorithm (http://www.syfpeithi.de) (23), b-actin contains 1) or 10 pmol (run 2) of spiked calibrating peptides several peptide motifs that match criteria for HLA-A*02 binding. b Sequence Run 1 Run 2 However, none of these putative HLA-A*02–restricted -actin peptides was ever detected in the cell isolates analyzed, neither in EC donor 1 EC nor in BLC (Supplemental Tables I, II). SLSEKTVLL 3396.37 2864.74 SLLDKIIGA 355.31 836.80 Peptide affinity for HLA-A*02 AIVDKVPSV 347.41 114.09 ILDQKINEV 335.29 121.39 High peptide affinity for HLA-A*02 may further contribute to YLLPAIVHI 11.15 62.07 the dominant representation of certain peptides in EC-derived BLC donor 1 profiles. We compared the endothelial peptides PTRF56–64 AIVDKVPSV 369,079.91 128,173.75 and CD59106–114, as well as the ubiquitous DDX5168–176 and YLLPAIVHI 54,923.29 23,609.07 SMCY , for binding to HLA-A*02. A functional competition ILDQKINEV 209,735.02 23,182.60 311–319 SLSEKTVLL 10,387.56 2764.77 assay was performed to measure possible differences in peptide SLLDKIIGA 30.47 157.82 affinity for HLA-A*02. In this assay, the peptide concentration was determined at which target cell killing by Flu58–66-specific CTL in 28 the presence of 10 MFlu58–66 peptide was inhibited by 50% (Fig. 6 3 25 was expressed both at the RNA and protein levels exclusively in 3A, 3B, Table III). SMCY311–319 required 0.3 0.2 10 M Downloaded from EC and was not found in any other cell line analyzed (Fig. 2). peptide concentration to reduce Flu58–66-specific killing by 50%. 6 3 25 6 3 CD59 was expressed at the RNA level in all cell lines analyzed For DDX5168–176 0.8 0.4 10 M, for PTRF56–64 1.5 0.7 25 6 3 25 (Fig. 2A). At the protein level, CD59 was expressed at highest 10 M, and for CD59106–114 2.1 1.1 10 M peptide was level in EC and faintly in the two BLC lines analyzed, but not in necessary to inhibit Flu58–66-specific lysis by 50%. This indicates T2 or LS174 colon cancer cells (Fig. 2B). DDX5, the ubiquitously that peptide affinity for HLA-A*02 is lowest for the two most abundant endothelial peptides PTRF56–64 and CD59106–114.This expressed protein that is the source of the allotypic reference http://www.jimmunol.org/ unexpected result was endorsed by a second independent experi- peptide YLLPAIVHI (DDX5168–176), which is present at large quantities in the peptide binding groove of HLA-A*02 molecules mental approach, that is, a T2 binding assay (24). Surface expres- of most tissues (19), was found in all cell lines both at the RNA as sion of HLA-A*02 was determined by flow cytometry after 25 at the protein level in similar amounts (Fig. 2). Interestingly, the overnight pulsing of T2 cells by 10 M peptide (Fig. 3C). SMCY311–319 and DDX5168–176 were the strongest binders, increasing the fluorescence index 3.2 6 0.2- and 2.9 6 0.1-fold, respectively (Fig. 3C, Table III). PTRF56–64 and CD59106–114 showed significantly weaker binding, increasing HLA-A*02 expression

2.2 6 0.1- and 1.4 6 0.1-fold, respectively (p = 0.002 and p = by guest on September 24, 2021 0.003, respectively). These results confirmed the observations made in the competitive binding assay. Peptide concentrations could be adjusted according to the findings of the competitive Flu58–66-binding assay to similar surface levels of HLA-A*02 in T2 cells (Fig. 3D). Compared with SMCY311–319, a 2.4-fold higher concentration for DDX5168–176,a 4.4-fold higher concentration for PTRF56–64, and an 8-fold higher peptide concentration for CD59106–114 was necessary to compensate for the weaker binding of the respective peptides (Fig. 3D). Stability of the peptide/MHC class I complex Finally, the decay of the peptide/HLA-A*02 complexes could make a difference in surface peptide presentation of cells. Half- lives of peptide/HLA-A*02 complexes were determined by measur- ing the HLA-A*02 surface expression on T2 cells at indicated time points after pulsing with adjusted peptide concentrations (Fig. 4). T2 cells were loaded with the peptide of interest for 24 h, unbound peptide was washed away, and HLA-A*02 surface expression was measured by flow cytometry at different time points (0, 2, 6, 24 h). SMCY311–319, DDX5168–176, and PTRF56–64 showed similar kinetics with half-lives of 6.0, 5.8, and 7 h, respectively (Fig. 4). However, the half-life of CD59106–114 was only 2.8 h and therefore substantially shorter compared with the other peptides. Stability of FIGURE 2. mRNA expression and cellular representation of source proteins that contain the HLA-A*02 presented endothelial peptide HLA-A*02/peptide complexes on the cell surface does not consequences. (A) Total RNA for RT-PCR was obtained from cultured tribute to the dominant presentation of CD59106–114. HUVEC (EC), BLC, the B cell line JY, T2 cells, and the colon carcinoma Modulation of endothelial CTL susceptibility by source protein cell line LS174. Shown are the RT-PCR products of PTRF, CD59, DDX5, expression and GAPDH transcripts. (B) Western blot was performed with whole cell lysates from EC, BLC, JY, T2 cells, and LS174. All cells were analyzed The dominant presentation of certain cell-specific peptides on for the presence of the proteins PTRF, CD59, and DDX5. b-actin was EC could also compete with the surface expression of immuno- used as a loading control. dominant CTL epitopes, for example, the HLA-A*02–restricted The Journal of Immunology 7

FIGURE 3. Peptide affinity for HLA-A*02. Competitive binding and T2 peptide binding assay were performed for four different allo- and auto- peptides (SMCY311–319, DDX5168–176, PTRF56–64 and CD59106–114). (A) The competitive binding assay shows the peptide concentration (1025 M) in the 28 presence of 10 M Flu58–66, at which target killing by Flu58–66-specific CTL was inhibited by 50%. (B) 28 Specific lysis of T2 cells loaded with 10 MFlu58–66 and different concentrations of the peptide of interest (SMCY311–319, DDX5168–176,PTRF56–64, 24 28 and CD59106–114) ranging from 10 to 10 Mby Flu58–66-specific CTL. The dashed line indicates the peptide concentration at which target cell killing by

Flu58–66-speciﬁc CTL was inhibited by 50%. (C and D) For the T2 binding assay, HLA-A*02 positivity

of peptide-loaded T2 cells was analyzed by flow Downloaded from cytometry. The fluorescence index was calculated as ΔMFI with peptide/ΔMFI without peptide. (C) Fluorescence index and typical histograms with 1025 M peptide concentrations are shown, and (D) with peptide concentrations adjusted to binding differences calculated from the competitive binding assay. For each peptide three independent competi- http://www.jimmunol.org/ tion and binding assays were performed. **Signifi- cant changes compared with fluorescence index for SMCY, p , 0.05.

minor histocompatibility Ag SMCY311–319. By this mechanism cell line JY treated with either specific or mock siRNA remained EC could be protected at least partially from CTL-mediated lysis unchanged (Fig. 5C). The seemingly small 7% difference in by guest on September 24, 2021 (13). To test this speculative hypothesis, precursor proteins of the specific killing translates into a 90-fold higher amount of peptide abundant EC peptides were transiently eliminated by RNA inter- expressed by EC (0.9 3 102 M) when the cytolytic activity on ference and EC were then used as targets in a CTL assay. siRNA treated and untreated EC is compared with the peptide titration designed for DDX5, PTRF, and CD59 using a publically available curve using SMCY311–319-pulsed T2 cells as targets (Fig. 5D). algorithm (www.microsynth.ch) (25) were used in EC and BLC HLA-A*02 expression levels of target cells were not changed by and protein levels were assessed by Western blot (Fig. 5A). In EC, siRNA treatment after up to 72 h (Fig. 5E). DDX5 knockdown was complete after 24 h and lasted for ˃96 h. CD59 protein levels started to decrease at 48 h and were unde- Discussion tectable by this method after 72 h. PTRF elimination also started In this study, we analyzed the endothelial peptide repertoire pre- after 48 h. The protein reached minimal levels (20%) by 72 h and sented by HLA-A*02 and compared it with the repertoire pre- started to increase after 96 h. Based on these protein kinetics, cells sented by syngeneic B lymphoblastoid cells. We demonstrate that were used after 72 h as targets in cytotoxicity assays. Simulta- EC present a quantitatively different peptide repertoire that may neous knockdown of PTRF56–64, CD59106–114, and DDX5168–176 contribute to the protection of EC from CTL-mediated lysis by on male EC should render these cells more susceptible for killing competition with immunogenic peptides for the MHC class I by SMCY311–319-specific CTL. We found that after treatment of binding sites. EC with combined siRNAs for 72 h, specific killing raised from Compared to leukocyte-derived cells, the peptide repertoire 14 6 5to216 6% (p = 0.002; Fig. 5B). Killing of the male B presented by EC showed a consistent prevalence of two peptides,

Table III. Binding afﬁnity and half-life of various peptide/HLA-A*02 complexes

Competitive Binding Assay T2 Binding Assay (Peptide Concentration for (Fold Increase of 50% Inhibition, M) Fluorescence Index) Half-Life Assessment (h)

Flu58–66 Not applicable Not applicable Not applicable 25 SMCY311–319 0.3 6 0.2 3 10 3.2 6 0.2 6.0 25 DDX5168–176 0.8 6 0.4 3 10 2.9 6 0.1 5.8 25 PTRF56–64 1.5 6 0.7 3 10 ** 2.2 6 0.1** 7.0 25 CD59106–114 2.1 6 1.1 3 10 ** 1.4 6 0.1** 2.8 See also Fig. 3. **p , 0.05, signiﬁcantly different from SMCY311–319. 8 PROTEIN EXPRESSION PROTECTS ENDOTHELIAL CELLS FROM CTL Downloaded from

FIGURE 4. Half-life of HLA-A*02–bound peptides. T2 cells were loaded with the peptides (SMCY311–319, DDX5168–176,PTRF56–64,and CD59106–114) in adjusted concentrations according to the competitive binding assay and HLA-A*02 surface positivity was analyzed by ﬂow cytometry at indicated time points (0, 2, 6, 24 h). For each peptide three http://www.jimmunol.org/ independent experiments were performed.

PTRF56–64 and CD59106–114. We found that the predominance of these two endothelial peptides was caused by the abundant expression of the source proteins PTRF and CD59. PTRF has been identified as a major caveolae-associated protein (18), and caveolae are particularly abundant in endothelial cells and adipocytes. BLC and EC both express CD59 protein, although the amount is substantially higher in EC. This may explain the vast abundance by guest on September 24, 2021 of CD59106–114 peptide on EC compared with BLC. Note that SMCY311–319, an immunodominant HLA-A*02–bound peptide originally described as minor histocompatibility Ag (19), was never identified in the isolates from male cells, neither in BLC nor in EC. Similarly, we could never find peptides derived from b-actin in these isolates, although it is a strongly expressed structure protein, contains several possible HLA-A*02 binding motifs, and although YALPHAILRL and ALAPSTMKI (both b from -actin) were identified previously on tumor tissue. FIGURE 5. Knockdown of abundant endothelial peptides. (A) Western b We assume that for SMCY and particularly for -actin, rather blot showing protein levels of DDX5, PTRF, and CD59 in EC after low protein synthesis or degradation rates are involved as mech- treatment with either mock siRNA or specific siRNA at different time anisms for quantitatively low surface expression of these peptides points after knockdown (24, 48, 72, 96 h). b-actin is shown confirming on both EC and BLC (26, 27). Male BLC (but not male EC) are similar protein loads. (B and C) Specific lysis of male EC (B) or JY cells C recognized and killed by CTL specific for SMCY311–319 (13). This ( ) treated with mock or specific siRNA by SMCY311–319-specific CTL. confirms that the peptide isolation assay is substantially less Shown are the results of three independent experiments. (D) Peptide ti- sensitive than the calcein-release–based killing assay for the sur- tration of SMCY311–319-loaded T2 cells. The dotted line indicates the face detection of peptide motifs. It cannot be excluded that cys- specific lysis of EC treated with mock siRNA, the dashed line of EC treated with specific siRNA. (E) HLA-A*02 levels on EC after treatment teine containing peptide SMCY is more prone to chemical 311–319 with either mock or specific siRNA. modifications during the isolation process, which can lead to a decreased intensity of the unmodified peptide and this may in- terfere with peak annotation after analysis. However, even under Peptides with lower affinity for the MHC class I molecule may reducing conditions and subsequent modification of cysteinyl form less stable peptide/MHC complexes, leading to decreased residues with iodoacetamide, SMCY could not be detected in any immunogenicity. The presentation of particularly weak binding of the runs (data not shown). peptides may therefore be another mechanism to protect EC from We further found that the strong expression and putatively also CTL-mediated lysis. turnover of the precursor proteins of PTRF56–64 and CD59106–114 The immunological significance of cell type-specific peptide are able to compensate for the low binding affinity of the endo- presentation has been reported previously in the context of auto- thelial peptides to HLA-A*02 and their short half-life. It has been immune diseases. Cell type-specific Ag expression may play an shown that one important factor for the immunogenicity of important role in organ- or cell-specific T cell-mediated autoim- a peptide is the stability of the peptide/MHC complex (28, 29). mune diseases such as type 1 diabetes mellitus or multiple sclerosis. The Journal of Immunology 9

In a murine model of type 1 diabetes, the insulin-producing b cells of 3. Tang, E. H., and P. M. Vanhoutte. 2010. Endothelial dysfunction: a strategic + target in the treatment of hypertension? Pflugers Arch. 459: 995–1004. the pancreas are selectively targeted by autoreactive CD8 Tcells 4. Palmer, R. M., A. G. Ferrige, and S. Moncada. 1987. Nitric oxide release + (30). In murine multiple sclerosis, autoreactive CD8 T cells re- accounts for the biological activity of endothelium-derived relaxing factor. Na- sponsive to myelin-derived peptides have been reported that have ture 327: 524–526. 5. Fernandez-Borja, M., J. D. van Buul, and P. L. Hordijk. 2010. The regulation of the potential to kill selectively MHC class I-matched oligoden- leucocyte transendothelial migration by endothelial signalling events. Car- drocytes (31–33). In the context of allogeneic bone marrow trans- diovasc. Res. 86: 202–210. plantation it has been shown that the restricted expression of minor 6. Romanic, A. M., and J. A. Madri. 1994. The induction of 72-kD gelatinase in T cells upon adhesion to endothelial cells is VCAM-1 dependent. J. Cell Biol. histocompatibility Ags on hematopoietic cells, including leukemic 125: 1165–1178. cells, can separate the beneficial graft-versus-leukemia from the 7. Marelli-Berg, F. M., M. J. James, J. Dangerfield, J. Dyson, M. Millrain, D. Scott, harmful graft-versus-host effect of ubiquitously expressed minor E. Simpson, S. Nourshargh, and R. I. Lechler. 2004. Cognate recognition of the endothelium induces HY-specific CD8+ T-lymphocyte transendothelial migration histocompatibility Ags (34, 35). However, for endothelial cells, (diapedesis) in vivo. Blood 103: 3111–3116. the cell type selective peptide repertoire seems to have an opposite 8. Hart, D. N., and J. W. Fabre. 1981. Localization of MHC antigens in long sur- effect. We provide evidence that the predominant presentation viving rat renal allografts: probable implication of passenger leukocytes in graft adaptation. Transplant. Proc. 13: 95–99. + of the endothelium-specific Ags PTRF56–64 and CD59106–114 can 9. Inaba, K., J. W. Young, and R. M. Steinman. 1987. Direct activation of CD8 protect EC from CTL-mediated lysis by competing with the im- cytotoxic T lymphocytes by dendritic cells. J. Exp. Med. 166: 182–194. 10. Shulman, H. M., G. E. Sale, K. G. Lerner, E. A. Barker, P. L. Weiden, munodominant peptide SMCY311–319 for the binding site on HLA- K. Sullivan, B. Gallucci, E. D. Thomas, and R. Storb. 1978. Chronic cutaneous A*02 molecules. Knockdown of the abundant endothelial source graft-versus-host disease in man. Am. J. Pathol. 91: 545–570. proteins PTRF, CD59, and DDX5 by RNA interference led to an 11. Ball, L. M., R. M. Egeler, and E. P. W. Party; EBMT Paediatric Working Party. 2008. Acute GvHD: pathogenesis and classification. Bone Marrow Transplant. increased presentation of endogenously processed male-specific 41(Suppl. 2): S58–S64. Downloaded from peptide SMCY311–319, proving that without the abundant endothe- 12. Biedermann, B. C., and J. S. Pober. 1999. Human vascular endothelial cells favor lial peptides EC become better targets for SMCY -specific clonal expansion of unusual alloreactive CTL. J. Immunol. 162: 7022–7030. 311–319 13. Kummer, M., A. Lev, Y. Reiter, and B. C. Biedermann. 2005. Vascular endo- CTL. Importantly, this mechanism does not restore EC as CTL thelial cells have impaired capacity to present immunodominant, antigenic targets to the same level as BLC. Lack of activating costimulators peptides: a mechanism of cell type-specific immune escape. J. Immunol. 174: 1947–1953. (36) or production of inhibitory cytokines (37, 38) might further 14. Biedermann, B. C., and J. S. Pober. 1998. Human endothelial cells induce and contribute to the immune privilege of vascular endothelium. 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Stumm, S. Kayser, A. Moris, inhibition of immunodominant epitope presentation. We do not D. Wallwiener, H. G. Rammensee, and S. Stevanovic. 2001. A MAGE-A1 HLA- A A*0201 epitope identified by mass spectrometry. Cancer Res. 61: 4072–4077. know whether this surface peptide modulation is significant 18. Rufer, N., E. Wolpert, C. Helg, J. M. Tiercy, A. Gratwohl, B. Chapuis, in vivo, that is, whether it protects cells or tissues from immune- M. Jeannet, E. Goulmy, and E. Roosnek. 1998. HA-1 and the SMCY-derived by guest on September 24, 2021 mediated injury. However, Bolinger et al. (40) showed in a mouse peptide FIDSYICQV (H-Y) are immunodominant minor histocompatibility antigens after bone marrow transplantation. Transplantation 66: 910–916. model that vascular EC expressing minor histocompatibility Ags 19. Hillen, N., and S. Stevanovic. 2006. Contribution of mass spectrometry-based are immunologically ignored by Ag specific CD8+ T cells. This proteomics to immunology. Expert Rev. 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Regulatory control of the terminal complement proteins at the surface of human endothelial titatively different HLA-A*02–restricted peptide repertoire com- cells: neutralization of a C5b-9 inhibitor by antibody to CD59. Blood 76: 2572– pared with syngeneic BLC and by this feature are at least partially 2577. protected from CTL-mediated lysis. The presented peptide profile 23. Rammensee, H., J. Bachmann, N. P. Emmerich, O. A. Bachor, and S. Stevanovic´. 1999. SYFPEITHI: database for MHC ligands and peptide motifs. Immunoge- can be modulated by knocking down the source proteins in target netics 50: 213–219. cells. The interference with source protein expression may be 24. Sun, Y., M. Song, S. Stevanovic´, C. Jankowiak, A. Paschen, H. G. Rammensee, and D. Schadendorf. 2000. Identification of a new HLA-A*0201-restricted T-cell a novel approach to modify target cell properties. epitope from the tyrosinase-related protein 2 (TRP2) melanoma antigen. Int. J. Cancer 87: 399–404. 25. Reynolds, A., D. Leake, Q. Boese, S. Scaringe, W. S. Marshall, and Acknowledgments A. Khvorova. 2004. Rational siRNA design for RNA interference. Nat. Bio- We thank Ed Palmer, Christoph Dehio, and Reto Krapf for helpful dis- technol. 22: 326–330. 26. Khan, S., R. de Giuli, G. Schmidtke, M. Bruns, M. Buchmeier, M. van den cussions and Denise Dubler for excellent technical assistance. Broek, and M. Groettrup. 2001. Cutting edge: neosynthesis is required for the presentation of a T cell epitope from a long-lived viral protein. J. Immunol. 167: 4801–4804. Disclosures 27. Yewdell, J. W., E. Reits, and J. Neefjes. 2003. Making sense of mass destruction: quantitating MHC class I antigen presentation. Nat. Rev. Immunol. 3: 952–961. The authors have no conflicts of interest. 28. van der Burg, S. H., M. J. Visseren, R. M. Brandt, W. M. Kast, and C. J. Melief. 1996. Immunogenicity of peptides bound to MHC class I molecules depends on the MHC-peptide complex stability. J. Immunol. 156: 3308–3314. 29. Feltkamp, M. C., M. P. Vierboom, W. M. Kast, and C. J. Melief. 1994. Efficient References MHC class I-peptide binding is required but does not ensure MHC class I- 1. Bombeli, T., M. Mueller, and A. Haeberli. 1997. Anticoagulant properties of the restricted immunogenicity. Mol. Immunol. 31: 1391–1401. vascular endothelium. Thromb. Haemost. 77: 408–423. 30. Eneé, E., E. Martinuzzi, P. Blancou, J. M. Bach, R. Mallone, and P. van Endert. 2. Biedermann, B., A. Rosenmund, M. Muller, H. P. Kohler, A. Haeberli, and 2008. Equivalent specificity of peripheral blood and islet-infiltrating CD8+ P. W. Straub. 1994. Human endothelial cells suppress prothrombin activation in T lymphocytes in spontaneously diabetic HLA-A2 transgenic NOD mice. J. nonanticoagulated whole blood in vitro. J. Lab. Clin. Med. 124: 339–347. Immunol. 180: 5430–5438. 10 PROTEIN EXPRESSION PROTECTS ENDOTHELIAL CELLS FROM CTL

31. Babbe, H., A. Roers, A. Waisman, H. Lassmann, N. Goebels, R. Hohlfeld, tous versus restricted tissue distribution indicates heterogeneity among human M. Friese, R. Schro¨der, M. Deckert, S. Schmidt, et al. 2000. Clonal expansions cytotoxic T lymphocyte-defined non-MHC antigens. J. Immunol. 149: 1788– of CD8+ T cells dominate the T cell infiltrate in active multiple sclerosis lesions 1794. as shown by micromanipulation and single cell polymerase chain reaction. J. 36. Hicks, C., C. Cheung, and R. Lindeman. 2003. Restimulation of tumour-specific Exp. Med. 192: 393–404. immunity in a patient with AML following injection with B7-1 positive autol- 32. Sun, D., J. N. Whitaker, Z. Huang, D. Liu, C. Coleclough, H. Wekerle, and ogous blasts. Leuk. Res. 27: 1051–1061. C. S. Raine. 2001. Myelin antigen-specific CD8+ T cells are encephalitogenic 37. Thomas, D. A., and J. Massague´. 2005. TGF-b directly targets cytotoxic T cell and produce severe disease in C57BL/6 mice. J. Immunol. 166: 7579–7587. functions during tumor evasion of immune surveillance. Cancer Cell 8: 369– 33. Huseby, E. S., D. Liggitt, T. Brabb, B. Schnabel, C. Ohleń, and J. Goverman. 380. 2001. A pathogenic role for myelin-specific CD8(+) T cells in a model for 38. Mahboubi, K., B. C. Biedermann, J. M. Carroll, and J. S. Pober. 2000. IL-11 multiple sclerosis. J. Exp. Med. 194: 669–676. activates human endothelial cells to resist immune-mediated injury. J. Immunol. 34. Niederwieser, D., A. Grassegger, J. Aubo¨ck, M. Herold, D. Nachbaur, 164: 3837–3846. A. Rosenmayr, A. Ga¨chter, W. Nussbaumer, S. Gaggl, M. Ritter, et al. 1993. 39. Butz, E. A., and M. J. Bevan. 1998. Differential presentation of the same MHC Correlation of minor histocompatibility antigen-specific cytotoxic T lympho- class I epitopes by fibroblasts and dendritic cells. J. Immunol. 160: 2139–2144. cytes with graft-versus-host disease status and analyses of tissue distribution 40. Bolinger, B., P. Krebs, Y. Tian, D. Engeler, E. Scandella, S. Miller, D. C. Palmer, of their target antigens. Blood 81: 2200–2208. N. P. Restifo, P. A. Clavien, and B. Ludewig. 2008. Immunologic ignorance of 35. de Bueger, M., A. Bakker, J. J. Van Rood, F. Van der Woude, and E. Goulmy. vascular endothelial cells expressing minor histocompatibility antigen. Blood 1992. Tissue distribution of human minor histocompatibility antigens: ubiqui- 111: 4588–4595. Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 Supplementary Table SI: Peptides identified on EC (high confidence FDR <0,05, medium confidence FDR <0,15)

Confidence Also found Sequence GeneID Description Level on BLC Confirmed* AIVDKVPSV 22820 Coatomer subunit gamma yes Confirmed* NLAEDIMRL 7431 Vimentin yes Polymerase I and transcript Confirmed* SLLDKIIGA 284119 no release factor Confirmed* SLSEKTVLL 966 CD59 glycoprotein yes Probable ATP-dependent RNA Confirmed* YLLPAIVHI 1655 yes helicase DDX5 COP9 signalosome complex High ALATLIHQV 50813 yes subunit 7a High ALGTDAEGQKV 6814 Syntaxin-binding protein 3 yes Nuclear pore complex protein High ALLDRIVSV 23165 yes Nup205 High DVIRHGADAV 93099 Dermokine no High GLADASLLKKV 25814 Ataxin-10 yes High GLDDIKDLKV 4343 Putative helicase MOV-10 yes Eukaryotic translation initiation High IALNEKLVNL 8665 no factor 3 subunit F High ILDQKINEV 4953 Ornithine decarboxylase yes High ILTDITKGV 1938 Elongation factor 2 yes High KVLDFEHFL 4637 Myosin light polypeptide 6 no High KVVLTQANKLGV 22872 Protein transport protein Sec31A no High LIGQVHEV 10129 Protein furry homolog no High LLAAWTARA 351 Amyloid beta A4 protein no High NMLGGKYQGTIL 1828 Desmoglein-1 no High NVINGGSHAGNKL 2023 Alpha-enolase no High RVVGPISGADL 1828 Desmoglein-1 no High SLGSALRPST 7431 Vimentin no High TTGAGNPVGDKL 847 Catalase no High VLIDYQRNV 7514 Exportin-1 yes High VLLKARLVPA 165215 Protein FAM171B yes High VLSPADKTNVKA 3039 Hemoglobin subunit alpha no High YLDPAQRGV 84326 UPF0585 protein C16orf13 yes Medium ALSDHHIYL 226 Fructose-bisphosphate aldolase A yes Medium FLDNERHEV 5585 Serine/threonine-protein kinase no N1 Probable dolichyl pyrophosphate Medium FLDPNNIPKA 79053 Glc1Man9GlcNAc2 alpha-1,3- yes glucosyltransferase Medium FLIRESETL 4067 Tyrosine-protein kinase Lyn yes Ubiquitin-associated protein 2- Medium FLQEKSPAV 9898 yes like 26S proteasome non-ATPase Medium FVDDYTVRV 10213 no regulatory subunit 14 Medium GLDIDGIYRV 94134 Rho GTPase-activating protein no Medium GLGPPGRSV 144108 Protein SPT2 homolog no Transient receptor potential Medium GLmKYIGEV 79054 cation channel subfamily M yes member 8 Polymerase I and transcript Medium GLRRVDDFKKA 284119 no release factor Solute carrier family 46 member Medium IIAVSLAVNL 283537 no 3 26S proteasome non-ATPase Medium ILDAGGHNV 5707 yes regulatory subunit 1 Nuclear pore complex protein Medium ILDPHVVLL 4927 no Nup88 Medium IQDITQRL 4478 Moesin no Medium KILNGKGILPT 222255 Ataxin-7-like protein 1 no Glyceraldehyde-3-phosphate Medium KTVDGPSGKL 2597 no dehydrogenase Medium KVFDPVPVGV 1660 ATP-dependent RNA helicase A no Medium LIRGPAETEAT 5756 Twinfilin-1 no Transmembrane emp24 domain- Medium LLGPRLVLA 10972 no containing protein 10 Medium LVKGHAYSI 147968 Calpain-12 no Medium LVQPRVEFIL 3837 Importin subunit beta-1 no Medium LVRPGTAL 1832 Desmoplakin no Medium LVRPGTALEL 1832 Desmoplakin no Cytochrome c oxidase subunit Medium LVVRHQLLKT 1350 no 7C, mitochondrial Medium NQWDGTQHGV 7402 Utrophin no Medium PLKQGGAKA 8022 Putative myosin-XVB no Haloacid Dehalogenase-like Medium RALPDFKGI 505403 hydrolase domain-containing no protein Medium RIIDVVYNA 6202 40S ribosomal protein S8 no Medium TVADKIHSV 11072 Dual specificity protein yes phosphatase 14 Medium VLDDKLVFV 196527 Anoctamin-6 yes Mitogen-activated protein kinase Medium VLWGETVHL 1326 yes kinase kinase 8 Medium VMDSKIVQV 3841 Importin subunit alpha-6 yes Polycystic kidney disease protein Medium VTSLEKSLI 342372 no 1-like 3 Elongation of very long chain Medium YQYPVIIHL 64834 yes fatty acids protein

*Confirmation achieved by positive matching of fragment spectra with respective spectra of synthetic peptides.

Supplementary Table SII: Peptides identified on BLC (high confidence FDR <0,05, medium confidence FDR <0,15).

Confidence Sequence GeneID Description Level Confirmed* AIVDKVPSV 22820 Coatomer subunit gamma Confirmed* ILDQKINEV 4953 Ornithine decarboxylase Confirmed* ILMEHIHKL 6143 60S ribosomal protein L19 Confirmed* SLSEKTVLL 966 CD59 glycoprotein Confirmed* VLIPKLPQL 94103 ORM1-like protein 3 Probable ATP-dependent RNA helicase Confirmed* YLLPAIVHI 1655 DDX5 Interleukin-1 receptor-associated kinase- Confirmed* YLPEDFIRV 3656 like 2 High AIKKKDELERVA 6193 40S ribosomal protein S5 High AILETAPKEV 6238 Ribosome-binding protein 1 High AILTVVPKI 9730 Protein VPRBP High ALAAALAHI 79009 ATP-dependent RNA helicase DDX50 High ALAALHVTL 27173 Zinc transporter ZIP1 High ALADGVQKV 8542 Apolipoprotein L1 High ALADKELLPSV 84883 Apoptosis-inducing factor 2 High ALAEIAKAEL 6421 Splicing factor, proline- and glutamine-rich High ALAGHQDGITFI 80344 DDB1- and CUL4-associated factor 11 High ALAKLVEAI 6130 60S ribosomal protein L7a EH domain-containing protein 1/EH High ALANHLIKV 10938/30845 domain-containing protein 3 High ALAQRLLEV 161176 Nesprin-3 High ALATLIHQV 50813 COP9 signalosome complex subunit 7a Probable ATP-dependent RNA helicase High ALDEYNMKI 55601 DDX60 High ALDKALTSV 8453 Cullin-2 High ALDKATVLL 27250 Programmed cell death protein 4 High ALDPASLPRV 9473 Protein THEMIS2 FYVE, RhoGEF and PH domain-containing High ALDSQVPKV 89846 protein 3 High ALEEKIPNI 2941 Glutathione S-transferase A4 High ALFDQDKSSMRL 221184 Copine-2 High ALFQRPPLI 1736 H/ACA ribonucleoprotein complex subunit

1 4

High ALGAGIERMGL 4670 Heterogeneous nuclear ribonucleoprotein M High ALGPTGRGV 9517 Serine palmitoyltransferase 2 High ALGTDAEGQKV 6814 Syntaxin-binding protein 3 Apoptosis-associated speck-like protein High ALIARVTNV 29108 containing a CARD High ALIDRMVNL 1649 DNA damage-inducible transcript 3 protein High ALIEAEKVAQV 11160 Erlin-2 High ALIEKLVEL 23649 DNA polymerase alpha subunit B High ALITRIFGV 63892 Thyroid adenoma-associated protein High ALKDLINEA 5111 Proliferating cell nuclear antigen High ALLAGPLRPA 26071 Protein FAM127B Eukaryotic translation initiation factor 3 High ALLAGSEYLKL 8664 subunit D High ALLDGRLQVV 2194 Fatty acid synthase High ALLDQLHTL 8202 Nuclear receptor coactivator 3 High ALLDRIVSV 23165 Nuclear pore complex protein Nup205 High ALLDSAHLL 8943 AP-3 complex subunit delta-1 High ALLEDEERVVRL 57488 Extended synaptotagmin-2 High ALLEMDARL 54512 Exosome complex component RRP41 High ALLGDLTKA 30000 Transportin-2 High ALLGILQHV 23386 NudC domain-containing protein 3 High ALLGRIPSA 506 ATP synthase subunit beta, mitochondrial High ALLQQVHSA 80209 Proline and serine-rich protein 1 High ALLSGLREA 23028 Lysine-specific histone demethylase 1A High ALMDEVVKA 5230 Phosphoglycerate kinase 1 High ALMDLDVKKMPL 10039 Poly [ADP-ribose] polymerase 3 High ALMEQVAHQTI 11140 Hsp90 co-chaperone Cdc37 High ALNEEAGRLLL 27338 Ubiquitin-conjugating enzyme E2 S Eukaryotic translation initiation factor 3 High ALNEKLVNL 8665 subunit F High ALNELLQHV 7094 Talin-1 High ALNEQIARL 10403 Kinetochore protein NDC80 homolog High ALQEKLWNV 92105 Integrator complex subunit 4 High ALQEKVQAV 23172 BRISC complex subunit Abro1

2 High ALQEMVHQV 4739 Enhancer of filamentation 1 High ALSDHHIYL 226 Fructose-bisphosphate aldolase A High ALSEKLARL 115106 HAUS augmin-like complex subunit 1 High ALSKEGIVAL 908 T-complex protein 1 subunit zeta High ALSNLEVKL 83706 Fermitin family homolog 3 High ALTRLHITV 64121 Ras-related GTP-binding protein C Biogenesis of lysosome-related organelles High ALVDHLNVGV 2647 complex 1 subunit 1 High ALVSSLHLL 22820 Coatomer subunit gamma Guanine nucleotide-binding protein G(s) High ALWEDEGVRA 2778 subunit alpha isoforms XLas High ALWSVGGEVHV 115330 Probable G-protein coupled receptor 146 Mannose-1-phosphate guanyltransferase High ALYASRLYL 29926 alpha Structural maintenance of chromosomes High ALYDEVRTV 23347 flexible hinge domain-containing protein 1 Asparagine synthetase [glutamine- High ALYDNVEKL 440 hydrolyzing] High ALYHLAIKL 11215 A-kinase anchor protein 11 High AMAQEGLREV 3071 Nck-associated protein 1-like High AMFDHIPVGV 51493 tRNA-splicing ligase RtcB homolog Sister chromatid cohesion protein PDS5 High AMFGKLMTI 23244 homolog A High AMLAVLHTV 60673 Autophagy-related protein 101 ATP-binding cassette sub-family F member High AMLENASDIKL 23 1 Activating signal cointegrator 1 complex High AMLTVLHEI 10973 subunit 3 Phosphopantothenoylcysteine High AMWEHPITA 60490 decarboxylase High AMYEHKIFV 2821 Glucose-6-phosphate isomerase High AVTKTAGPIASA 4134 Microtubule-associated protein 4 High EITNVTQKI 10388 Synaptonemal complex protein 2 ADP-ribosylation factor GTPase-activating High FASHVSPEV 26286 protein 3 High FIFDVHVHEV 23654 Plexin-B2 High FIFSDTHEL 143 Poly [ADP-ribose] polymerase 4 High FIFSEKPVFV 6653 Sortilin-related receptor Signal transducer and activator of High FIIEKQPPQV 6777 transcription 5B

3 DNA-directed RNA polymerase I subunit High FIINGIEKV 84172 RPA2 High FIMEGPLTRI 6655 Son of sevenless homolog 2 High FIMEGTLTRV 6654 Son of sevenless homolog 1 Phosphoribosylformylglycinamidine High FINARNWTL 5198 synthase High FINIVVHSV 91010 Formin-like protein 3 High FIQEIEHAL 60412 Exocyst complex component 4 26S proteasome non-ATPase regulatory High FISEFEHRV 5719 subunit 13 High FIWENIHTL 667 Dystonin High FLADVDKLKL 9793 Cytoskeleton-associated protein 5 DNA dC->dU-editing enzyme APOBEC- High FLAEDPKVTL 200316 3G High FLAEHDYGL 25962 Protein virilizer homolog High FLAEHPNVTL 200316 DNA dC->dU-editing enzyme APOBEC-3F General transcription factor 3C polypeptide High FLAHDQAVRTL 2976 2 High FLASESLIKQI 4736 60S ribosomal protein L10a High FLAVRVQQV 57514 Rho GTPase-activating protein 31 Basic leucine zipper and W2 domain- High FLDASGAKL 9689 containing protein 1 Probable JmjC domain-containing histone High FLDDVVHSL 221037 demethylation protein 2C High FLDENVHFA 54878 Dipeptidyl peptidase 8 High FLDIHNIHV 4534 Myotubularin Ubiquitin fusion degradation protein 1 High FLDITNPKA 7353 homolog High FLDKNDHSL 11113 Citron Rho-interacting kinase Epidermal growth factor receptor substrate High FLDKQGFYV 58513 15-like 1 High FLDPGGPMMKL 55627 Sphingomyelin phosphodiesterase 4 Probable dolichyl pyrophosphate High FLDPNNIPKA 79053 Glc1Man9GlcNAc2 alpha-1,3- glucosyltransferase High FLDPRPLTV 1545 Cytochrome P450 1B1 High FLDPRPLTVV 1545 Cytochrome P450 1B1 Microtubule-actin cross-linking factor 1, High FLDQHGHNL 23499 isoform 4 High FLDVNSHKI 114791 Gamma-tubulin complex component 5 High FLFDRPMHV 50804 Myelin expression factor 2

4 Serine/threonine-protein phosphatase 4 High FLFEPVVKA 55671 regulatory subunit 3A High FLFGEVHKA 55388 Protein MCM10 homolog High FLFNKVVNL 51646 Protein yippee-like 5 High FLFQEPRSI 23272 Protein FAM208A High FLGEKIASV 283635 Protein FAM177A1 High FLHDDNMNFRV 79591 UPF0668 protein C10orf76 High FLHDHQAEL 259197 Natural cytotoxicity triggering receptor 3 High FLHSLNIEM 23133 Histone lysine demethylase PHF8 High FLIEGTISRA 55171 TBCC domain-containing protein 1 High FLIEPEHVNTV 80124 Deubiquitinating protein VCIP135 TFIIH basal transcription factor complex High FLIGPKLYEA 2071 helicase XPB subunit High FLIRESETL 4067 Tyrosine-protein kinase Lyn High FLKEHMDEV 5108 Pericentriolar material 1 protein High FLLDKKIGV 10576 T-complex protein 1 subunit beta High FLLDPVKGERL 5339 Plectin High FLLEGIRSL 3071 Nck-associated protein 1-like High FLLEKGYEV 2762 GDP-mannose 4,6 dehydratase High FLLEKSRVV 4643 Unconventional myosin-Ie High FLLENSSKV 9873 FCH and double SH3 domains protein 2 High FLLETVVRV 9910 Rab GTPase-activating protein 1-like High FLLGPEGQHLL 84875 Poly [ADP-ribose] polymerase 10 Transmembrane and coiled-coil domain- High FLLGSVVHEV 79613 containing protein 7 High FLLPHPGLKV 51062 Atlastin-1 High FLLPKVQSI 23240 Transmembrane protein 131-like High FLLQHQTFL 23345 Nesprin-1 Nucleotide-binding oligomerization High FLLQHVQEL 64127 domain-containing protein 2 High FLMDFIHQV 55746 Nuclear pore complex protein Nup133 High FLMEEVHMI 10057 Multidrug resistance-associated protein 5 Caspase recruitment domain-containing High FLMNEVIKL 84433 protein 11 High FLNGEVIRL 3028 3-hydroxyacyl-CoA dehydrogenase type-2 High FLNKMKIMV 10575 T-complex protein 1 subunit delta

5 High FLPETRIMTSV 11319 Protein SGT1 High FLQEHNTTL 440193 Protein Daple AT-rich interactive domain-containing High FLQEKLQEI 5926 protein 4A Probable DNA dC->dU-editing enzyme High FLQENTHVRL 9582 APOBEC-3B High FLQEYTVKL 2941 Glutathione S-transferase A4 MMS19 nucleotide excision repair protein High FLQLAVDKV 64210 homolog High FLQPHLDAV 55610 Coiled-coil domain-containing protein 132 cAMP-dependent protein kinase type I- High FLREYFERL 5573 alpha regulatory subunit 6-phosphofructo-2-kinase/fructose-2,6- High FLSEEGGHVAV 5210 biphosphatase 4 Probable DNA dC->dU-editing enzyme High FLSEHPNVTL 9582 APOBEC-3B High FLTDSNNIKEV 3735 Lysine--tRNA ligase Zinc finger SWIM domain-containing High FLVDGPRVQL 90204 protein 1 High FLVEHVLTL 25800 Zinc transporter ZIP6 Ribosome biogenesis protein BRX1 High FLVQNIHTL 55299 homolog High FLVTVIHTL 10154 Plexin-C1 High FLWPKEVEL 146206 Leucine-rich repeat-containing protein 16C High FLYAGHIFL 90379 DDB1- and CUL4-associated factor 15 High FLYDDNQRV 7153 DNA topoisomerase 2-alpha Alpha-ketoglutarate-dependent dioxygenase High FMDPQKMPYL 79068 FTO Serine/threonine-protein phosphatase 6 High FMFDEKLVTV 5537 catalytic subunit High FMFGQKLNV 3191 Heterogeneous nuclear ribonucleoprotein L High FMIDASVHPTL 221960 Vacuolar fusion protein CCZ1 homolog B Proline synthase co-transcribed bacterial High FMLETVDSVKL 11212 homolog protein U4/U6 small nuclear ribonucleoprotein High FMNDAIEKA 9129 Prp3 Multiple C2 and transmembrane domain- High FTFNIKDIHSV 79772 containing protein 1 High FVDERPEEV 84811 BUD13 homolog High FVMEGEPPKL 348654 Flap endonuclease GEN homolog 1 High GIFGGHIRSV 9100 Ubiquitin carboxyl-terminal hydrolase 10 High GILTKELLHSV 9107 Myotubularin-related protein 6

6 High GLADASLLKKV 25814 Ataxin-10 High GLADKVYFL 790 CAD protein High GLADNTVIAKV 6897 Threonine--tRNA ligase, cytoplasmic High GLDDIKDLKV 4343 Putative helicase MOV-10 High GLDHYVYKV 9931 Probable helicase with zinc finger domain High GLDPNKPPEL 57479 Proline-rich protein 12 High GLDPQGDRSFL 57189 Protein LCHN High GLDPSGARLV 55100 WD repeat-containing protein 70 High GLDRLNVTV 3098 Hexokinase-1 High GLDVDGIYRV 55843 Rho GTPase-activating protein 15 High GLFDQHFRL 11188 Nischarin High GLFQGKTPL 22974 Targeting protein for Xklp2 High GLFSNDIPHV 134430 WD repeat-containing protein 36 High GLGPTFKL 582 Bardet-Biedl syndrome 1 protein High GLIDGRLTI 9789 Signal peptidase complex subunit 2 Microtubule-actin cross-linking factor 1, High GLIDRQVTV 23499 isoform 4 Ubiquitin-like domain-containing CTD High GLIDVKPLGV 134510 phosphatase 1 High GLIEILKKV 9141 Programmed cell death protein 5 Na(+)/H(+) exchange regulatory cofactor High GLLAGDRLVEV 9368 NHE-RF1 Trifunctional purine biosynthetic protein High GLLENIPRV 2618 adenosine-3 High GLLETTVQKV 9092 U4/U6.U5 tri-snRNP-associated protein 1 Microtubule-actin cross-linking factor 1, High GLMDEKLLHNV 23499 isoform 4 Glucocorticoid modulatory element-binding High GLMDTVKKV 10691 protein 1 Hydroxyacyl-coenzyme A dehydrogenase, High GLMGAGIAQV 3033 mitochondrial High GLMKKAYEL 4207 Myocyte-specific enhancer factor 2B Glyceraldehyde-3-phosphate High GLMTTVHAI 2597 dehydrogenase High GLQEGTHEL 6731 Signal recognition particle 72 kDa protein High GLSTEGIYRV 2909 Rho GTPase-activating protein 35 High GLTGQRLLGV 55147 Probable RNA-binding protein 23 High GLVDKLQAL 57019 Anamorsin

7 High GLVGALMHV 7454 Wiskott-Aldrich syndrome protein Cyclic AMP-responsive element-binding High GLWEDGRSTLL 90993 protein 3-like protein 1 High GLWEEAYRV 26160 Intraflagellar transport protein 172 homolog High GLWGPVHEL 9747 Protein FAM115A Ribonucleases P/MRP protein subunit High GLWSGPLPRV 10940 POP1 High GLYDGPVHEV 10570 Dihydropyrimidinase-related protein 4 High GLYDSQNPPTV 23408 Formin-binding protein 1 High GMYGKIAVMEL 1642 DNA damage-binding protein 1 High GMYIFLHTV 94103 ORM1-like protein 3 Eukaryotic translation initiation factor 2 High GVAGGSILKGV 1968 subunit 3 High GVYGGSVHEA 55748 Cytosolic non-specific dipeptidase High HLAEVLERV 56254 E3 ubiquitin-protein ligase BRE1A High HLAIKLEQV 9730 Protein VPRBP High HLANIVERV 10346 E3 ubiquitin-protein ligase TRIM22 Integrin-alpha FG-GAP repeat-containing High HLFDLTPAKV 55846 protein 2 High HLLEHSVEL 54914 Uncharacterized protein KIAA1797 High HLLERVDQV 51199 Ninein High HLLSKLISV 285527 Protein furry homolog-like High HLMEIQVNGGTV 6122 60S ribosomal protein L3 High HLSELNTKL 2803 Golgin subfamily A member 4 High HLTDITLKV 3735 Lysine--tRNA ligase Multiple C2 and transmembrane domain- High HLWRGIVSI 79772 containing protein 1 Nucleoside diphosphate-linked moiety X High HLYDIHVTV 390916 motif 19, mitochondrial High IILEEGKEILV 1072 Cofilin-1 High ILAAHVPTL 513 ATP synthase subunit delta, mitochondrial 26S proteasome non-ATPase regulatory High ILDAGGHNVTI 5707 subunit 1 High ILDDIGHGV 10152 Abl interactor 2 High ILDDSPKEI 26269 F-box only protein 8 High ILDDTAKNLRV 375056 Melanoma inhibitory activity protein 3 High ILDEAIYKV 115811 IQ domain-containing protein D High ILDEHVQRV 8312 Axin-1

8 Dihydroxyacetone phosphate High ILDEMSHKLRL 8443 acyltransferase High ILDISEHTL 10926 Protein DBF4 homolog A High ILDKIVQKV 26260 F-box only protein 25 HSP90AB1 heat shock protein 90kDa alpha High ILDKKVEKV 3326 (cytosolic), class B member 1 High ILDSGIYRI 11234 Hermansky-Pudlak syndrome 5 protein Guanine nucleotide-binding protein subunit High ILFGHENRV 10681 beta-5 High ILFNRVLGV 5321 Cytosolic phospholipase A2 High ILHDRLYYL 51449 Prenylcysteine oxidase 1 High ILIEKEYLERV 8454 Cullin-1 Mitogen-activated protein kinase kinase High ILINDAGEVRL 11184 kinase kinase 1 High ILISKLLGA 51284 Toll-like receptor 7 High ILLDDHGHIRI 2870 G protein-coupled receptor kinase 6 Dual specificity mitogen-activated protein High ILLDERGQIKL 5609 kinase kinase 7 High ILLDNDHYAM 83752 Lon protease homolog 2, peroxisomal High ILLDQTVRV 25962 Protein virilizer homolog Histone-lysine N-methyltransferase High ILLEHNYAL 9739 SETD1A High ILLNNSGQIKL 51755 Cyclin-dependent kinase 12 High ILLPEPSIRSV 156 Beta-adrenergic receptor kinase 1 High ILLQGRLYL 54762 GRAM domain-containing protein 1C High ILLSEPGLVKL 9344 Serine/threonine-protein kinase TAO2 High ILMEHIHKLKA 6143 60S ribosomal protein L19 BTB/POZ domain-containing protein High ILMHHPPQV 222658 KCTD20 High ILMIFSHQI 10735 Cohesin subunit SA-2 Epidermal growth factor receptor substrate High ILNKQEFFV 2060 15 High ILNNKIPEA 80208 Spatacsin High ILQDRLNQV 990 Cell division control protein 6 homolog AT-rich interactive domain-containing High ILQEKLQEI 51742 protein 4B Eukaryotic translation initiation factor 2 High ILQNKIDLV 1968 subunit 3 High ILQQHIATV 51130 Ankyrin repeat and SOCS box protein 3 High ILQSTRLPLI 5591 DNA-dependent protein kinase catalytic

9 subunit

High ILSEVQQAV 142 Poly [ADP-ribose] polymerase 1 High ILSGVIRSV 6813 Syntaxin-binding protein 2 High ILTDITKGV 1938 Elongation factor 2 Ribonucleases P/MRP protein subunit High ILTEAIKAA 10940 POP1 High ILTNVVPKM 9317 Phosphotriesterase-related protein High ILVKHLTNV 79658 Rho GTPase-activating protein 10 High ILYDKLEKI 10574 T-complex protein 1 subunit eta High ILYEHQLGQA 23215 Protein PRRC2C Methionine adenosyltransferase 2 subunit High ILYGEVEKL 27430 beta Chromosome transmission fidelity protein 8 High ILYGKIIHL 54921 homolog High IMAQLPQEQKA 1495 Catenin alpha-1 Small nuclear ribonucleoprotein G-like High IMLEALERV 6637 protein High IMLSEKHLISV 92799 SH3KBP1-binding protein 1 Staphylococcal nuclease domain-containing High ITDDLHFYV 27044 protein 1 HLA class II histocompatibility antigen, High IVADHVASYGV 3117 DQ alpha 1 chain Probable JmjC domain-containing histone High IVLPAGALHQV 221037 demethylation protein 2C High IVMEHVVFL 203859 Anoctamin-5 High KIADFGWSV 9212 Aurora kinase B High KIFDEILVNA 7153 DNA topoisomerase 2-alpha High KIFSGVFVKV 285855 60S ribosomal protein L7-like 1 High KIFTEDLPEV 23339 Vam6/Vps39-like protein High KIIDFIYTA 55958 Kelch-like protein 9 High KILDLETQL 4957 Outer dense fiber protein 2 High KILDYEVTL 3572 Interleukin-6 receptor subunit beta Ankyrin repeat domain-containing protein High KILEELQKV 26057 17 High KILQELPSV 55677 Protein IWS1 homolog G2/M phase-specific E3 ubiquitin-protein High KILSELFTV 55632 ligase High KIMEKIRNV 146857 Schlafen family member 13 High KIMESLPQI 23224 Nesprin-2

10 High KIMTEKELLAV 2319 Flotillin-2 High KIYEGQVEV 6125 60S ribosomal protein L5 High KLADFGDAVQL 7204 Triple functional domain protein High KLADFGLARA 983 Cyclin-dependent kinase 1 High KLAEGVLDV 253143 Protein PRR14L High KLAEIVKQV 8550 MAP kinase-activated protein kinase 5 High KLAESVEKA 11198 FACT complex subunit SPT16 High KLAKLQAQV 91408 Transcription factor BTF3 homolog 4 Mediator of RNA polymerase II High KLDDKDLPSV 51586 transcription subunit 15 High KLDDLTQDLTV 11116 FGFR1 oncogene partner High KLDEHIAYL 54821 DNA excision repair protein ERCC-6-like High KLDETGNSLKV 7153 DNA topoisomerase 2-alpha Structural maintenance of chromosomes High KLDQDLNEV 9126 protein 3 High KLDQVIIHV 80896 N-acetylneuraminate lyase High KLFDRPQEL 55667 DENN domain-containing protein 4C High KLFDRPQELKL 55667 DENN domain-containing protein 4C High KLFEKVKEV 55711 Fatty acyl-CoA reductase 2 Putative homeodomain transcription factor High KLFGHLTSA 57157 2 High KLGHTDILVGV 23016 Exosome complex component RRP42 High KLHDINAQL 162 AP-1 complex subunit beta-1 Guanine nucleotide-binding protein subunit High KLHDVELHQV 10681 beta-5 High KLHGVNINV 83759 RNA-binding protein 4B High KLIDGQVIQL 7702 Zinc finger protein 143 High KLIDRTESL 4046 Lymphocyte-specific protein 1 High KLIEKLDIKL 1938 Elongation factor 2 High KLILLITQV 23160 WD repeat-containing protein 43 High KLIPFLEKL 51496 CTD small phosphatase-like protein 2 High KLIQNVFEI 10640 Exocyst complex component 5 DNA repair and recombination protein High KLLAVIHEL 25788 RAD54B High KLLDEDEIRGYKL 27336 HIV Tat-specific factor 1 26S proteasome non-ATPase regulatory High KLLDIRSYL 5713 subunit 7

11 High KLLDISELDMV 7469 Negative elongation factor A High KLLDLENENMMRV 91010 Formin-like protein 3 High KLLDLMPRL 92105 Integrator complex subunit 4 High KLLDLQVRV 161176 Nesprin-3 High KLLDLSDSTSV 6093 Rho-associated protein kinase 1 High KLLDQMPSL 51191 E3 ISG15--protein ligase HERC5 High KLLDVVHPA 10574 T-complex protein 1 subunit eta High KLLEATSAV 201725 Uncharacterized protein C4orf46 High KLLEEQGIFL 23352 E3 ubiquitin-protein ligase UBR4 High KLLEIDIDGV 5422 DNA polymerase alpha catalytic subunit High KLLEKAFSI 9420 25-hydroxycholesterol 7-alpha-hydroxylase High KLLELDPEHQRA 5033 Prolyl 4-hydroxylase subunit alpha-1 High KLLELEKHIRV 51478 3-keto-steroid reductase High KLLEVQPQV 23301 EH domain-binding protein 1 FYVE, RhoGEF and PH domain-containing High KLLEVYEQL 89846 protein 3 High KLLEYIEEI 3161 Hyaluronan mediated motility receptor High KLLGYDVHV 835 Caspase-2 Disintegrin and metalloproteinase domain- High KLLTEVHAA 101 containing protein 8 Conserved oligomeric Golgi complex High KLMDHIYAV 10466 subunit 5 High KLMDKVVRL 867 E3 ubiquitin-protein ligase CBL High KLMDLDVEQL 5111 Proliferating cell nuclear antigen High KLMDQLEAL 51534 Vps20-associated 1 homolog (S. cerevisiae) High KLPPPPPQA 23028 Lysine-specific histone demethylase 1A High KLQDQIFNL 6001 Regulator of G-protein signaling 10 High KLQELENRL 23224 Nesprin-2 Signal transducer and activator of High KLQELNYNL 6772 transcription 1-alpha/beta High KLQEQIFHL 23338 Protein Jade-2 DNA-directed RNA polymerase II subunit High KLSDLQTQL 5432 RPB3 High KLSEVLQAV 7874 Ubiquitin carboxyl-terminal hydrolase 7 High KLVKEVIAV 2109 Electron transfer flavoprotein subunit beta High KLWGQSIQL 54823 Transcriptional protein SWT1

12 High KLYDIDVAKV 6147 60S ribosomal protein L23a High KLYEEINQV 57404 Cytochrome P450 20A1 High KLYEQHHVV 1718 Delta(24)-sterol reductase High KLYGPEGLELV 3394 Interferon regulatory factor 8 High KLYPQLPAEI 57724 Ectopic P granules protein 5 homolog High KLYQHEINL 10153 CCAAT/enhancer-binding protein zeta High KLYQRDLEV 10635 RAD51-associated protein 1 High KLYTGLREV 8452 Cullin-3 High KMAEEKLTHKMEA 3925 Stathmin High KMAEMLVEL 114932 MORF4 family-associated protein 1-like 1 High KMDPIISRV 7832 Protein BTG2 High KMFDLNGDGEV 10367 Calcium uptake protein 1, mitochondrial ATP-binding cassette sub-family F member High KMLEKLPEL 55324 3 High KMMDVTVTI 408050 Nodal modulator 3 Homocysteine-responsive endoplasmic High KMPEINAKV 9709 reticulum-resident ubiquitin-like domain member 1 protein High KMSEKILLL 5690 Proteasome subunit beta type-2 High KMWEEAISL 1794 Dedicator of cytokinesis protein 2 cAMP-dependent protein kinase type I- High KMYEEFLSKV 5573 alpha regulatory subunit Rap1 GTPase-GDP dissociation stimulator High KVANIIAEV 5910 1 High KVGPVPVLV 10952 Protein transport protein Sec61 subunit beta High KVIEINPYL 5693 Proteasome subunit beta type-5 Probable E3 ubiquitin-protein ligase High KVLEILHRV 26091 HERC4 High KVLERVNAV 5721 Proteasome activator complex subunit 2 CCR4-NOT transcription complex subunit High KVLGIVVGV 23019 1 High KVMDEVEKA 51631 Putative RNA-binding protein Luc7-like 2 Proteasome-associated protein ECM29 High KVMELLVHL 23392 homolog High LADETLLKV 4001 Lamin-B1 High LLADLLHNV 1677 DNA fragmentation factor subunit beta High LLADRSWLL 55732 Uncharacterized protein C1orf112 BTB/POZ domain-containing adapter for High LLAEAKYYL 83892 CUL3-mediated RhoA degradation protein

13 3

High LLAEKIYKI 1387 CREB-binding protein High LLAETHYQL 4678 Nuclear autoantigenic sperm protein High LLAILPEAARA 51282 SCAN domain-containing protein 1 High LLAKEVQLV 57724 Ectopic P granules protein 5 homolog Arf-GAP with coiled-coil, ANK repeat and High LLDATQHTL 9744 PH domain-containing protein 1 High LLDEEISRV 9444 Protein quaking ATP-binding cassette sub-family F member High LLDEPTNHL 10061/55324 2/3 High LLDIIKSTV 9896 Polyphosphoinositide phosphatase High LLDKKIGV 10576 T-complex protein 1 subunit beta High LLDPNVKSIFV 79033 ERI1 exoribonuclease 3 High LLDQTKTLA 7094 Talin-1 High LLDRFLATV 10983 Cyclin-I High LLDSQSHHL 56905 Uncharacterized protein C15orf39 High LLDVTPKAV 9414 Tight junction protein ZO-2 High LLFDRPMHV 4670 Heterogeneous nuclear ribonucleoprotein M DNA-directed RNA polymerase II subunit High LLFEGEKITI 548644 RPB11-b1 Conserved oligomeric Golgi complex High LLFEGIARI 25839 subunit 4 High LLGPPPVGV 25792 Cip1-interacting zinc finger protein Protein phosphatase 1 regulatory subunit High LLIASGANLLA 84988 16A High LLIDDKGTIKL 983 Cyclin-dependent kinase 1 High LLIGHLERV 23165 Nuclear pore complex protein Nup205 RCC1 and BTB domain-containing protein High LLIKQVVEV 55213 1 High LLKVDVQGEI 9179 AP-4 complex subunit mu-1 High LLLDRIASV 79675 FAST kinase domain-containing protein 1 High LLLPEHVLVKI 23403 F-box only protein 46 High LLLPGELAKHAV 8348 Histone H2B Ankyrin repeat domain-containing protein High LLLQHSPAL 51239 39 Eukaryotic translation initiation factor 3 High LLMDRVDEM 8667 subunit H Nuclear receptor subfamily 2 group C High LLMEHIFKL 7181 member 1

14 CDK5 regulatory subunit-associated protein High LLMEHIQEI 55755 2 DNA-directed RNA polymerase II subunit High LLNSEVHML 5433 RPB4 High LLQDAIREV 9181 Rho guanine nucleotide exchange factor 2 High LLQDKQFEL 23339 Vam6/Vps39-like protein High LLQEVEHQL 4591 E3 ubiquitin-protein ligase TRIM37 High LLQPHLERV 55635 DEP domain-containing protein 1A High LLVDVEPKV 6632 Small nuclear ribonucleoprotein Sm D1 High LLVGCTECALLA 120775 Olfactory receptor 2D3 Lipopolysaccharide-responsive and beige- High LLYDGKLSSA 987 like anchor protein Electron transfer flavoprotein subunit alpha, High LLYDLADQLHA 2108 mitochondrial High LMAEMGVHSV 54963 Uridine-cytidine kinase-like 1 High LMIENLPKL 54536 Exocyst complex component 6 High LMQTEVHHV 23370 Rho guanine nucleotide exchange factor 18 High LMVDHVTEV 10011 Steroid receptor RNA activator 1 High LVIDVIHEV 57140 Arginyl aminopeptidase-like 1 High MLANDIARL 10938 EH domain-containing protein 1 Cytochrome c oxidase subunit 4 isoform 1, High MLATRVFSL 1327 mitochondrial High MLFGHPLLV 8237 Ubiquitin carboxyl-terminal hydrolase 11 High MLLDTVQKV 54892 Condensin-2 complex subunit G2 High MLLEKLPQV 10211 Flotillin-1 High MLNEHDFEV 672 Breast cancer type 1 susceptibility protein High MLQDSIHVV 4999 Origin recognition complex subunit 2 High MLQEKLKEL 66008 Trafficking kinesin-binding protein 2 High NIIELVHQV 6850 Tyrosine-protein kinase SYK High NLAEKLIGV 57148 Ral GTPase-activating protein subunit beta High NLAENISRV 5836 Glycogen phosphorylase, liver form High NLFNRYPAL 3936 Plastin-2 Glycerol-3-phosphate dehydrogenase, High NLLEIAPHL 2820 mitochondrial High NLLSFTYKL 79172 Centromere protein O High NLMDDIERA 10694 T-complex protein 1 subunit theta Bifunctional purine biosynthesis protein High NLYPFVKTV 471 PURH

15 High NMVAKVDEV 4736 60S ribosomal protein L10a Propionyl-CoA carboxylase beta chain, High NMVDIIHSV 5096 mitochondrial High QILENELQI 11124 FAS-associated factor 1 Transmembrane and coiled-coil domains High QLAEDIEALKV 57458 protein 3 Putative ATP-dependent RNA helicase High QLAQFVHEV 1663 DDX11-like protein 8 High QLDDLKVEL 11224 Ribosomal protein L35 High QLIDIIHTA 54881 Testis-expressed sequence 10 protein High QLIDKVWQL 6397 SEC14-like protein 1 High QLLQHVQAV 54919 HEAT repeat-containing protein 2 High QLQEGKNVIGL 8407 Transgelin-2 High QLQGYLRSV 6128 60S ribosomal protein L6 High QLVDIIEKV 10197 Proteasome activator complex subunit 3 High QLVESLHKV 53371 Nucleoporin p54 High QLVQRVASV 5683 Proteasome subunit alpha type-2 High QMISRIEYI 1453 Casein kinase I isoform delta High QMLDVAIRV 8943 AP-3 complex subunit delta-1 High QMLEAIKALEV 7690 Zinc finger protein 131 High QVFPGLLERV 25814 Ataxin-10 CCR4-NOT transcription complex subunit High RLAEVGQYEQV 23019 1 Cyclin-D-binding Myb-like transcription High RLAEVVHEL 9988 factor 1 High RLAQHITYV 4176 DNA replication licensing factor MCM7 High RLAVYIDKV 4001 Lamin-B1 High RLAVYIDRV 4000 Prelamin-A/C Vacuolar protein sorting-associated protein High RLDDNIRTV 55275 53 homolog High RLFGILAHV 54839 Leucine-rich repeat-containing protein 49 High RLFNHPNILRL 8576 Serine/threonine-protein kinase 16 Putative sodium-coupled neutral amino acid High RLGEAEGLMKV 124565 transporter 10 High RLLDEEISRV 9444 Protein quaking High RLLDGAFKL 23332 CLIP-associating protein 1 High RLLDSEIKI 5702 26S protease regulatory subunit 6A High RLLDSVRAI 50628 Gem-associated protein 4

16 KN motif and ankyrin repeat domain- High RLLDYVVNI 25959 containing protein 2 Pyridoxal-dependent decarboxylase High RLLEEGVLRQI 23024 domain-containing pro Mediator of DNA damage checkpoint High RLLEGYEIYV 9656 protein 1 High RLLEIDPYL 2632 1,4-alpha-glucan branching enzyme High RLLEQLQEI 56850 GRIP1-associated protein 1 High RLMGSILGV 9321 Thyroid receptor-interacting protein 11 High RLMNLPLHSV 116496 Protein Niban High RLQDAIAKV 23384 Cytospin-A High RLQEDPPAGV 7319 Ubiquitin-conjugating enzyme E2 A High RLQEDPPVGV 7320 Ubiquitin-conjugating enzyme E2 B Inhibitor of nuclear factor kappa-B kinase- High RLQEEINEV 121457 interacting protein High RLSELGITQA 79801 SHC SH2 domain-binding protein 1 High RLTDYISKV 10550 PRA1 family protein 3 High RLVEIQYEL 9815 ARF GTPase-activating protein GIT2 High RLVQGSILKKV 5111 Proliferating cell nuclear antigen High RLYDGLFKV 1642 DNA damage-binding protein 1 ATP-binding cassette sub-family B member High RLYDPASGTISL 23456 10, mitochondrial High RLYPEGLAQL 9114 V-type proton ATPase subunit d 1 High RLYPWGVVEV 4735 Septin-2 High RMFDGKFVVA 127933 Serine/threonine-protein kinase Kist High RMLDSVEKL 3071 Nck-associated protein 1-like High RVITEEEKNFKA 6137 60S ribosomal protein L13 DNA-directed RNA polymerase I subunit High RVLEVGALQAV 25885 RPA1 High RVLPPSALQSV 9212 Aurora kinase B High RVPPPPQSV 6464 SHC-transforming protein 1 Serine/threonine-protein phosphatase PP1- High SIIQRLLEV 5501 gamma catalytic subunit Eukaryotic translation initiation factor 2 High SILRHVAEV 1965 subunit 1 High SLADLYFRA 51434 Anaphase-promoting complex subunit 7 High SLAEGLRTV 4940 2'-5'-oligoadenylate synthase 3 High SLAEKIQAL 140459 Ankyrin repeat and SOCS box protein 6

17 DNA polymerase epsilon catalytic subunit High SLAELKGFEV 5426 A High SLAELVHAV 27244 Sestrin-1 High SLAETDKITL 523 V-type proton ATPase catalytic subunit A Phosphatidylinositol 3-kinase regulatory High SLAQYNPKL 8503 subunit gamma ADP-ribosylation factor-binding protein High SLASIHVPL 23163 GGA3 High SLASLLAKV 57448 Baculoviral IAP repeat-containing protein 6 High SLATSLPRL 9675 TEL2-interacting protein 1 homolog Vacuolar protein sorting-associated protein High SLDESGEHMGV 27072 41 homolog Leucine-rich repeat and coiled-coil domain- High SLDSTLHAV 85444 containing protein 1 Transmembrane emp24 domain-containing High SLFAGGMLRV 54732 protein 9 High SLFDLDGPKV 79142 PHD finger protein 23 High SLFEKGLKNV 26234 F-box/LRR-repeat protein 5 High SLFERLVKV 57169 NFX1-type zinc finger-containing protein 1 High SLFGGSVKL 10015 Programmed cell death 6-interacting protein High SLFQSRISL 9873 FCH and double SH3 domains protein 2 High SLFRVITEV 2132 Exostosin-2 High SLGKVFGV 641 Bloom syndrome protein High SLGRFEITV 10947 AP-3 complex subunit mu-2 High SLIAGVIRV 79735 TBC1 domain family member 17 High SLIEKIPTA 58517 RNA-binding protein 25 High SLIEKLWQT 5991 Transcription factor RFX3 High SLIEKYFSV 3838 Importin subunit alpha-2 High SLIQRLMSV 9736 Ubiquitin carboxyl-terminal hydrolase 34 High SLLEDKGLAEV 59342 Retinoid-inducible serine carboxypeptidase Eukaryotic translation initiation factor 2- High SLLEHLSHV 27102 alpha kinase 1 High SLLEKQQIYL 23370 Rho guanine nucleotide exchange factor 18 Eukaryotic translation elongation factor 1 High SLLEKSLGL 9521 epsilon-1 Mediator of RNA polymerase II High SLLENIAKA 9968 transcription subunit 12 Heterogeneous nuclear ribonucleoprotein High SLLENLEKI 343069 C-like 1 High SLLGGDVVSVKL 1831 TSC22 domain family protein 3

18 High SLLKEPQKVQL 23154 Neurochondrin High SLLPTEQPRL 65057 Adrenocortical dysplasia protein homolog High SLLQTLYKV 5905 Ran GTPase-activating protein 1 High SLLQTQHAL 23647 Arfaptin-2 Heterogeneous nuclear ribonucleoprotein High SLLRVGWSV 221092 U-like protein 2 High SLLTSTVQV 3431 Sp110 nuclear body protein High SLMEKISKL 23268 Dynamin-binding protein High SLMEKVRNMAL 57706 DENN domain-containing protein 1A High SLMGPVVHEV 5116 Pericentrin High SLNGLEVHL 89781 Hermansky-Pudlak syndrome 4 protein High SLPDHLPSV 1120 Choline/ethanolamine kinase High SLPKKLALL 28974 Leydig cell tumor 10 kDa protein homolog High SLQEKLWAI 11264 Peroxisomal membrane protein 4 High SLSFMNPRL 26985 AP-3 complex subunit mu-1 Eukaryotic translation initiation factor 3 High SLVNQVPKI 8665 subunit F Branched-chain-amino-acid High SLWANPKYV 586 aminotransferase, cytosolic High SLYDWNVKL 55585 Ubiquitin-conjugating enzyme E2 Q1 Mediator of RNA polymerase II High SLYSQVHQI 9968 transcription subunit 12 High SMLDDLRNV 3689 Integrin beta-2 High SMLEDVQRA 55131 RNA-binding protein 28 High SMLQKTWLL 26015 RNA polymerase II-associated protein 1 Isochorismatase domain-containing protein High SMMDRMFAL 51015 1 General transcription factor 3C polypeptide High SMYDKVLML 9328 5 High STLHLVLRL 7316 Polyubiquitin-C High SVLGKIWKL 30845 EH domain-containing protein 3 Ribosomal RNA processing protein 1 High TIAQLVHAV 23076 homolog B High TLAAAVPKI 3064 Huntingtin Transformation/transcription domain- High TLADLVHHV 8295 associated protein Set1/Ash2 histone methyltransferase High TLADVLYHV 9070 complex subunit ASH2 Non-POU domain-containing octamer- High TLAEIAKVEL 4841 binding protein

19 High TLAQRVKEV 550 Ancient ubiquitous protein 1 High TLDDKKVYL 54520 Coiled-coil domain-containing protein 93 Probable ATP-dependent RNA helicase High TLDEKIEKV 55661 DDX27 High TLDITPHTV 2177 Fanconi anemia group D2 protein High TLFDYEVRL 29128 E3 ubiquitin-protein ligase UHRF1 High TLHRETFYL 9134 G1/S-specific cyclin-E2 High TLIDLPGITKV 26052 Dynamin-3 Interferon-induced GTP-binding protein High TLIDLPGITRV 4599 Mx1 Cyclic AMP-dependent transcription factor High TLIEELKTL 466 ATF-1 Mitotic spindle-associated MMXD complex High TLIGLSIKV 51647 subunit MIP18 High TLLDRMVHL 51497 Negative elongation factor C/D NmrA-like family domain-containing High TLLEDGTFKV 57407 protein 1 High TLLERAFSL 79809 Tetratricopeptide repeat protein 21B High TLLNVIKSV 3709 Inositol 1,4,5-trisphosphate receptor type 2 High TLLSNIQGV 123 Perilipin-2 High TLLVVVPKL 528 V-type proton ATPase subunit C 1 High TLMERTVSL 92689 Protein NOXP20 High TLNEKLFLL 7220 Short transient receptor potential channel 1 High TLQEQLEKA 5411 Pinin High TLQKEGVIGV 25777 SUN domain-containing protein 2 High TLSDLRVYL 140809 Sulfiredoxin-1 High TLVYHVVGV 23450 Splicing factor 3B subunit 3 High TLYDIAHTPGV 4191 Malate dehydrogenase, mitochondrial High TLYEAVREV 4736 60S ribosomal protein L10a High TMAKESSIIGV 1429 Quinone oxidoreductase High TMVDRIEEV 5927 Lysine-specific demethylase 5A High TVADKIHSV 11072 Dual specificity protein phosphatase 14 Cell division cycle and apoptosis regulator High TVMDEIHTV 55749 protein 1 High VIIEKTYSL 219988 Protein PAT1 homolog 1 Phosphoinositide 3-kinase regulatory High VIIPLLHTV 146850 subunit 6 High VILEGELERA 7171 Tropomyosin alpha-4 chain

20 High VIMDRAPSV 64397 Zinc finger protein 106 homolog High VIWGKVTRA 6165 60S ribosomal protein L35a High VLADLKVQL 5203 Prefoldin subunit 4 High VLADQEVRL 10636 Regulator of G-protein signaling 14 High VLAEQLHQA 23218 Neurobeachin-like protein 2 High VLAHTILGV 55325 Ufm1-specific protease 2 High VLDDKDYFL 3336 10 kDa heat shock protein, mitochondrial High VLDDKLVFV 196527 Anoctamin-6 High VLDNVKMNL 55027 HEAT repeat-containing protein High VLDSVDVRL 113189 Carbohydrate sulfotransferase 14 High VLFEKEVNEV 116496 Protein Niban High VLFENTDSVHL 23029 RNA-binding protein 34 Isochorismatase domain-containing protein High VLFGVETHV 51015 1 High VLFNKLTYL 9910 Rab GTPase-activating protein 1-like High VLFTGVKEV 27131 Sorting nexin-5 High VLGGKAFLEHL 79959 Centrosomal protein of 76 kDa High VLHKELPEV 11343 Monoglyceride lipase High VLIDYQRNV 7514 Exportin-1 High VLKEIVERV 4076 Caprin-1 High VLLDGIHRV 23078 Uncharacterized protein KIAA0564 Cyclic AMP-responsive element-binding High VLLELQQHL 64764 protein 3-like protein 2 High VLLGESAVGKSSL 5868 Ras-related protein Rab-5A High VLLGHIFYV 23383 MAU2 chromatid cohesion factor homolog High VLLSDSNLHDA 57019 Anamorsin High VLLSIYPRV 6890 Antigen peptide transporter 1 High VLLTGLHAV 29799 Protein yippee-like 1 Bromo adjacent homology domain- High VLLTRLENV 22893 containing 1 protein Inhibitor of nuclear factor kappa-B kinase High VLMDHILNL 1147 subunit alpha Nucleolar pre-ribosomal-associated protein High VLMDRLPSL 9875 1 Probable ATP-dependent RNA helicase High VLMEKPDVV 54606 DDX56 High VLMEMSYRL 55159 E3 ubiquitin-protein ligase RFWD3

21 High VLMEYIRSL 29919 Uncharacterized protein C18orf8 High VLMKDVQEI 84298 Protein LLP homolog High VLMQDSRLYL 983 Cyclin-dependent kinase 1 Eukaryotic translation initiation factor 4 High VLMTEDIKL 1981 gamma 1 High VLNEYFHNV 1175 AP-2 complex subunit sigma High VLNNVVIHL 3064 Huntingtin High VLQEKVPEI 727957 HEAT repeat-containing protein 7A High VLQGRLPAV 7036 Transferrin receptor protein 2 Pumilio domain-containing protein High VLQNLKGQYV 161424 C14orf21 High VLSELAARL 159013 Uncharacterized protein CXorf38 HLA class II histocompatibility antigen, High VLSSRLAFA 3125 DR beta 3 chain High VLTEFTREV 55705 Importin-9 High VLTSEVHSV 55726 Cell cycle regulator Mat89Bb homolog High VLVDDDGIKVV 79022 Transmembrane protein 106C High VLVDQTTGL 1994 ELAV-like protein 1 High VLVDRTIYI 10247 Ribonuclease UK114 Zinc finger BED domain-containing protein High VLVEDSNNHHL 9189 1 High VLVPGHLQSV 25833 POU domain, class 2, transcription factor 3 Signal transducer and activator of High VLWDRTFSL 6772 transcription 1-alpha/beta Mitogen-activated protein kinase kinase High VLWGETVHL 1326 kinase 8 Mediator of RNA polymerase II High VLYDQPRHV 9968 transcription subunit 12 High VLYEKDIQL 64400 AKT-interacting protein High VLYGPKIDRA 3385 Intercellular adhesion molecule 3 Set1/Ash2 histone methyltransferase High VLYHVETEV 9070 complex subunit ASH2 High VLYPHEPTAV 29980 Protein downstream neighbor of Son High VLYTGDFRL 64421 Protein artemis High VLYTGVVRV 64682 Anaphase-promoting complex subunit 1 High VMDSKIVQV 3841 Importin subunit alpha-6 High VMFGGKQVVV 10768 Putative adenosylhomocysteinase 2 Ligand-dependent nuclear receptor- High VMFNGKVYL 55791 interacting factor 1

22 High VMFRTPLASV 26271 F-box only protein 5 High VMGKIFAV 57563 Kelch-like protein 8 High VMLEYVERA 8453 Cullin-2 DNA-directed RNA polymerases I, II, and High VMYGKVYRI 5437 III subunit RPABC3 RNA pseudouridylate synthase domain- High VTLNKPQGL 285367 containing protein 3 High WLAEKLPTL 9793 Cytoskeleton-associated protein 5 High WLEENVHEV 22980 Transcription factor 25 High WLIEDGKVVTV 10726 Nuclear migration protein nudC Serine/threonine-protein phosphatase 2A 65 High WLVDHVYAI 5518 kDa regulatory subunit A alpha isoform High YALNHTLSV 5450 POU domain class 2-associating factor 1 HLA class I histocompatibility antigen, A- High YAYDGKDYIAL 3105 23 alpha chain Probable E3 ubiquitin-protein ligase High YAYEKPHVV 8925 HERC1 High YIFDNVAKV 5954 Reticulocalbin-1 High YIMDNKLAQI 6596 Helicase-like transcription factor High YLADPAKFPEA 29088 39S ribosomal protein L15, mitochondrial High YLAERIPTL 7135 Troponin I, slow skeletal muscle High YLAHAIHQV 2806 Aspartate aminotransferase, mitochondrial Zinc finger and BTB domain-containing High YLAKHIENI 22890 protein 1 CCR4-NOT transcription complex subunit High YLAPFLRNV 23019 1 High YLAPHVRTL 2873 COP9 signalosome complex subunit 1 26S proteasome non-ATPase regulatory High YLASLIRSV 5713 subunit 7 S-adenosylmethionine synthase isoform High YLDEDTIYHL 4144 type-2 High YLDEIVKEV 7175 Nucleoprotein TPR High YLDEYIARM 7249 Tuberin High YLDFTNPKV 2595 Neutral alpha-glucosidase C High YLDLSNNRL 81793 Toll-like receptor 10 High YLDPAQRGV 84326 UPF0585 protein C16orf13 Putative ATP-dependent RNA helicase High YLDQTLPRA 1663 DDX11-like protein 8 High YLDRFLAGV 894 G1/S-specific cyclin-D2 High YLDVSVGKIVA 9277 WD repeat-containing protein 46

23 BTB/POZ domain-containing protein High YLDWTIERV 51133 KCTD3 High YLEDKSYKL 57683 DBF4-type zinc finger-containing protein 2 High YLEEYKFQV 8835 Suppressor of cytokine signaling 2 High YLEPYLKEV 7381 Cytochrome b-c1 complex subunit 7 High YLFERIKEL 84928 Transmembrane protein 209 NADH dehydrogenase [ubiquinone] iron- High YLFREPATI 4728 sulfur protein 8, mitochondrial High YLGPHIASV 81671 Vacuole membrane protein 1 High YLGQVTTI 10054 SUMO-activating enzyme subunit 2 Mitotic checkpoint serine/threonine-protein High YLHNQGIGV 701 kinase BUB1 beta High YLIDGTHKI 2946 Glutathione S-transferase Mu 2 High YLINEIDRI 667 Dystonin High YLISQVEGHQV 51002 TP53RK-binding protein High YLITHPLAV 85440 Dedicator of cytokinesis protein 7 Cyclic AMP-dependent transcription factor High YLKDLIEEV 468 ATF-4 High YLLDHAPPEI 8525 Diacylglycerol kinase zeta High YLLDKETLRL 10444 Protein zer-1 homolog Phosphatidylinositol-3,4,5-trisphosphate 5- High YLLDQHILI 3635 phosphatase 1 High YLLEKSRAI 4629 Myosin-11 High YLLEKSRAV 4628 Myosin-10 High YLLEKSRVI 4625 Myosin-7 High YLLESVNKL 5055 Plasminogen activator inhibitor 2 Alpha-1,6-mannosylglycoprotein 6-beta-N- High YLLGHDIRI 4249 acetylglucosaminyltransferase A High YLLHEKLNL 55010 PCNA-interacting partner Probable ATP-dependent RNA helicase High YLLLKGVEAV 51428 DDX41 High YLLLKTHQL 55795 PCI domain-containing protein 2 High YLLPFSKSI 148870 Coiled-coil domain-containing protein 27 High YLLPKDQGITL 23582 Cyclin-D1-binding protein 1 High YLLQEPPRTV 5074 PRKC apoptosis WT1 regulator protein High YLLQRAVEV 6894 Probable methyltransferase TARBP1 High YLLRSLDYV 55915 LanC-like protein 2 High YLLTESSKL 51122 COMM domain-containing protein 2

24 High YLLTHPPPIM 10075 E3 ubiquitin-protein ligase HUWE1 High YLLTRNPHL 4199 NADP-dependent malic enzyme 26S proteasome non-ATPase regulatory High YLMEGSYNKV 5714 subunit 8 High YLMEQNVTKL 55209 SET domain-containing protein 5 High YLMEVTHDL 51520 Leucine--tRNA ligase, cytoplasmic High YLMVENIRL 57464 Protein FAM40B High YLNDFTHEI 64376 Zinc finger protein Pegasus High YLNDLIHSV 11216 A-kinase anchor protein 10, mitochondrial High YLNDRVDEL 51251 Cytosolic 5'-nucleotidase 3 RNA polymerase II subunit A C-terminal High YLNKEIEEA 9150 domain phosphatase DNA repair protein complementing XP-G High YLNQHIEHV 2073 cells Alpha-ketoglutarate-dependent dioxygenase High YLNTRLFTV 79068 FTO High YLPDIIKDQKA 5496 Protein phosphatase 1G High YLQEKLHAL 80184 Centrosomal protein of 290 kDa High YLQQVNHKL 6721 Sterol regulatory element-binding protein 2 High YLSAVRATL 10093 Actin-related protein 2/3 complex subunit 4 High YLSDNVHLV 11140 Hsp90 co-chaperone Cdc37 High YLSPDLSKV 673 Serine/threonine-protein kinase B-raf Serine/threonine-protein phosphatase 2A 55 High YLSVKVWDL 55844 kDa regulatory subunit B delta isoform High YLTDRVMTV 8841 Histone deacetylase 3 High YLTNEGIQYL 6204 40S ribosomal protein S10 High YLVAEKVTV 3320 Heat shock protein HSP 90-alpha Meiotic recombination protein REC8 High YLVEDIQHI 9985 homolog Translation initiation factor eIF-2B subunit High YLWQGVRVA 8893 epsilon High YLWRTSLYL 5250 Phosphate carrier protein, mitochondrial High YLYDLNHTL 81622 Protein unc-93 homolog B1 1-acyl-sn-glycerol-3-phosphate High YLYGIRVEV 10554 acyltransferase alpha Sodium- and chloride-dependent taurine High YLYPDITRL 6533 transporter High YLYTKEQLL 10939 AFG3-like protein 2 High YMADRLLGV 9820 Cullin-7

25 High YMAELIERL 51053 Geminin High YMGEEKLIASV 23404 Exosome complex component RRP4 High YMIAHITGL 7298 Thymidylate synthase High YMLEHVITL 9735 Kinetochore-associated protein 1 Cyclic AMP-dependent transcription factor High YMLGLEARL 22926 ATF-6 alpha High YTYDEAIHSV 134957 Syntaxin-binding protein 5 High YVFEGKMLEA 6305 Myotubularin-related protein 5 High YVIDPIKGLKL 5339 Plectin High YVLEGLKSV 26098 Erythroid differentiation-related factor 1 Translation initiation factor eIF-2B subunit High YVLPEVSKV 8890 delta High YVYDKDIEQI 389856 Ubiquitin carboxyl-terminal hydrolase 27 Sarcoplasmic/endoplasmic reticulum Medium AIVRSLPSV 487 calcium ATPase 1 Medium ALAKEIDSV 5880 Ras-related C3 botulinum toxin substrate 2 Medium ALFDSGLHPA 55247 Endonuclease 8-like 3 Medium ALFEGKVQL 57728 WD repeat-containing protein 19 Medium ALFKAWAL 3662 Interferon regulatory factor 4 Medium ALFQPHLINV 10097 Actin-related protein 2 Medium ALLKGLAAV 57558 Ubiquitin carboxyl-terminal hydrolase 35 Medium ALLKQVEI 84070 Protein FAM186B Mitogen-activated protein kinase-binding Medium ALMSRPAQV 23005 protein 1 Medium ALNGKLYIV 10625 Influenza virus NS1A-binding protein LisH domain and HEAT repeat-containing Medium ALQSLIPSL 57614 protein KIAA1468 Medium ALRDVSEEL 200081 Alpha-taxilin SET and MYND domain-containing protein Medium ALREENEQL 114826 4 Leucine-rich PPR motif-containing protein, Medium ALRGEIETV 10128 mitochondrial Medium ALSSLIHAL 7520 X-ray repair cross-complementing protein 5 FERM, RhoGEF and pleckstrin domain- Medium ALWEGPSKA 9855 containing protein 2 Vacuolar protein sorting-associated protein Medium ALYGKLLKL 157680 13B Medium ALYGRAEAAEV 3090 Hypermethylated in cancer 1 protein Medium ALYNDISHMKI 135112 Nuclear receptor coactivator 7

26 Ankyrin repeat and KH domain-containing Medium AMNGHVPAV 54882 protein 1 Beta-1,3-galactosyl-O-glycosyl- Medium ATIQRIPEV 2650 glycoprotein beta-1,6-N- acetylglucosaminyltransferase Bifunctional methylenetetrahydrofolate Medium AVIDVGINRV 10797 dehydrogenase/cyclohydrolase, mitochondrial Leucine-rich repeat flightless-interacting Medium DLKDQIQDV 9209 protein 2 Mitotic checkpoint serine/threonine-protein Medium EMEKKLKEI 701 kinase BUB1 beta Medium EVGGEALGRL 3043 Hemoglobin subunit beta HLA class I histocompatibility antigen, Medium FAYDGKDYI 3107 Cw-4 alpha chain Ankyrin repeat and LEM domain- Medium FIFEKKLAQA 23141 containing protein 2 Medium FIYHGEVPQA 4261 MHC class II transactivator Medium FLANIGTSV 26060 DCC-interacting protein 13-alpha Medium FLAVKPDGV 4832 Nucleoside diphosphate kinase 3 ATP-binding cassette sub-family F member Medium FLDEPTNHL 10061 2 Medium FLDHIIASV 51780 Lysine-specific demethylase 3B Medium FLDSGTIRGV 2268 Tyrosine-protein kinase Fgr Medium FLEEKIPSI 85476 Elongation factor G, mitochondrial Bromodomain adjacent to zinc finger Medium FLEPVNPRL 11176 domain protein 2A Zinc finger and SCAN domain-containing Medium FLGEINDRL 80345 protein 16 Medium FLGSFIDHV 91147 Meckelin Medium FLIEEQKIVV 6164 60S ribosomal protein L34 Medium FLLEKPFSV 10196 Protein arginine N-methyltransferase 3 Medium FLLGKEVSL 23061 TBC1 domain family member 9B Medium FLLHIQQQV 10802 Protein transport protein Sec24A Medium FLLPIKTVGV 8833 GMP synthase [glutamine-hydrolyzing] Medium FLMKNSDLYGA 79801 SHC SH2 domain-binding protein 1 Translation initiation factor eIF-2B subunit Medium FLNKEITSV 8890 delta Medium FLPRKFPSL 23246 Ribosome biogenesis protein BOP1 Medium FLQEKSPAV 9898 Ubiquitin-associated protein 2-like Medium FLQPELVKL 23198 Proteasome activator complex subunit 4

27 Medium FLTKQEILL 10519 Calcium and integrin-binding protein 1 Medium FLVEHNLVL 55818 Lysine-specific demethylase 3A 1-phosphatidylinositol-4,5-bisphosphate Medium FLYDTHQNL 5336 phosphodiesterase gamma-2 Medium FLYFEDHGL 55255 WD repeat-containing protein 41 Cation-dependent mannose-6-phosphate Medium FLYQRLVVGA 4074 receptor Medium FMMPRIVNV 23269 MAX gene-associated protein Medium FMVDRLESL 51324 Maspardin Medium FVDGLTFKV 4343 Putative helicase MOV-10 Medium GILSHINTV 2983 Guanylate cyclase soluble subunit beta-1 Medium GLAPHLEQI 79711 Importin-4 Medium GLAPLEVRV 2317 Filamin-B Medium GLDRNAPSV 1479 Cleavage stimulation factor subunit 3 Transcription initiation factor TFIID Medium GLHFVVPSV 6873 subunit 2 Medium GLLREQVAQL 3726 Transcription factor jun-B Tumor necrosis factor receptor superfamily Medium GLMDNEIKV 8795 member 10B Medium GLMQEKIYI 1786 DNA (cytosine-5)-methyltransferase 1 Medium GLNEEIARV 10403 Kinetochore protein NDC80 homolog Medium GLPRFGIEMV 64397 Zinc finger protein 106 homolog ATPase family AAA domain-containing Medium GLSNHIAAL 29028 protein 2 Medium GLVGSLQEV 64682 Anaphase-promoting complex subunit 1 Medium GLWGPEEEPHL 84726 Protein PRRC2B tRNA (adenine(58)-N(1))-methyltransferase Medium GLYEFPLNKV 51605 non-catalytic subunit T Medium GMAERIPEL 2181 Long-chain-fatty-acid--CoA ligase 3 Medium GVAESIHLWEV 57418 WD repeat-containing protein 18 Medium HLAEALHQA 66005 Chitinase domain-containing protein 1 Arf-GAP with SH3 domain, ANK repeat Medium HLAVKVANQA 55616 and PH domain-containing protein 3 Medium HLDATKLLL 55761 Tetratricopeptide repeat protein 17 Medium HLDPTEMEKV 22824 Heat shock 70 kDa protein 4L Medium HLIDTNKIQL 55619 Dedicator of cytokinesis protein 10 Phosphatidylinositol-4-phosphate 3-kinase Medium HLIHAPPEV 5287 C2 domain-containing subunit beta

28 Medium HLYARQLTL 84309 Protein syndesmos Medium HLYSEVKEV 23064 Probable helicase senataxin Medium IAAQDLLLAV 55705 Importin-9 Medium IIADMDKWTV 96459 Folliculin-interacting protein 1 Medium IINFSLLLV 81309 Olfactory receptor 4C15 Medium ILAAVETRL 23219 F-box only protein 28 Ras GTPase-activating-like protein Medium ILAPVVKEI 8826 IQGAP1 26S proteasome non-ATPase regulatory Medium ILDAGGHNV 5707 subunit 1 Medium ILDGNQLHI 7812 Cold shock domain-containing protein E1 Medium ILDSRGNPTV 2026 Gamma-enolase Medium ILEEGKEILV 1072 Cofilin-1 Medium ILEKKVEKV 3320 Heat shock protein HSP 90-alpha Medium ILGDRFSWNV 10985 Translational activator GCN1 Medium ILHDDEVTV 6176 60S acidic ribosomal protein P1 Medium ILHHKVYDL 1528 Cytochrome b5 ATP-binding cassette sub-family B member Medium ILIRPLVSV 22 7, mitochondrial Medium ILLPDNVHYV 23517 Superkiller viralicidic activity 2-like 2 Medium ILLPYVSKV 84286 Transmembrane protein 175 Medium ILLRDLPTL 400451 Membrane protein FAM174B Putative pre-mRNA-splicing factor ATP- Medium ILMGVLKEV 1665 dependent RNA helicase DHX15 Medium ILQEREYRL 6597 Transcription activator BRG1 Medium ILTESEIKL 7257 Translin-associated protein X WD repeat- and FYVE domain-containing Medium ILWDLDHLTHV 57705 protein 4 Ras GTPase-activating-like protein Medium IMDDKSLNI 8826 IQGAP1 Medium ITAKQLETL 89884 LIM/homeobox protein Lhx4 Medium ITQTVIHTV 3965 Galectin-9 Medium IVDKTASFV 10291 Splicing factor 3A subunit 1 Medium KIGDFGLATV 673 Serine/threonine-protein kinase B-raf Medium KILEDVVGV 22974 Targeting protein for Xklp2 Medium KIMDQVQQA 324 Adenomatous polyposis coli protein Medium KLAAAISEV 831 Calpastatin

29 Medium KLADDVDLEQV 7415 Transitional endoplasmic reticulum ATPase Medium KLADISINYV 26046 E3 ubiquitin-protein ligase listerin Guanosine-3',5'-bis(diphosphate) 3'- Medium KLADKLYNL 374659 pyrophosphohydrolase MESH1 Medium KLAEKLTIL 3071 Nck-associated protein 1-like Medium KLAENIDAQL 23636 Nuclear pore glycoprotein p62 Kelch repeat and BTB domain-containing Medium KLDPATSAVTV 55709 protein 4 LETM1 and EF-hand domain-containing Medium KLFEDELTL 3954 protein 1, mitochondrial Medium KLFSELPLA 51650 28S ribosomal protein S33, mitochondrial Medium KLGELTMQL 124944 Chromatin complexes subunit BAP18 Medium KLGLKPLEV 9092 U4/U6.U5 tri-snRNP-associated protein 1 Signal recognition particle receptor subunit Medium KLIDDVHRL 6734 alpha Medium KLIEELETL 1390 cAMP-responsive element modulator Medium KLIPQLPTL 64121 Ras-related GTP-binding protein C Medium KLLEPVPVSV 51321 Archaemetzincin-2 Medium KLLERLPEA 5980 DNA polymerase zeta catalytic subunit Medium KLLERVSAI 25920 Negative elongation factor B Medium KLLKDLPEL 27250 Programmed cell death protein 4 Cytosolic acyl coenzyme A thioester Medium KLMDEVAGI 11332 hydrolase Dolichyl-diphosphooligosaccharide-- Medium KLNPQQFEV 3703 protein glycosyltransferase subunit STT3A Medium KLPEKWESV 26156 Ribosomal L1 domain-containing protein 1 Probable tRNA Medium KLVELPYTV 55644 threonylcarbamoyladenosine biosynthesis protein OSGEP Medium KLVMEIIKL 23064 Probable helicase senataxin Medium KLWEAESKL 54069 Protein Mis18-alpha Medium KLYDGFQYL 5298 Phosphatidylinositol 4-kinase beta Medium KMDEVLYSI 10907 Thioredoxin-like protein 4A Transmembrane emp24 domain-containing Medium KMGLIFEV 10959 protein 2 Medium KVLEPSETLVV 6651 Protein SON Medium LIEECGGLEKI 3840 Importin subunit alpha-4 Medium LLAPRPVAV 1028 Cyclin-dependent kinase inhibitor 1C Medium LLDVVHPAA 10574 T-complex protein 1 subunit eta

30 Medium LLFDKFNAV 60412 Exocyst complex component 4 Medium LLFSGNIQEA 57560 Intraflagellar transport protein 80 homolog Medium LLGRVYDV 124936 Neuferricin Nicotinate-nucleotide pyrophosphorylase Medium LLLGERVAL 23475 [carboxylating] Medium LLLGHVAQV 4686 Alpha-N-acetylgalactosaminidase Zinc finger SWIM domain-containing Medium LLNDRIWLA 90204 protein 1 Medium LLQRKVEEV 23636 Nuclear pore glycoprotein p62 Signal transducer and activator of Medium LLSDTVQHL 6778 transcription 6 Medium LLYEKGISL 29015 Solute carrier family 43 member 3 Medium LLYQGPHNTL 3676 Integrin alpha-4 KH domain-containing, RNA-binding, Medium LMAHAMEEV 10657 signal transduction-associated protein 1 Medium LmLAGGQITGL 5451 POU domain, class 2, transcription factor 1 Medium LTAAQLLDTL 286077 Protein FAM83H Medium MAELKGYNL 55567 Dynein heavy chain 3, axonemal Alanine--glyoxylate aminotransferase 2-like Medium MLKEAHIEL 64850 1 Medium MLLEGLREL 8409 Protein UXT Medium NILEKGGDPL 9169 Protein SCAF11 Medium NIVEKLREV 85363 Tripartite motif-containing protein 5 Medium NIVERVKEV 10346 E3 ubiquitin-protein ligase TRIM22 Proteasome-associated protein ECM29 Medium NLAEKPKTV 23392 homolog Medium NLAEVVERV 26263 F-box only protein 22 Medium NLASRIPAA 23524 Serine/arginine repetitive matrix protein 2 Medium NLVEKTPAL 267020 ATP synthase subunit g 2, mitochondrial Medium NMYGKVVTV 6829 Transcription elongation factor SPT5 Medium QLDSHVHAV 23557 SNARE-associated protein Snapin Enoyl-CoA hydratase domain-containing Medium QLLEKVIEL 55862 protein 1 Medium QLTNVILHL 54471 Mitochondrial dynamic protein MID51 Medium RLAGADVVLGV 790 CAD protein Medium RLFHYRITV 29102 Ribonuclease 3 Medium RLIDLHTNV 23256 Sec1 family domain-containing protein 1 Medium RLLEQKVEL 5411 Pinin

31 Medium RLLEVVTSI 83440 ADP-dependent glucokinase Medium RLLQEQHQL 57674 RING finger protein 213 Medium RLNEKNYEL 51606 V-type proton ATPase subunit H Double-strand-break repair protein rad21 Medium RLQESVMEA 5885 homolog Interferon-induced protein with Medium RLSDVQIYV 3433 tetratricopeptide repeats 2 Eukaryotic translation initiation factor 3 Medium RLTPKLMEV 8667 subunit H Medium RLWNETVEL 55789 DEP domain-containing protein 1B Medium RMFDGKFVV 127933 Serine/threonine-protein kinase Kist Medium RMLPHAPGV 3065 Histone deacetylase 1 Serine/threonine-protein phosphatase PP1- Medium SIIGRLLEV 5499 alpha catalytic subunit TATA-binding protein-associated factor Medium SLADVHIEV 9044 172 Medium SLAEGRLYL 80210 LisH domain-containing protein ARMC9 Trinucleotide repeat-containing gene 6A Medium SLAHELWKV 27327 protein Medium SLANETHQL 79155 TNFAIP3-interacting protein 2 Voltage-dependent L-type calcium channel Medium SLAPIIVHV 785 subunit beta-4 ATP-binding cassette sub-family A member Medium SLDNRINEV 10349 10 Medium SLFPGKLEV 2314 Protein flightless-1 homolog Medium SLFPHNPQFI 80135 Ribosome production factor 1 Medium SLIDKTTAA 6631 U1 small nuclear ribonucleoprotein C Medium SLIGHLQTL 1847 Dual specificity protein phosphatase 5 RNA polymerase-associated protein CTR9 Medium SLIGNLHLA 9646 homolog Medium SLKNRIESV 9824 Rho GTPase-activating protein 11A Developmentally-regulated GTP-binding Medium SLLEKELESV 1819 protein 2 Medium SLLESVQKL 545 Serine/threonine-protein kinase ATR Probable ATP-dependent RNA helicase Medium SLLKRDFGA 1655 DDX5 Vacuolar protein sorting-associated protein Medium SLLQKQIML 157680 13B Small glutamine-rich tetratricopeptide Medium SLNKHVEAV 6449 repeat-containing protein alpha Medium SLPDIKVYL 10636 Regulator of G-protein signaling 14 Medium SLQDEIQRV 10885 WD repeat-containing protein 3

32 Mitogen-activated protein kinase kinase Medium SLREACETV 4217 kinase 5 Medium SLSDKTPSV 22993 HMG domain-containing protein 3 Medium SLSDTVEKL 1639 Dynactin subunit 1 Medium SLSSFLHGV 57679 Alsin Medium SLVENIERLKV 7726 Tripartite motif-containing protein 26 Medium SLYEMVSRV 6749 FACT complex subunit SSRP1 Ribosome biogenesis protein BMS1 Medium SLYGYLRGA 9790 homolog Medium SMLNRILAV 9203 ZMYM3 zinc finger, MYM-type 3 Medium SMTLAIHEI 8560 Sphingolipid delta(4)-desaturase DES1 Pre-mRNA-processing factor 40 homolog Medium TLDAGNIKL 55660 A Medium TLDEYTTRV 4899 Nuclear respiratory factor 1 Staphylococcal nuclease domain-containing Medium TLFTKELVL 27044 protein 1 CPSF1 cleavage and polyadenylation Medium TLITDGMRSV 29894 specific factor 1 Medium TLLDSPYARV 54497 HEAT repeat-containing protein 5B Medium TLLGHEFVL 996 Cell division cycle protein 27 homolog Medium TLLGHLDYI 1314 Coatomer subunit alpha Medium TLLIQVPRI 55172 Protein kintoun Medium TLNDREYQL 790 CAD protein Mitochondrial import receptor subunit Medium TLSERLWGL 56993 TOM22 homolog Medium1 TLSFMNPRL 10947 AP-3 complex subunit mu-2 Medium TLTEEGVIKV 23560 Nucleolar GTP-binding protein 1 Medium TLVQSTLTLQL 81470 Olfactory receptor 2G2 Medium TVLGKIWKL 10938 EH domain-containing protein 1 Medium VIDKFGSDII 313 Acyloxyacyl hydrolase Medium VIMKSSPEV 54108 Chromatin accessibility complex protein 1 Medium VLADALKSI 6210 40S ribosomal protein S15a Medium VLAPLPLHGV 55819 E3 ubiquitin-protein ligase RNF130 Calmodulin-regulated spectrin-associated Medium VLDASVKEV 157922 protein 1 Medium VLFEGRTVQL 23287 Cytosolic carboxypeptidase 1 Medium VLFHNLPSL 963 Leukocyte surface antigen CD53 Medium VLIGHSYGV 79575 Abhydrolase domain-containing protein 8

33 Asparagine synthetase [glutamine- Medium VLLDTANKKVFL 440 hydrolyzing] Medium VLLGKVYVV 54800 Kelch-like protein 24 Medium VLLKARLVPA 165215 Protein FAM171B Medium VLLSTIHEL 80145 THO complex subunit 7 homolog Medium VLQQHLETL 10363 High mobility group protein 20A Medium VLTEHSEEL 8874 Rho guanine nucleotide exchange factor 7 Medium VLVESEHQV 3716 Tyrosine-protein kinase JAK1 Medium VLYDRPLKI 64783 Putative RNA-binding protein 15 Medium VLYENKVAV 54468 WD repeat-containing protein mio Medium VLYGKLVEA 7486 Werner syndrome ATP-dependent helicase Medium VMIAGKVAVV 191 Adenosylhomocysteinase Medium YGVKAGPGV 170954 Phostensin Medium YIAKITEGV 55293 Ubiquitin-conjugating enzyme E2 variant 3 Medium YIMSYISRV 4643 Unconventional myosin-Ie Medium YIWDRHYNI 54461 F-box/WD repeat-containing protein 5 Medium YLDLSENRL 2314 Protein flightless-1 homolog Pre-rRNA-processing protein TSR1 Medium YLFAHAVDFV 55720 homolog Medium YLFERTFNL 9394 Heparan-sulfate 6-O-sulfotransferase 1 Medium YLFTSPQRV 25972 Protein unc-50 homolog Medium YLHRQVAAV 6890 Antigen peptide transporter 1 Medium YLPSQVSRV 9276 Coatomer subunit beta' Medium YLPYPIHQV 1196 Dual specificity protein kinase CLK2 ETS domain-containing transcription factor Medium YLQAHTQSV 2077 ERF Pumilio domain-containing protein Medium YLQEHAQEV 9933 KIAA0020 Medium YLQPKLLGI 545 Serine/threonine-protein kinase ATR Nuclease-sensitive element-binding protein Medium YLRSVGDGETV 4904 1 Medium YLSDIPLHDA 2983 Guanylate cyclase soluble subunit beta-1 Medium YLSKIIPAL 5985 Replication factor C subunit 5 Medium YLSPKLWAL 55323 La-related protein 6 Medium YLSSLQPRL 8563 THO complex subunit 5 homolog Medium YLVDFTQKI 51244 Uncharacterized protein C3orf19

34 Medium YMNHIMVSV 2120 Transcription factor ETV6 Medium YMYEKESEL 22806 Zinc finger protein Aiolos Medium YTTDRVMTV 3065 Histone deacetylase 1 Medium YVFPGVTRL 84221 Uncharacterized protein C21orf56 CCR4-NOT transcription complex subunit Medium YVVPFVAKV 23019 1

*Confirmation achieved by positive matching of fragment spectra with respective spectra of synthetic peptides

35 Supplementary Table SIII: AUC and retention time of peaks originating from calibrating peptides labeled with deuterated nicotinic acid (dNIC) and corresponding nicotinylated peptides isolated from EC and BLC (both from donor 1). Two LC-MS/MS runs were performed. To run#1, 0.3 pmol of dNIC peptides were spiked for calibration, to run#2, 10 pmol of dNIC peptides were spiked.

EC 0.3 pmol 10 pmol run#1 run#2 dNIC RT dNIC RT NIC RT NIC RT Sequence (AUC) [min] (AUC) [min] (AUC) [min] (AUC) [min] SLSEKTVLL 1.28 x 106 92.13 5.68 x 106 91 1.45 x 107 92.44 1.63 x 107 91.34 SLLDKIIGA 7.20 x 106 134.58 1.14 x 108 133.37 8.53 x 106 134.86 9.53 x 106 133.61 AIVDKVPSV 5.76 x 105 84.77 1.59 x 108 130.31 6.67 x 105 85.11 2.83 x 106 130.61 ILDQKINEV 1.02 x 106 90.76 9.32 x 107 89.72 1.14 x 106 91.13 1.13 x 106 90.03 YLPEDFIRV 7.77 x 106 131.63 9.01 x 107 83.74 2.68 x 106 131.96 1.03 x 106 84.13 YLLPAIVHI 4.54 x 107 149.73 4.14 x 108 148.09 1.69 x 107 150.03 2.57 x 106 148.38

BLC 0.3 pmol 10 pmol run#1 run#2 dNIC RT dNIC RT NIC RT NIC RT Sequence (AUC) [min] (AUC) [min] (AUC) [min] (AUC) [min] AIVDKVPSV 5,72 x 105 84,37 9,04 x 107 83,86 7,04 x 108 84,74 1,16 x 109 84,21 YLLPAIVHI 5,19 x 106 148,98 4,97 x 108 146,46 9,50 x 108 149,23 1,17 x 109 146,72 ILDQKINEV 3,72 x 105 90,4 1,27 x 108 89,79 2,60 x 108 90,7 2,93 x 108 90,16 YLPEDFIRV 1,52 x 106 130,38 1,45 x 108 130,02 7,43 x 107 130,72 8,42 x 107 130,31 SLSEKTVLL 4,13 x 105 91,61 6,34 x 107 91,06 1,43 x 107 91,95 1,75 x 107 91,44 SLLDKIIGA 2,28 x 107 133,57 1,83 x 108 133,19 2,31 x 106 133,77 2,88 x 106 133,39