Supplementary Data to the Manuscript

In vivo biotinylation and mass spectrometry identify the extra-domain A of fibronectin as antigen for -based targeting of primary tumors and metastastic lesions

Jascha-N. Rybak1*, Christoph Roesli1*, Manuela Kaspar2, Alessandra Villa2 and Dario Neri1§

1Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland 2Philochem AG, c/o ETH Zurich, 8093 Zurich, Switzerland *The two authors contributed equally to the work. §Corresponding author: Prof. Dr. Dario Neri ETH Zurich Institute of Pharmaceutical Sciences Wolfgang-Pauli-Strasse 10, HCI G396 8093 Zurich, Switzerland

Runnning Title: EDA as antigen for antibody-based tumor targeting

These Supplementary Data contain:

- Supplementary Experimental Procedures

- Supplementary Table 1

- Supplementary Table 2

- Supplementary Table 3

- Supplementary Figure 1

Supplementary Data to Rybak et al. Page 1 of 21 Supplementary Experimental Procedures

In vivo biotinylation

In vivo biotinylation experiments were performed as described previously (Roesli et al., 2006;

Rybak et al., 2005). In brief, the chest of the anesthetized mouse was opened through a

median sternotomy. The left heart ventricle was punctured with a perfusion needle and a

small cut was made in the right atrium to allow the outflow of the perfusion solutions.

Immediately after, perfusion of the systemic circulation was performed with a pressure of 100

mm Hg at a flow rate of 1.5 ml/min. In a first step, perfusion was carried out with 15 ml

biotinylation solution (pre-warmed to 38°C), containing 1 mg/ml sulfo-NHS-LC-biotin

(Pierce, Rockford, IL, USA) in PBS, pH 7.4, supplemented with 10% (w/v) dextran-40

(Amersham Biosciences, Uppsala, Sweden) as plasma expander. Thereby, blood components,

which could compete with the biotinylation reaction, were eliminated from circulation within

the first few minutes of perfusion and accessible primary amine-containing (and

certain glycolipids and phospholipids) in the different tissues could be covalently modified

with biotin. To neutralize unreacted biotinylation reagent, the in vivo biotinylation was

followed by a 10 min washing step with 50 mM Tris, 10% (w/v) dextran-40, in PBS, pH 7.4,

prewarmed to 38°C. During perfusion with biotinylation reagent (and during the first three

minutes of the following perfusion with quenching solution) the region around the heart was

washed with 50 mM Tris in PBS, pH 7.4 (38°C), to quench out-flowing unreacted

biotinylation reagent and avoid undesired labeling of molecules at the organ surfaces.

MALDI-TOF/TOF mass spectrometry

MALDI-TOF and MALDI-TOF/TOF mass spectrometric analyses were carried out using the

4700 Proteomics Analyzer (Applied Biosystems). Peptide masses were acquired over a range

from 750 to 4000 m/z, with a focus mass of 2000 m/z. MS spectra were summed from 2000

Supplementary Data to Rybak et al. Page 2 of 21 laser shots from an Nd:YAG laser operating at 355 nm and 200 Hz. An automated plate

calibration was performed using five peptide standards (masses 900−2400 m/z; Applied

Biosystems) in six calibration wells. This plate calibration was used to update the instrument

default mass calibration, which was applied to all MS and MS/MS spectra. Furthermore, an

internal calibration of each MS spectrum using the internal standard peptide added to the

MALDI matrix was performed. A maximum of 15 precursors per sample well with a

signal-to-noise ratio of >100 was automatically selected for subsequent fragmentation by

collision induced dissociation. MS/MS spectra were summed from 2500 to 5000 laser shots.

Spectra were processed and analyzed by the Global Server Workstation (Applied

Biosystems), which uses internal MASCOT (Matrix Science) software for matching MS and

MS/MS data against databases of in-silico digested proteins. The MASCOT search

parameters were (i) a mouse database downloaded from the European Bioinformatics Institute

(EBI) homepage on 9/9/2006 (ftp.ebi.ac.uk/pub/databases/SPproteomes/fasta/proteomes/

59.M_musculus.fasta.gz); (ii) enzyme: trypsin and semi-trypsin; (iii) allowed number of

missed cleavages: 1; (iv) variable posttranslational modifications: methionine oxidation; (v)

peptide tolerance: ±30 ppm; (vi) MS/MS tolerance: ± 0.2 Da; (vii) peptide charge: + 1; (viii)

minimum ion score C.I. % for peptides: 95 and (ix) maximum peptide rank: 1. Furthermore,

an MS/MS peak filtering with the following parameters was used: (i) mass range: 60 Da to 20

Da below precursor mass; (ii) minimum signal-to-noise ratio: 6; (iii) peak density filter:

maximum 30 peaks per 200 Da and (iv) maximum number of peaks per spectrum: 65.

Supplementary Data to Rybak et al. Page 3 of 21 Supplementary Table 1. List of all proteins identified in the proteomic analysis with a Mascot confidence level of >95%.

†Neg. †Neg. *Peptide † †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 1 Q8R0Y6 10-formyltetrahydrofolate dehydrogenase 5 0 0 0 2 Q61176 Arginase 1 5 0 0 0 3 P16015 Carbonic anhydrase 3 (EC 4.2.1.1) (Carbonic 5 0 0 0 4 P50247 Adenosylhomocysteinase (EC 3.3.1.1) 5 0 0 0 5 P52760 Ribonuclease UK114 (EC 3.1.-.-) 5 0 0 0 6 Q9WVT6 Carbonic anhydrase 14 precursor (EC 5 0 0 0 7 P24270 Catalase (EC 1.11.1.6) 5 0 0 0 8 Q64374 Regucalcin (RC) (Senescence marker protein 5 0 0 0 9 P25688 Uricase (EC 1.7.3.3) (Urate oxidase) 5 0 0 0 10 P00186 P450 1A2 (EC 1.14.14.1) 5 0 0 0 11 P41216 Long-chain-fatty-acid--CoA ligase 1 (EC 5 0 0 0 Q6GTG6 Acyl-CoA synthetase long-chain family 5 0 0 0 12 P49429 4-hydroxyphenylpyruvate dioxygenase (EC 5 0 0 0 13 O78207 H-2D cell surface glycoprotein (Fragment) 5 0 0 0 P01899 H-2 class I histocompatibility antigen, D-B 5 0 0 0 14 P10518 Delta-aminolevulinic acid dehydratase (EC 5 0 0 0 15 Q9JII6 Alcohol dehydrogenase [NADP+] (EC 1.1.1.2) 5 0 0 0 16 Q05421 2E1 (EC 1.14.14.1) 5 0 0 0 Q0PGA1 Cytochrome P450 2E1 5 0 0 0 17 Q9WVL0 Maleylacetoacetate isomerase (EC 5.2.1.2) 5 0 0 0 18 Q8VCW9 Cytochrome P450, family 2, subfamily a, 5 0 0 0 19 O35215 D-dopachrome decarboxylase (EC 4.1.1.84) 5 0 0 0 20 Q6XL44 UDP glycosyltransferase 1 family polypeptide 5 0 0 0 21 Q62452 UDP-glucuronosyltransferase 1-7 precursor 5 0 0 0 22 Q9QXD6 Fructose-1,6-bisphosphatase 1 (EC 5 0 0 0 23 Q91VS7 Microsomal glutathione S-transferase 1 (EC 5 0 0 0 24 Q31168 H-2D cell surface glycoprotein (Fragment) 5 0 0 0 25 Q91YI0 Argininosuccinate lyase (EC 4.3.2.1) 5 0 0 0 26 P00329 Alcohol dehydrogenase 1 (EC 1.1.1.1) 5 0 0 0 27 Q91WL5 Cytochrome P450 4A12 (EC 1.14.14.1) 5 0 0 0 28 Q61133 Glutathione S-transferase theta-2 (EC 4 0 0 0 Q91VB0 Adult male cDNA, RIKEN full-length 4 0 0 0 29 Q8CIM7 Cytochrome P450 2D26 (EC 1.14.14.1) 4 0 0 0 30 Q91W49 Enoyl-Coenzyme A, hydratase/3-hydroxyacyl 4 0 0 0 Q9DBM2 Peroxisomal bifunctional enzyme (PBE) 4 0 0 0 31 P35505 Fumarylacetoacetase (EC 3.7.1.2) 4 0 0 0 Q3TY87 Visual cortex cDNA, RIKEN full-length 4 0 0 0 32 Q91W31 Transmembrane protein TIARP 4 0 0 0 33 Q561M6 UDP glucuronosyltransferase 1 family, 4 0 0 0 34 Q63886 UDP-glucuronosyltransferase 1-1 precursor 4 0 0 0 35 Q3UAV7 Bone marrow macrophage cDNA, RIKEN 4 0 0 0 Q8C338 0 day neonate lung cDNA, RIKEN full-length 4 0 0 0 36 Q80X81 Acetyl-Coenzyme A acetyltransferase 3 4 0 0 0 Q8CAY6 Acetyl-CoA acetyltransferase, cytosolic (EC 4 0 0 0 Q8R4V3 Acetyl CoA transferase-like protein 4 0 0 0 37 P56593 Cytochrome P450 2A12 (EC 1.14.14.1) 4 0 0 0 38 P55264 Adenosine kinase (EC 2.7.1.20) (AK) 4 0 0 0 39 Q8R084 UDP glucuronosyltransferase 2 family, 4 0 0 0 40 P56528 ADP-ribosyl cyclase 1 (EC 3.2.2.5) (Cyclic 4 0 0 0 41 O70475 UDP-glucose 6-dehydrogenase (EC 1.1.1.22) 4 0 0 0

Supplementary Data to Rybak et al. Page 4 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 42 P14152 Malate dehydrogenase, cytoplasmic (EC 4 0 0 0 43 Q31167 H-2D cell surface glycoprotein (Fragment) 4 0 0 0 Q3KQJ3 H2-L protein (Fragment) 4 0 0 0 44 P62991 Ubiquitin 4 0 0 0 Q3TH47 17 days embryo kidney cDNA, RIKEN 4 0 0 0 Q5SX22 Ubiquitin B 4 0 0 0 Q642L7 Ribosomal protein S27a 4 0 0 0 Q66JP1 Uba52 protein (Ubiquitin A-52 residue 4 0 0 0 Q6NZC5 Ubiquitin B 4 0 0 0 Q8R0Z9 Ubc protein (13 days embryo liver cDNA, 4 0 0 0 Q8VC46 Ubc protein 4 0 0 0 Q922B0 Ubc protein 4 0 0 0 45 P01897 H-2 class I histocompatibility antigen, L-D 4 0 0 0 46 Q4FJV1 Lap3 protein 4 0 0 0 47 Q61009 Scavenger receptor class B member 1 4 0 0 0 48 Q99P30 Peroxisomal coenzyme A diphosphatase 4 0 0 0 49 Q791V5 Mitochondrial carrier homolog 2 4 0 0 0 50 Q6PDD0 UDP glucuronosyltransferase 2 family, 4 0 0 0 Q80X89 Ugt2a1 protein (16 days embryo head cDNA, 4 0 0 0 Q8BWQ1 Adult male liver tumor cDNA, RIKEN 4 0 0 0 51 P33267 Cytochrome P450 2F2 (EC 1.14.14.-) 4 0 0 0 52 Q9DCG6 Probable isomerase MAWBP-2 (EC 5.1.-.-) 4 0 0 0 53 Q64442 Sorbitol dehydrogenase (EC 1.1.1.14) (L-iditol 4 0 0 0 54 Q64459 Cytochrome P450 3A11 (EC 1.14.14.1) 4 0 0 0 55 Q9EQF5 Dihydropyrimidinase (EC 3.5.2.2) (DHPase) 3 0 0 0 56 P01901 H-2 class I histocompatibility antigen, K-B 3 0 0 0 Q3UBW0 Bone marrow macrophage cDNA, RIKEN 3 0 0 0 57 P50172 Corticosteroid 11-beta-dehydrogenase 3 0 0 0 58 P34927 Asialoglycoprotein receptor 1 (ASGPR 1) 3 0 0 0 Q91Y84 Asialoglycoprotein receptor major subunit 3 0 0 0 59 O09173 Homogentisate 1,2-dioxygenase (EC 3 0 0 0 60 Q9JK53 Prolargin precursor (Proline-arginine-rich end 3 0 0 0 61 Q3THH1 CRL-1722 L5178Y-R cDNA, RIKEN 3 0 0 0 Q3TML0 Lung RCB-0558 LLC cDNA, RIKEN 3 0 0 0 62 P16331 Phenylalanine-4-hydroxylase (EC 1.14.16.1) 3 0 0 0 63 Q9DBB8 Adult male liver cDNA, RIKEN full-length 3 0 0 0 64 P70691 UDP-glucuronosyltransferase 1-2 precursor 3 0 0 0 Q5FW80 UDP glycosyltransferase 1 family, 3 0 0 0 Q64435 UDP-glucuronosyltransferase 1-6 precursor 3 0 0 0 Q6XL45 UDP glycosyltransferase 1 family polypeptide 3 0 0 0 Q6XL46 UDP glycosyltransferase 1 family polypeptide 3 0 0 0 Q6XL48 UDP glycosyltransferase 1 family polypeptide 3 0 0 0 Q8R0P3 UDP glucuronosyltransferase 1 family, 3 0 0 0 65 P00184 Cytochrome P450 1A1 (EC 1.14.14.1) 3 0 0 0 66 O08807 Peroxiredoxin-4 (EC 1.11.1.15) (Prx-IV) 3 0 0 0 67 Q91W64 Cytochrome P450 2C70 (EC 1.14.14.1) 3 0 0 0 68 O08709 Peroxiredoxin-6 (EC 1.11.1.15) (Antioxidant 3 0 0 0 Q6GT24 Peroxiredoxin 6 (8 days embryo whole body 3 0 0 0 69 P34914 Epoxide hydrolase 2 (EC 3.3.2.10) (Soluble 3 0 0 0 70 Q8QZR3 Carboxylesterase 6 (Adult male cecum 3 0 0 0 71 Q64481 Cytochrome P450 3A16 (EC 1.14.14.1) 3 0 0 0 72 Q9D050 10 days embryo whole body cDNA, RIKEN 3 0 0 0 73 Q61735 Leukocyte surface antigen CD47 precursor 2 0 0 0 74 P97807 Fumarate hydratase, mitochondrial precursor 2 0 0 0

Supplementary Data to Rybak et al. Page 5 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 75 P51660 Peroxisomal multifunctional enzyme type 2 2 0 0 0 76 P14429 H-2 class I histocompatibility antigen, Q7 2 0 0 0 P14430 H-2 class I histocompatibility antigen, Q8 2 0 0 0 P79567 Class Ib MHC antigen Qa-2 precursor 2 0 0 0 P79568 Class Ib MHC antigen Qa-2 2 0 0 0 77 Q5EBQ0 Voltage-dependent anion channel 3 (17 days 2 0 0 0 78 Q8BYC3 0 day neonate thymus cDNA, RIKEN 2 0 0 0 Q8C168 10 days neonate skin cDNA, RIKEN 2 0 0 0 Q9R0H0 Acyl-coenzyme A oxidase 1, peroxisomal 2 0 0 0 79 P42925 Peroxisomal membrane protein 2 (22 kDa 2 0 0 0 Q5D073 Pxmp2 protein 2 0 0 0 80 P09103 Protein disulfide-isomerase precursor (EC 2 0 0 0 Q3TF72 17 days embryo kidney cDNA, RIKEN 2 0 0 0 81 O09131 Glutathione transferase omega-1 (EC 2 0 0 0 82 Q9DBJ1 Phosphoglycerate mutase 1 (EC 5.4.2.1) (EC 2 0 0 0 83 P08228 Superoxide dismutase [Cu-Zn] (EC 1.15.1.1) 2 0 0 0 84 Q8VDC3 Cytoplasmic aconitase 2 0 0 0 85 O55022 Membrane-associated progesterone receptor 2 0 0 0 86 O19453 L(W16) protein (H-2L(Q) antigen) (M. 2 0 0 0 P01896 H-2 class I histocompatibility antigen, alpha 2 0 0 0 87 Q31209 MHC class I H2 (haplotype d), 2 0 0 0 Q61891 MRNA, . precursor 2 0 0 0 Q61892 MRNA, . precursor 2 0 0 0 88 Q3UVJ5 Adult male urinary bladder cDNA, RIKEN 2 0 0 0 89 P17182 Alpha-enolase (EC 4.2.1.11) 2 0 0 0 Q5XJG8 LOC433182 protein (Fragment) 2 0 0 0 Q6PHC1 Eno1 protein 2 0 0 0 90 Q8BW75 Amine oxidase [flavin-containing] B (EC 2 0 0 0 91 Q8CF78 Adult male kidney cDNA, RIKEN full-length 2 0 0 0 92 P05201 Aspartate aminotransferase, cytoplasmic (EC 2 0 0 0 93 Q8C7E5 Adult male liver tumor cDNA, RIKEN 2 0 0 0 Q91XE3 Camello-like 1 2 0 0 0 Q9JIY7 Putative N-acetyltransferase Camello 4 2 0 0 0 94 P62897 Cytochrome c, somatic 2 0 0 0 95 P40142 Transketolase (EC 2.2.1.1) (TK) (P68) 2 0 0 0 96 Q8BS01 Adult male aorta and vein cDNA, RIKEN 2 0 0 0 97 Q4U105 NF-kB2 splice variant 4 2 0 0 0 98 Q9D4V3 Adult male testis cDNA, RIKEN full-length 2 0 0 0 99 Q4FJX7 Lcn7 protein 2 0 0 0 Q99JR5 Tubulointerstitial nephritis antigen-like 2 0 0 0 100 O09172 Glutamate--cysteine ligase regulatory subunit 2 0 0 0 101 Q91X34 Bile acid CoA:amino acid N-acyltransferase 2 0 0 0 102 Q9R257 -binding protein 1 (p22HBP) 2 0 0 0 103 Q8BPD3 18 days pregnant adult female placenta and 2 0 0 0 104 Q61830 Macrophage mannose receptor 1 precursor 2 0 0 0 105 P51885 Lumican precursor (Keratan sulfate 2 0 0 0 106 O35488 Very-long-chain acyl-CoA synthetase (EC 2 0 0 0 Q91WV6 Slc27a2 protein 2 0 0 0 107 Q6V956 Aldehyde oxidase 1 1 0 0 0 108 Q3TVD9 Adult male tongue cDNA, RIKEN full-length 1 0 0 0 109 Q8VCN5 Cystathionine gamma-lyase (EC 4.4.1.1) 1 0 0 0 110 Q9QUM9 Proteasome subunit alpha type 6 (EC 1 0 0 0 111 Q6WG34 Glucose 6-phosphate transporter 1 0 0 0 112 Q9JLJ4 Elongation of very long chain fatty acids 1 0 0 0

Supplementary Data to Rybak et al. Page 6 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 113 Q5SXR6 Clathrin, heavy polypeptide (Hc) 1 0 0 0 Q68FD5 Clathrin heavy chain 1 0 0 0 114 Q9CPU0 Lactoylglutathione lyase (EC 4.4.1.5) 1 0 0 0 115 P02762 Major urinary protein 6 precursor (MUP 6) 1 0 0 0 P04938 Major urinary proteins 11 and 8 (MUP11 and 1 0 0 0 P11589 Major urinary protein 2 precursor (MUP 2) 1 0 0 0 Q58EV3 Major urinary protein 1 1 0 0 0 Q9CXU6 13 days embryo head cDNA, RIKEN 1 0 0 0 Q9D0H8 10 days embryo whole body cDNA, RIKEN 1 0 0 0 116 Q99J08 SEC14-like protein 2 1 0 0 0 117 P14431 H-2 class I histocompatibility antigen, Q9 1 0 0 0 Q31199 MHC class I Q8/9d cell surface antigen 1 0 0 0 Q9CZ20 10, 11 days embryo whole body cDNA, 1 0 0 0 118 Q9DCY1 Adult male kidney cDNA, RIKEN full-length 1 0 0 0 119 O88451 Retinol dehydrogenase 7 (EC 1.1.1.105) 1 0 0 0 Q3V1Y2 17 days embryo head cDNA, RIKEN 1 0 0 0 Q8CGV4 Retinol dehydrogenase type 1 (Adult male 1 0 0 0 Q8K3L9 Truncated cis-retinol/3alpha-hydroxysterol 1 0 0 0 Q8K3M9 Retinol dehydrogenase similar protein 2 1 0 0 0 Q9R092 Trans-retinol/estrogen dehydrogenase 1 0 0 0 120 O19440 Major histocompatibility complex class I b 1 0 0 0 O19441 Major histocompability complex Q1b 1 0 0 0 O78206 H-2D cell surface glycoprotein (Fragment) 1 0 0 0 121 P45591 Cofilin-2 (Cofilin, muscle isoform) 1 0 0 0 122 Q31152 MHC class I Q4 beta-2-microglobulin (Qb-1) 1 0 0 0 Q31214 MHC (Qa) Q10-k class I antigen (Adult male 1 0 0 0 Q4KN81 MHC class I antigen 1 0 0 0 Q4VCN4 Truncated MHC class I antigen splice variant 1 0 0 0 Q8HWB2 16 days neonate thymus cDNA, RIKEN 1 0 0 0 Q8HWL1 H2-Q10 protein (Fragment) 1 0 0 0 Q95H92 H2-Q1 protein 1 0 0 0 Q95HC3 H2-Q10 protein 1 0 0 0 123 Q9CX22 12 days embryo embryonic body between 1 0 0 0 124 O35660 Glutathione S-transferase Mu 6 (EC 2.5.1.18) 1 0 0 0 P19639 Glutathione S-transferase Mu 3 (EC 2.5.1.18) 1 0 0 0 125 P50236 Bile salt sulfotransferase 2 (EC 2.8.2.14) 1 0 0 0 P52843 Bile salt sulfotransferase 1 (EC 2.8.2.14) 1 0 0 0 126 Q8C0Z1 Protein ITFG3 1 0 0 0 127 P33622 Apolipoprotein C-III precursor (Apo-CIII) 1 0 0 0 128 P35279 Ras-related protein Rab-6A (Rab-6) 1 0 0 0 P61294 Ras-related protein Rab-6B 1 0 0 0 129 Q3TQ93 16 days embryo head cDNA, RIKEN 1 0 0 0 130 Q8BHD0 Ras-related protein Rab-39A (Rab-39) 1 0 0 0 131 Q8K386 Ras-related protein Rab-15 1 0 0 0 Q91YW0 Rab15 protein 1 0 0 0 132 P35276 Ras-related protein Rab-3D 1 0 0 0 P35283 Ras-related protein Rab-12 (Rab-13) 1 0 0 0 P55258 Ras-related protein Rab-8A (Oncogene c-mel) 1 0 0 0 P56371 Ras-related protein Rab-4A 1 0 0 0 P61027 Ras-related protein Rab-10 1 0 0 0 P61028 Ras-related protein Rab-8B 1 0 0 0 P62821 Ras-related protein Rab-1A (YPT1-related 1 0 0 0 P62823 Ras-related protein Rab-3C 1 0 0 0 P63011 Ras-related protein Rab-3A 1 0 0 0

Supplementary Data to Rybak et al. Page 7 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) Q3UHW5 17 days pregnant adult female amnion 1 0 0 0 Q3USC3 10 days neonate cerebellum cDNA, RIKEN 1 0 0 0 Q504M8 Ras-related protein Rab-26 1 0 0 0 Q6PHN9 Ras-related protein Rab-35 1 0 0 0 Q8BHC1 Ras-related protein Rab-39B 1 0 0 0 Q8BKQ0 12 days embryo spinal ganglion cDNA, 1 0 0 0 Q8BNU1 10 days neonate cortex cDNA, RIKEN 1 0 0 0 Q8BQX0 Adult male corpora quadrigemina cDNA, 1 0 0 0 Q8BRM8 10 days neonate cortex cDNA, RIKEN 1 0 0 0 Q8CG50 Ras-related protein Rab-43 1 0 0 0 Q91V41 Ras-related protein Rab-14 1 0 0 0 Q91ZR1 Ras-related protein Rab-4B 1 0 0 0 Q923S9 Ras-related protein Rab-30 1 0 0 0 Q9CZT8 Ras-related protein Rab-3B 1 0 0 0 Q9D1G1 Ras-related protein Rab-1B 1 0 0 0 Q9JKM7 Ras-related protein Rab-37 1 0 0 0 133 O54754 Aldehyde oxidase (EC 1.2.3.1) (Retinal 1 0 0 0 Q8R387 Aldehyde oxidase 1 1 0 0 0 134 P08071 Lactotransferrin precursor (EC 3.4.21.-) 1 0 0 0 Q8CBA0 Adult male bone cDNA, RIKEN full-length 1 0 0 0 135 Q3TJQ7 13 days pregnant adult female placenta 1 0 0 0 Q9ET01 Glycogen phosphorylase, liver form (EC 1 0 0 0 136 Q810X5 Aldehyde reductase (Fragment) 1 0 0 0 137 Q3UEI8 Adult male liver tumor cDNA, RIKEN 1 0 0 0 Q3UEP4 Adult male liver tumor cDNA, RIKEN 1 0 0 0 Q3UWB9 Adult male colon cDNA, RIKEN full-length 1 0 0 0 Q8VCN3 UDP glucuronosyltransferase 2 family, 1 0 0 0 Q91WH2 UDP glucuronosyltransferase 2 family, 1 0 0 0 138 P11352 Glutathione peroxidase 1 (EC 1.11.1.9) 1 0 0 0 139 Q31145 MHC class I D-q alpha-chain (H2 haplotype), 1 0 0 0 Q31151 MHC class I L-q alpha-chain gene (H2 1 0 0 0 Q4KN89 MHC class I antigen 1 0 0 0 140 P70168 Importin beta-1 subunit (Karyopherin beta-1 1 0 0 0 Q3UHW8 17 days embryo heart cDNA, RIKEN 1 0 0 0 141 Q03249 Galactose-1-phosphate uridylyltransferase 1 0 0 0 142 Q5RI75 Novel protein similar to RAB member RAS 1 0 0 0 Q5RI76 Novel protein similar to RAB member RAS 1 0 0 0 Q5SYL4 RAB34, member of RAS oncogene family 1 0 0 0 Q5SYL5 RAB34, member of RAS oncogene family 1 0 0 0 Q5SYL6 RAB34, member of RAS oncogene family 1 0 0 0 Q5SYM6 RAB34, member of RAS oncogene family 1 0 0 0 Q64008 Ras-related protein Rab-34 (Rab-39) 1 0 0 0 Q8CB87 Adult male bone cDNA, RIKEN full-length 1 0 0 0 Q99P58 Ras-related protein Rab-27B 1 0 0 0 Q9ERI2 Ras-related protein Rab-27A 1 0 0 0 143 Q8BHH2 Ras-related protein Rab-9B 1 0 0 0 Q91Z34 1810048P08Rik protein 1 0 0 0 144 Q61045 Single-minded homolog 1 (mSIM1) 1 0 0 0 Q61079 Single-minded homolog 2 (SIM transcription 1 0 0 0 Q9QZQ0 Neuronal PAS domain-containing protein 3 1 0 0 0 145 P15105 Glutamine synthetase (EC 6.3.1.2) 1 0 0 0 Q3TRK7 Adult female vagina cDNA, RIKEN full-length 1 0 0 0 146 P62962 -1 (Profilin I) 1 0 0 0 Q5SX49 1 0 0 0

Supplementary Data to Rybak et al. Page 8 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 147 Q6P6I4 Glutathione S-transferase, theta 3 1 0 0 0 148 Q64471 Glutathione S-transferase theta-1 (EC 1 0 0 0 149 Q91X50 Glutathione S-transferase, theta 1 1 0 0 0 150 P61458 Pterin-4-alpha-carbinolamine dehydratase 1 0 0 0 151 O08573 Galectin-9 1 0 0 0 152 Q9D826 Peroxisomal sarcosine oxidase (EC 1.5.3.1) 1 0 0 0 153 P34884 Macrophage migration inhibitory factor (MIF) 1 0 0 0 154 P37913 DNA ligase 1 (EC 6.5.1.1) (DNA ligase I) 1 0 0 0 Q3U4X8 2 days neonate thymus thymic cells cDNA, 1 0 0 0 155 Q3V366 4 days neonate thymus cDNA, RIKEN 1 0 0 0 156 Q8K229 Procollagen, type VI, alpha 2 1 0 0 0 157 Q9QYG0 Protein NDRG2 (Protein Ndr2) 1 0 0 0 158 P28654 precursor (Bone proteoglycan II) 1 0 0 0 159 Q01768 Nucleoside diphosphate kinase B (EC 1 0 0 0 160 Q149K3 2310038E17Rik protein 1 0 0 0 161 Q3TN34 B cells CRL-1669 BCL1 Clone 13.20-3B3 1 0 0 0 162 Q6AW46 Carboxylesterase-like urinary excreted 1 0 0 0 163 Q6PDB7 CDNA sequence BC015286 1 0 0 0 Q8BK48 11 days pregnant adult female ovary and 1 0 0 0 164 Q8CEB6 10 days neonate skin cDNA, RIKEN 1 0 0 0 165 Q8R097 RIKEN cDNA 2210023G05 gene 1 0 0 0 166 Q8R4B4 Down syndrome cell adhesion molecule-like 1 0 0 0 167 Q91VC3 Probable ATP-dependent RNA helicase 1 0 0 0 168 Q91WG0 Carboxylesterase 2 (Carboxylesterase ML3 1 0 0 0 Q9D998 Adult male testis cDNA, RIKEN full-length 1 0 0 0 169 Q9DCT1 Aldo-keto reductase family 1 member E1 1 0 0 0 170 Q8BTY1 Kynurenine--oxoglutarate transaminase 1 1 0 0 0 171 Q9DD20 Adult male kidney cDNA, RIKEN full-length 1 0 0 0 172 P14211 Calreticulin precursor (CRP55) (Calregulin) 1 0 0 0 173 P14824 Annexin A6 (Annexin VI) (Lipocortin VI) (P68) 1 0 0 0 174 Q3UDK4 Bone marrow macrophage cDNA, RIKEN 1 0 0 0 175 Q3V2Z4 13 days embryo forelimb cDNA, RIKEN 1 0 0 0 176 Q6XVG2 Cytochrome P450 1 0 0 0 177 Q3UK06 TIB-55 BB88 cDNA, RIKEN full-length 1 0 0 0 178 Q99KY6 Cytochrome P450, family 4, subfamily f, 1 0 0 0 Q99N16 Cytochrome P450 4F3 (EC 1.14.13.30) 1 0 0 0 Q99N18 Cytochrome P450 CYP4F15 1 0 0 0 Q9EP75 Cytochrome P450 4F14 (EC 1.14.13.30) 1 0 0 0 179 P11609 T-cell surface glycoprotein CD1d1 precursor 1 0 0 0 P11610 T-cell surface glycoprotein CD1d2 precursor 1 0 0 0 180 P14246 Solute carrier family 2, facilitated glucose 1 0 0 0 181 P35564 Calnexin precursor 1 0 0 0 182 Q62383 Transcription elongation factor SPT6 1 0 0 0 183 Q76LV0 Nucleolar phospholipid hydroperoxide 1 0 0 0 184 Q7TQC0 DNA polymerase Q 1 0 0 0 185 Q80X52 Slc25a22 protein 1 0 0 0 186 Q8CGS6 DNA polymerase theta short isoform 1 0 0 0 187 Q91XR9 Phospholipid hydroperoxide glutathione 1 0 0 0 188 Q9D6M3 Mitochondrial glutamate carrier 1 (GC-1) 1 0 0 0 189 P12970 60S ribosomal protein L7a (Surfeit locus 1 0 0 0 190 Q3UQS0 13 days embryo heart cDNA, RIKEN 1 0 0 0 191 Q6GQT0 Dual specificity protein phosphatase 1 0 0 0 192 Q8BMG8 Mitochondrial folate transporter/carrier 1 0 0 0 193 Q91XD4 Formimidoyltransferase-cyclodeaminase 1 0 0 0

Supplementary Data to Rybak et al. Page 9 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 194 P10126 Elongation factor 1-alpha 1 (EF-1-alpha-1) 6 7 0 0 195 Q3TIT9 14 days embryo liver cDNA, RIKEN full-length 5 1 0 0 Q8BWT1 3-ketoacyl-CoA thiolase, mitochondrial (EC 5 1 0 0 196 P19157 Glutathione S-transferase P 1 (EC 2.5.1.18) 5 1 0 0 197 Q9QXX4 Calcium-binding mitochondrial carrier protein 5 1 0 0 198 P35700 Peroxiredoxin-1 (EC 1.11.1.15) (Thioredoxin 5 1 0 0 199 Q8BGL3 Adult male aorta and vein cDNA, RIKEN 5 1 0 0 200 Q64458 Cytochrome P450 2C29 (EC 1.14.14.1) 5 1 0 0 201 P12710 Fatty acid-binding protein, liver (L-FABP) (14 5 1 0 0 202 P28474 Alcohol dehydrogenase class 3 (EC 1.1.1.1) 5 1 0 0 203 Q5U677 Fcgr2b protein (Fragment) 5 1 0 0 204 Q61490 CD166 antigen precursor (Activated 5 1 0 0 205 P10649 Glutathione S-transferase Mu 1 (EC 2.5.1.18) 5 2 0 0 206 Q91X83 S-adenosylmethionine synthetase isoform 5 2 0 0 207 P14094 Sodium/potassium-transporting ATPase 5 2 0 0 208 P30115 Glutathione S-transferase A3 (EC 2.5.1.18) 5 2 0 0 209 P58710 L-gulonolactone oxidase (EC 1.1.3.8) (LGO) 5 2 0 0 Q8K152 Gulonolactone (L-) oxidase (14 days 5 2 0 0 210 Q91Y97 Fructose-bisphosphate aldolase B (EC 5 3 0 0 211 Q921H8 3-ketoacyl-CoA thiolase A, peroxisomal 5 3 0 0 212 Q8VCH0 3-ketoacyl-CoA thiolase B, peroxisomal 5 3 0 0 213 Q9EP89 Serine beta-lactamase-like protein LACTB 5 3 0 0 214 Q91XE8 CDNA sequence BC010787 5 3 0 0 215 P01887 Beta-2-microglobulin precursor 5 3 0 0 Q3U679 Bone marrow macrophage cDNA, RIKEN 5 3 0 0 216 Q8VEM8 Phosphate carrier protein, mitochondrial 5 3 0 0 217 P24549 Retinal dehydrogenase 1 (EC 1.2.1.36) 5 4 0 0 218 O35490 Betaine--homocysteine S-methyltransferase 5 4 0 0 219 P70694 Estradiol 17 beta-dehydrogenase 5 (EC 5 4 0 0 220 P32020 Nonspecific lipid-transfer protein (EC 5 4 0 0 221 Q8VCT4 Carboxylesterase 3 precursor (EC 3.1.1.1) 5 4 0 0 222 Q9QXZ6 Solute carrier organic anion transporter 5 7 0 0 223 Q921I1 Serotransferrin precursor (Transferrin) 5 8 0 0 224 P11087 alpha-1(I) chain precursor 5 8 0 0 225 P60710 , cytoplasmic 1 (Beta-actin) 5 8 0 0 P63260 Actin, cytoplasmic 2 (Gamma-actin) 5 8 0 0 Q3TSB7 In vitro fertilized eggs cDNA, RIKEN 5 8 0 0 Q3U804 Bone marrow macrophage cDNA, RIKEN 5 8 0 0 226 P11499 Heat shock protein HSP 90-beta 5 8 0 0 227 Q9QZ83 Gamma actin-like protein 5 8 0 0 228 Q63880 Liver carboxylesterase 31 precursor (EC 4 1 0 0 Q6P8Z0 Es31 protein 4 1 0 0 229 P70296 Phosphatidylethanolamine-binding protein 1 4 1 0 0 230 Q8VCU1 Esterase 31-like 4 1 0 0 231 P17751 Triosephosphate isomerase (EC 5.3.1.1) 4 1 0 0 232 Q4LDG0 Bile acyl-CoA synthetase (EC 6.2.1.7) 4 1 0 0 233 Q91XD5 LOC13909 protein (Fragment) 4 1 0 0 234 Q920E5 Farnesyl pyrophosphate synthetase (FPP 4 1 0 0 235 O70503 Estradiol 17-beta-dehydrogenase 12 (EC 4 1 0 0 236 P28843 Dipeptidyl peptidase 4 (EC 3.4.14.5) 4 1 0 0 237 P55096 ATP-binding cassette sub-family D member 3 4 1 0 0 Q3U645 Bone marrow macrophage cDNA, RIKEN 4 1 0 0 238 Q9CY27 Synaptic glycoprotein SC2 4 1 0 0 239 Q9DCP2 System N amino acid transporter 1 (SN1) 4 1 0 0

Supplementary Data to Rybak et al. Page 10 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 240 Q5F277 Fatty acid Coenzyme A ligase, long chain 6 4 1 0 0 Q5F279 Fatty acid Coenzyme A ligase, long chain 6 4 1 0 0 Q5F281 Fatty acid Coenzyme A ligase, long chain 6 4 1 0 0 Q8BYJ0 Adult male spinal cord cDNA, RIKEN 4 1 0 0 Q8R1X1 Acyl-CoA synthetase long-chain family 4 1 0 0 Q91WC3 Long-chain-fatty-acid--CoA ligase 6 (EC 4 1 0 0 241 Q9R0P3 S-formylglutathione hydrolase (EC 3.1.2.12) 4 2 0 0 242 Q3UWB1 Adult male colon cDNA, RIKEN full-length 4 2 0 0 243 P47911 60S ribosomal protein L6 (TAX-responsive 4 2 0 0 244 Q3TCF1 NOD-derived CD11c +ve dendritic cells 4 3 0 0 245 Q9JJL3 Solute carrier organic anion transporter family 4 3 0 0 246 Q8BLD7 Adult male corpora quadrigemina cDNA, 4 3 0 0 247 Q3UA95 Bone marrow macrophage cDNA, RIKEN 4 3 0 0 Q64522 Histone H2A type 2-B (H2a-613A) 4 3 0 0 248 Q01279 Epidermal growth factor receptor precursor 4 6 0 0 249 P0C0S6 Histone H2A.Z (H2A/z) 4 6 0 0 P22752 Histone H2A type 1 4 6 0 0 P27661 Histone H2A.x (H2a/x) 4 6 0 0 Q3THW5 Histone H2AV (H2A.F/Z) 4 6 0 0 Q64523 Histone H2A type 2-C (H2a-613B) 4 6 0 0 Q6GSS7 Histone H2A type 2-A (H2A.2) (H2a-614) 4 6 0 0 Q8BFU2 Histone H2A type 3 4 6 0 0 Q8CGP4 Histone protein Hist1h2aa (Histone 1, H2aa) 4 6 0 0 Q8CGP5 Histone H2A type 1-F 4 6 0 0 Q8CGP6 Histone H2A type 1-H 4 6 0 0 Q8CGP7 Histone H2A type 1-K 4 6 0 0 Q8R1M2 H2afj protein (H2A histone family, member 4 6 0 0 250 Q03734 Serine protease inhibitor A3M precursor 4 7 0 0 251 P11499 Heat shock protein HSP 90-beta (HSP 84) 4 7 0 0 252 Q4V783 LOC654472 protein 4 7 0 0 253 Q6P287 Glyceraldehyde-3-phosphate dehydrogenase 4 7 0 0 254 Q4VBD1 AK190093 protein 4 7 0 0 255 Q01149 Collagen alpha-2(I) chain precursor 4 7 0 0 256 Q3UHL6 CDNA, RIKEN full-length enriched library, 4 8 0 0 257 P07758 Alpha-1-antitrypsin 1-1 precursor (Serine 4 8 0 0 P81105 Alpha-1-antitrypsin 1-6 precursor (Serine 4 8 0 0 258 Q00896 Alpha-1-antitrypsin 1-3 precursor (Serine 4 8 0 0 259 P07759 Serine protease inhibitor A3K precursor 4 8 0 0 260 Q04857 Collagen alpha-1(VI) chain precursor 4 8 0 0 261 Q6YK32 Histidine-rich glycoprotein HRG 4 8 0 0 262 O55107 Basigin (Fragment) 3 1 0 0 P18572 Basigin precursor (Basic immunoglobulin 3 1 0 0 263 Q8R1S9 Sodium-coupled neutral amino acid 3 1 0 0 264 Q8CJ71 Heterogeneous nuclear ribonucleoprotein 3 1 0 0 265 P11276 Fibronectin precursor (FN) 3 2 0 0 266 Q64310 Surfeit locus protein 4 3 2 0 0 267 P10853 Histone H2B type 1-F/J/L (H2B 291A) 3 2 0 0 P10854 Histone H2B type 1-M (H2B 291B) 3 2 0 0 Q64475 Histone H2B type 1-B (h2B-143) 3 2 0 0 Q64478 Histone H2B type 1-H (h2B-221) 3 2 0 0 Q64524 Histone H2B type 2-E (H2b 613) 3 2 0 0 Q64525 Histone H2B type 2-B (H2b 616) 3 2 0 0 Q6ZWY9 Histone H2B type 1-C/E/G 3 2 0 0 Q8CBB6 Adult male bone cDNA, RIKEN full-length 3 2 0 0

Supplementary Data to Rybak et al. Page 11 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) Q8CGP0 Histone H2B type 3-B 3 2 0 0 Q8CGP1 Histone H2B type 1-K 3 2 0 0 Q8CGP2 Histone H2B type 1-P 3 2 0 0 Q8VEE3 Similar to H2B histone family, member J 3 2 0 0 Q921L4 Hist1h2bc protein 3 2 0 0 Q9D2U9 Histone H2B type 3-A 3 2 0 0 268 P10648 Glutathione S-transferase A2 (EC 2.5.1.18) 3 2 0 0 P13745 Glutathione S-transferase A1 (EC 2.5.1.18) 3 2 0 0 Q6P8Q0 Glutathione S-transferase, alpha 1 (Ya) 3 2 0 0 269 Q64176 Liver carboxylesterase 22 precursor (EC 3 2 0 0 270 Q8VCX1 3-oxo-5-beta-steroid 4-dehydrogenase (EC 3 2 0 0 271 P20029 78 kDa glucose-regulated protein precursor 3 2 0 0 272 Q91WT7 Aldo-keto reductase family 1, member C14 3 3 0 0 273 P23953 Liver carboxylesterase N precursor (EC 3 4 0 0 274 P58252 Elongation factor 2 (EF-2) 3 6 0 0 275 P07901 Heat shock protein HSP 90-alpha (HSP 86) 3 8 0 0 276 Q60932 Voltage-dependent anion-selective channel 2 1 0 0 277 P58735 Sulfate anion transporter 1 (SAT-1) (Solute 2 1 0 0 278 Q6P8Q1 Glutathione S-transferase, alpha 2 (Yc2) 2 1 0 0 279 Q78PY7 Staphylococcal nuclease domain-containing 2 1 0 0 280 P08101 Low affinity immunoglobulin gamma Fc region 2 1 0 0 P08508 Low affinity immunoglobulin gamma Fc region 2 1 0 0 281 Q69ZD6 MKIAA1675 protein (Fragment) 2 1 0 0 Q6PB79 Prdm16 protein 2 1 0 0 Q7TPF4 Transcription factor MEL1 2 1 0 0 282 Q9D8N0 Elongation factor 1-gamma (EF-1-gamma) 2 1 0 0 283 O70324 Monocarboxylate transporter 8 (MCT 8) 2 1 0 0 284 Q6P9L6 family member 15 2 1 0 0 285 Q9Z0I9 Collagen alpha3(VI) precursor (Fragment) 2 4 0 0 286 P63017 Heat shock cognate 71 kDa protein (Heat 2 5 0 0 287 Q9WVF5 Epidermal growth factor receptor (Adult 2 5 0 0 288 P68372 beta-2C chain 2 6 0 0 289 P07309 precursor (Prealbumin) 2 7 0 0 290 Q99K47 Fibrinogen, alpha polypeptide 2 8 0 0 291 O88493 Type VI collagen alpha 3 subunit 2 8 0 0 292 P08122 Collagen alpha-2(IV) chain precursor 2 8 0 0 293 Q3UEP0 Adult male liver tumor cDNA, RIKEN 1 1 0 0 294 P15626 Glutathione S-transferase Mu 2 (EC 2.5.1.18) 1 1 0 0 Q3TRV7 12 days embryo embryonic body between 1 1 0 0 Q8R5I6 Glutathione transferase GSTM7-7 (EC 1 1 0 0 295 P84443 Small inducible cytokine A21a precursor 1 1 0 0 296 P10630 Eukaryotic initiation factor 4A-II (EC 3.6.1.-) 1 1 0 0 P60843 Eukaryotic initiation factor 4A-I (EC 3.6.1.-) 1 1 0 0 297 Q8C850 Scavenger receptor class A member 3 1 1 0 0 298 Q00897 Alpha-1-antitrypsin 1-4 precursor (Serine 1 2 0 0 299 Q6IWE2 Beta-actin (Fragment) 1 2 0 0 300 P09803 Epithelial-cadherin precursor (E-cadherin) 1 2 0 0 301 Q8BFZ3 10 days neonate skin cDNA, RIKEN 1 2 0 0 302 P62631 Elongation factor 1-alpha 2 (EF-1-alpha-2) 1 2 0 0 303 Q5RKM8 Hspg2 protein (Fragment) 1 2 0 0 304 Q8VCC2 Liver carboxylesterase 1 precursor (EC 1 2 0 0 305 P62737 Actin, aortic smooth muscle (Alpha-actin-2) 1 3 0 0 P63268 Actin, gamma-enteric smooth muscle 1 3 0 0 P68033 Actin, alpha cardiac (Alpha-cardiac actin) 1 3 0 0

Supplementary Data to Rybak et al. Page 12 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) P68134 Actin, alpha skeletal muscle (Alpha-actin-1) 1 3 0 0 Q3TG92 17 days embryo heart cDNA, RIKEN 1 3 0 0 Q3UIJ3 17 days embryo heart cDNA, RIKEN 1 3 0 0 Q61852 Smooth muscle gamma-actin 1 3 0 0 Q9CXK3 14, 17 days embryo head cDNA, RIKEN 1 3 0 0 306 P09055 Integrin beta-1 precursor (Fibronectin receptor 1 4 0 0 307 Q05793 Perlecan 1 4 0 0 308 P10493 Nidogen-1 precursor (Entactin) 1 4 0 0 309 P57780 Alpha--4 (Non-muscle alpha-actinin 4) 1 4 0 0 Q7TPR4 Alpha-actinin-1 (Alpha-actinin cytoskeletal 1 4 0 0 310 Q58E39 Hnrpa3 protein (Fragment) 1 4 0 0 Q6P6I7 Hnrpa3 protein 1 4 0 0 Q8BG05 Heterogeneous nuclear ribonucleoprotein A3 1 4 0 0 311 P68433 Histone H3.1 1 4 0 0 P84228 Histone H3.2 1 4 0 0 P84244 Histone H3.3 1 4 0 0 Q3TW79 Osteoclast-like cell cDNA, RIKEN full-length 1 4 0 0 Q569U8 H3 histone, family 3B 1 4 0 0 Q8CGN9 Histone protein Hist2h3c1 (Histone 2, H3c1) 1 4 0 0 Q9D0H3 10 days embryo whole body cDNA, RIKEN 1 4 0 0 312 P22599 Alpha-1-antitrypsin 1-2 precursor (Serine 1 5 0 0 Q91X22 Serpina1b protein 1 5 0 0 313 P62806 Histone H4 1 5 0 0 Q6PDS7 Hist1h4h protein (Fragment) 1 5 0 0 Q80WC8 Hist2h4 protein (Fragment) 1 5 0 0 Q811M0 Hist1h4h protein (Fragment) 1 5 0 0 Q9D0C9 10 days embryo whole body cDNA, RIKEN 1 5 0 0 314 Q3TPN0 9 days embryo whole body cDNA, RIKEN 1 7 0 0 315 O88322 Nidogen-2 precursor (NID-2) (Entactin-2) 1 7 0 0 316 Q8K0E8 Fibrinogen beta chain precursor [Contains: 1 8 0 0 317 Q3UHH3 14 days pregnant adult female placenta 1 8 0 0 318 P01868 Ig gamma-1 chain C region secreted form 1 8 0 0 P01869 Ig gamma-1 chain C region, 1 8 0 0 Q99LC4 Igh-4 protein 1 8 0 0 319 P28665 Murinoglobulin-1 precursor (MuG1) 0 8 0 0 320 Q6PEM2 Pregnancy zone protein 0 8 0 0 321 P01027 Complement C3 precursor (HSE-MSF) 0 8 0 0 322 P99024 Tubulin beta-5 chain 0 8 0 0 323 Q80X19 Collagen alpha-1(XIV) chain precursor 0 8 0 0 324 Q3UNE7 CDNA, RIKEN full-length enriched library, 0 7 0 0 325 Q91X72 Hemopexin precursor 0 7 0 0 326 Q06890 Clusterin precursor (Sulfated glycoprotein 2) 0 7 0 0 327 P05213 Tubulin alpha-2 chain (Alpha-tubulin 2) 0 6 0 0 P68369 Tubulin alpha-1 chain (Alpha-tubulin 1) 0 6 0 0 P68373 Tubulin alpha-6 chain (Alpha-tubulin 6) 0 6 0 0 Q3TGF0 16 days embryo heart cDNA, RIKEN 0 6 0 0 Q3TPJ1 12 days embryo spinal ganglion cDNA, 0 6 0 0 Q3ULN1 5 days embryo whole body cDNA, RIKEN 0 6 0 0 328 Q60849 CD98 heavy chain 0 6 0 0 329 Q9CQN1 Heat shock protein 75 kDa, mitochondrial 0 6 0 0 330 Q00623 Apolipoprotein A-I precursor (Apo-AI) 0 6 0 0 331 P06728 Apolipoprotein A-IV precursor (Apo-AIV) 0 6 0 0 Q91XF8 Apolipoprotein A-IV (17 days pregnant adult 0 6 0 0 332 Q3TVV6 Osteoclast-like cell cDNA, RIKEN full-length 0 5 0 0

Supplementary Data to Rybak et al. Page 13 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 333 P29788 precursor (Serum-spreading 0 5 0 0 334 Q9JK98 Sulfated glycoprotein-2 isoform 2 0 5 0 0 335 P29699 Alpha-2-HS-glycoprotein precursor (Fetuin-A) 0 5 0 0 336 O35945 Aldehyde dehydrogenase Ahd-2-like 0 4 0 0 337 P05214 Tubulin alpha-3/alpha-7 chain (Alpha-tubulin 0 4 0 0 338 P68368 Tubulin alpha-4 chain (Alpha-tubulin 4) 0 4 0 0 Q9D9Y2 Adult male testis cDNA, RIKEN full-length 0 4 0 0 339 P06909 Complement factor H precursor (Protein 0 4 0 0 340 Q811L7 Heterogeneous nuclear ribonucleoprotein H1 0 4 0 0 Q8BVU8 0 day neonate head cDNA, RIKEN full-length 0 4 0 0 Q8C2Q7 2 days neonate thymus thymic cells cDNA, 0 4 0 0 341 Q62362 Testicular alpha tubulin (Fragment) 0 4 0 0 342 Q3UQ28 15 days embryo head cDNA, RIKEN 0 4 0 0 343 P52480 Pyruvate kinase isozyme M2 (EC 2.7.1.40) 0 4 0 0 344 Q9JJ59 ATP-binding cassette sub-family B member 9 0 4 0 0 345 Q61703 Inter-alpha-trypsin inhibitor heavy chain H2 0 4 0 0 346 O08677 Kininogen-1 precursor [Contains: Kininogen-1 0 4 0 0 Q32MX7 Kng1 protein 0 4 0 0 Q6S9I1 HMW kininogen-I variant (High molecular 0 4 0 0 347 P20918 Plasminogen precursor (EC 3.4.21.7) 0 4 0 0 348 Q8K023 Aldo-keto reductase family 1 member C18 0 3 0 0 349 P21995 Embigin precursor (Teratocarcinoma 0 3 0 0 350 Q61247 Alpha-2-antiplasmin precursor 0 3 0 0 351 Q6P8V0 Aldo-keto reductase family 1, member C21 0 3 0 0 Q8VC77 Akr1c20 protein (Aldo-keto reductase) 0 3 0 0 Q9CZU0 10 days embryo whole body cDNA, RIKEN 0 3 0 0 352 Q01339 Beta-2-glycoprotein 1 precursor 0 3 0 0 Q9CY42 13 days embryo liver cDNA, RIKEN 0 3 0 0 353 P27659 60S ribosomal protein L3 (J1 protein) 0 3 0 0 Q91VJ6 Ribosomal protein L3 (NOD-derived CD11c 0 3 0 0 354 P06684 Complement C5 precursor (Hemolytic 0 3 0 0 355 P01873 Ig mu chain C region membrane-bound form 0 3 0 0 Q4KML5 Igh-6 protein 0 3 0 0 Q4V9V8 Igh-6 protein 0 3 0 0 Q7TMT6 MGC60843 protein 0 3 0 0 Q8VCX7 Igh-6 protein 0 3 0 0 356 O09130 Nuclear protein NIP45 (Nuclear factor of 0 3 0 0 357 P28653 Biglycan precursor (Bone/cartilage 0 3 0 0 358 Q3TG26 13 days embryo liver cDNA, RIKEN 0 2 0 0 Q9CWF2 ES cells cDNA, RIKEN full-length enriched 0 2 0 0 359 Q5XJF8 Tubulin, alpha 1 0 2 0 0 360 P01029 Complement C4-B precursor [Contains: 0 2 0 0 361 P02468 gamma-1 chain precursor (Laminin 0 2 0 0 362 Q9CXW4 60S ribosomal protein L11 0 2 0 0 363 Q3UHL7 CDNA, RIKEN full-length enriched library, 0 2 0 0 364 Q08879 Fibulin-1 precursor (Basement-membrane 0 2 0 0 365 Q923I7 Sodium/glucose cotransporter 2 0 2 0 0 366 Q3TLT4 RCB-0526 Jyg-MC(A) 0 2 0 0 367 Q8BKG3 Tyrosine-protein kinase-like 7 0 2 0 0 368 Q03265 ATP synthase subunit alpha, mitochondrial 0 2 0 0 369 P21614 Vitamin D-binding protein precursor (DBP) 0 2 0 0 370 Q6VGS5 DVL-binding protein DAPLE 0 2 0 0 Q80XQ1 0610010D24Rik protein 0 2 0 0 371 Q61838 Alpha-2-macroglobulin precursor (Alpha-2-M) 0 8 0 0

Supplementary Data to Rybak et al. Page 14 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 372 P46425 Glutathione S-transferase P 2 (EC 2.5.1.18) 0 1 0 0 373 P01863 Ig gamma-2A chain C region, A allele 0 1 0 0 P01865 Ig gamma-2A chain C region, 0 1 0 0 Q505N9 Igh-1a protein 0 1 0 0 Q569W9 Igh-1a protein 0 1 0 0 Q66K04 Igh-1a protein 0 1 0 0 Q6PF95 Igh-1a protein 0 1 0 0 Q6PIP8 Igh-1a protein 0 1 0 0 Q6PJA7 Igh-1a protein 0 1 0 0 Q6PJB2 Igh-1a protein 0 1 0 0 374 Q8BP96 8 days embryo whole body cDNA, RIKEN 0 1 0 0 375 Q9QXK3 Coatomer subunit gamma-2 (Gamma-2 coat 0 1 0 0 376 P06802 Ectonucleotide 0 1 0 0 Q924C4 Ectonucleotide 0 1 0 0 377 Q5ND35 Serine (Or cysteine) proteinase inhibitor, 0 1 0 0 378 P02463 Collagen alpha-1(IV) chain precursor 0 1 0 0 379 Q8K159 Itih1 protein 0 1 0 0 380 Q5NCJ0 Novel protein similar to human PDZ domain 0 1 0 0 Q5NCJ1 Novel protein similar to human PDZ domain 0 1 0 0 Q5NCJ4 Novel protein similar to human PDZ domain 0 1 0 0 Q5NCJ5 Novel protein similar to human PDZ domain 0 1 0 0 381 Q61739 Integrin alpha-6 precursor (VLA-6) (CD49f 0 1 0 0 382 P39061 Collagen alpha-1(XVIII) chain precursor 0 1 0 0 Q61434 Collagen (Fragment) 0 1 0 0 Q6P1Y4 Procollagen, type XVIII, alpha 1 0 1 0 0 383 Q5EBP9 Tripartite motif protein 28 (7 days embryo 0 1 0 0 Q62318 Transcription intermediary factor 1-beta 0 1 0 0 384 O88207 Collagen alpha-1(V) chain precursor 0 1 0 0 385 P29621 Serine protease inhibitor A3C precursor 0 1 0 0 Q3U1B7 Activated spleen cDNA, RIKEN full-length 0 1 0 0 Q4V9W0 Serpina3g protein 0 1 0 0 Q5I2A0 Serine protease inhibitor A3G (Serine 0 1 0 0 Q80X76 Serine protease inhibitor A3F (Serpin A3F) 0 1 0 0 Q91WP6 Serine protease inhibitor A3N precursor 0 1 0 0 386 Q9D2L4 0 day neonate head cDNA, RIKEN full-length 0 1 0 0 Q9Z0T9 Integrin beta-6 precursor 0 1 0 0 387 Q3U8Y3 Bone marrow macrophage cDNA, RIKEN 0 1 0 0 Q4PJX1 Olfactory specific odr-4 0 1 0 0 Q99J66 CDNA sequence BC003331 0 1 0 0 388 O88411 Female sterile homeotic-related protein Frg-1 0 1 0 0 Q99PC5 Ring 3 (Fragment) 0 1 0 0 389 Q3U7M5 Bone marrow macrophage cDNA, RIKEN 0 1 0 0 Q8C0M2 Adult male testis cDNA, RIKEN full-length 0 1 0 0 390 Q6NXK7 Inactive dipeptidyl peptidase 10 (Dipeptidyl 0 1 0 0 391 P42228 Signal transducer and activator of 0 1 0 0 Q3V157 Adult male testis cDNA, RIKEN full-length 0 1 0 0 392 P16627 Heat shock 70 kDa protein 1L (Heat shock 70 0 1 0 0 P17156 Heat shock-related 70 kDa protein 2 (Heat 0 1 0 0 P17879 Heat shock 70 kDa protein 1B (HSP70.1) 0 1 0 0 393 Q61696 Heat shock 70 kDa protein 1A (Heat shock 70 0 1 0 0 Q99KD7 Heat shock protein 2 0 1 0 0 Q9R2A1 Spermatid-specific heat shock protein 70 0 1 0 0 394 Q61645 Protein hairless 0 1 0 0 395 P48774 Glutathione S-transferase Mu 5 (EC 2.5.1.18) 0 1 0 0

Supplementary Data to Rybak et al. Page 15 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 396 Q80YV3 Transformation/transcription 0 1 0 0 397 Q8BVW3 Tripartite motif-containing protein 14 0 1 0 0 398 Q7TQK5 Coiled-coil domain-containing protein 93 0 1 0 0 399 P70404 Isocitrate dehydrogenase [NAD] subunit 0 1 0 0 400 Q80UK7 Spindle assembly abnormal protein 6 0 1 0 0 401 Q9WU42 Nuclear receptor corepressor 2 (N-CoR2) 0 1 0 0 402 Q6P5C8 0 1 0 0 403 O54882 PK-120 0 1 0 0 404 P62717 60S ribosomal protein L18a 0 1 0 0 405 Q3TFU6 13 days embryo liver cDNA, RIKEN 0 1 0 0 406 Q3U560 2 days neonate thymus thymic cells cDNA, 0 1 0 0 407 Q3UAM9 Bone marrow macrophage cDNA, RIKEN 0 1 0 0 408 Q5NC91 Novel histone H2A family member 0 1 0 0 409 Q60847 Collagen alpha-1(XII) chain precursor 0 1 0 0 410 Q63961 Endoglin precursor (Cell surface MJ7/18 0 1 0 0 411 Q8K100 Endoglin 0 1 0 0 412 Q9DBK8 Adult male liver cDNA, RIKEN full-length 0 1 0 0 413 P62245 40S ribosomal protein S15a 0 1 0 0 414 Q3UT79 2 cells egg cDNA, RIKEN full-length enriched 0 1 0 0 415 Q9CQF0 39S ribosomal protein L11, mitochondrial 0 1 0 0 416 P19221 Prothrombin precursor (EC 3.4.21.5) 0 1 0 0 417 P24472 Glutathione S-transferase A4 (EC 2.5.1.18) 0 1 0 0 418 Q3URQ7 7 days embryo whole body cDNA, RIKEN 0 1 0 0 419 Q5FWI3 Transmembrane protein 2 0 1 0 0 420 Q80WS4 Mthfsd protein 0 1 0 0 421 Q8BI46 13 days embryo heart cDNA, RIKEN 0 1 0 0 422 Q8VDD5 -9 (Myosin heavy chain, nonmuscle 0 1 0 0 423 Q9CSJ3 10, 11 days embryo whole body cDNA, 0 1 0 0 424 O35227 ADAM 7 precursor (A disintegrin and 0 1 0 0 425 Q6S9I2 HMW-kininogen-II variant 0 1 0 0 Q6S9I3 HMW kininogen-II 0 1 0 0 426 Q80Y86 Mitogen-activated protein kinase 15 (EC 0 1 0 0 427 Q811K4 Alanine-glyoxylate aminotransferase 2-like 1 0 1 0 0 428 Q8BZ13 Adult female vagina cDNA, RIKEN full-length 0 1 0 0 429 Q8C8C2 10 days neonate cerebellum cDNA, RIKEN 0 1 0 0 430 Q3V0M8 Adult male testis cDNA, RIKEN full-length 0 1 0 0 431 Q641K3 Col2a1 protein 0 1 0 0 Q80VY3 Col2a1 protein 0 1 0 0 Q80X38 Col2a1 protein 0 1 0 0 432 Q80XH2 Interphotoreceptor matrix proteoglycan 2 0 1 0 0 Q810Y3 Sialoprotein associated with cones and rods 0 1 0 0 Q8C8X6 Adult retina cDNA, RIKEN full-length 0 1 0 0 433 Q01320 DNA topoisomerase 2-alpha (EC 5.99.1.3) 0 1 0 0 434 Q64511 DNA topoisomerase 2-beta (EC 5.99.1.3) 0 1 0 0 435 Q61543 Golgi apparatus protein 1 precursor (Golgi 0 1 0 0 436 Q6JIZ0 Acetyl-CoA carboxylase 2 1 0 2 3 437 Q3T9S7 Activated spleen cDNA, RIKEN full-length 6 8 2 3 438 Q91ZA3 Propionyl-CoA carboxylase alpha chain, 5 8 2 3 439 Q6JIZ1 Acetyl-CoA carboxylase 1 1 8 2 3 440 Q99MR8 Methylcrotonoyl-CoA carboxylase subunit 5 8 2 3 441 P02088 subunit beta-1 (Hemoglobin 1 6 2 2 Q9R0S6 Beta-1- (Fragment) 1 6 2 2 442 P02089 -2 (Hemoglobin 1 5 2 2 443 Q5D0E8 Hbb-b2 protein 1 5 2 1

Supplementary Data to Rybak et al. Page 16 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 444 Q91V86 11 days embryo whole body cDNA, RIKEN 1 3 2 1 445 P02104 Hemoglobin subunit epsilon-Y2 (Hemoglobin 1 2 2 1 446 P16460 Argininosuccinate synthase (EC 6.3.4.5) 5 7 2 0 447 Q8C196 Carbamoyl-phosphate synthase [ammonia], 5 1 2 0 448 Q63836 Selenium-binding protein 2 (56 kDa 5 1 2 0 449 P17563 Selenium-binding protein 1 (56 kDa 3 1 2 0 Q91X87 Selenium binding protein 1 3 1 2 0 450 P04104 , type II cytoskeletal 1 (-1) 2 8 1 3 451 Q6IFZ6 Keratin, type II cytoskeletal 1b (Type II keratin 2 8 1 3 Q6IFZ9 Type II keratin Kb37 2 8 1 3 Q6NXH9 Keratin 73 (Type II keratin Kb36) 2 8 1 3 Q9D2K8 0 day neonate head cDNA, RIKEN full-length 2 8 1 3 Q9R0H5 Keratin, type II cytoskeletal 6G 2 8 1 3 452 Q9D518 Uncharacterized protein C15orf33 homolog 1 2 1 1 453 P01942 (Hemoglobin alpha 0 6 1 1 Q9CY10 13 days embryo liver cDNA, RIKEN 0 6 1 1 454 Q6U1M1 Potassium channel KCNH3 (Fragment) 0 2 1 1 Q9WVJ0 Potassium voltage-gated channel subfamily 0 2 1 1 455 P54869 Hydroxymethylglutaryl-CoA synthase, 4 0 1 0 456 Q2MHN3 ZFH-5 0 0 1 0 457 Q9QXF8 Glycine N-methyltransferase (EC 2.1.1.20) 5 2 1 0 458 Q2MHN3 ZFH-5 0 0 1 0 459 P11679 Keratin, type II cytoskeletal 8 (Cytokeratin-8) 0 0 0 3 460 Q0VDN0 Krt78 protein (Fragment) 0 0 0 3 Q6IFT3 Keratin Kb40 0 0 0 3 461 P11679 Keratin, type II cytoskeletal 8 (Cytokeratin-8) 0 0 0 3 462 Q0VDN0 Krt78 protein (Fragment) 0 0 0 3 Q6IFT3 Keratin Kb40 0 0 0 3 463 Q3TTL3 Adult male testis cDNA, RIKEN full-length 0 1 0 3 Q80VP2 Spermatogenesis-associated protein 7 0 1 0 3 464 P47738 Aldehyde dehydrogenase, mitochondrial 2 0 0 1 465 Q80VP7 Similar to keratin complex 2, basic, gene 6b 1 0 0 1 466 Q99K87 Serine hydroxymethyl transferase 2 0 0 0 1 467 P50446 Keratin, type II cytoskeletal 6A 0 0 0 1 Q0VDR7 0 0 0 1 Q8BGZ7 10 days neonate skin cDNA, RIKEN 0 0 0 1 Q922U2 Keratin, type II cytoskeletal 5 (Cytokeratin-5) 0 0 0 1 Q9Z331 Keratin, type II cytoskeletal 6B 0 0 0 1 468 P02535 Keratin, type I cytoskeletal 10 0 0 0 1 Q8BVU3 0 day neonate head cDNA, RIKEN full-length 0 0 0 1 469 P38647 Stress-70 protein, mitochondrial precursor (75 0 0 0 1 470 Q31119 E beta 2 precursor (Fragment) 0 0 0 1 Q3U060 Activated spleen cDNA, RIKEN full-length 0 0 0 1 471 Q3TTY5 10 days neonate skin cDNA, RIKEN 0 0 0 1 472 Q6EJB6 U3 small nucleolar RNA-associated protein 0 0 0 1 473 Q99KG7 Hermansky-Pudlak syndrome 4 protein 0 0 0 1 474 Q9QY56 Transcription factor phi AP3 0 0 0 1 475 Q8K2E4 CDNA sequence BC031593 0 0 0 1 476 Q3UVR3 Tau-tubulin kinase 2 0 0 0 1 477 Q61414 Keratin, type I cytoskeletal 15 0 0 0 1 Q61781 Keratin, type I cytoskeletal 14 0 0 0 1 478 Q8VCW2 Keratin 25 0 0 0 1 479 Q9EQD6 Keratin 16a 0 0 0 1 Q9Z320 C29 0 0 0 1

Supplementary Data to Rybak et al. Page 17 of 21 †Neg. †Neg. *Peptide †Liver †Tumor control control Acc. # Protein name set no. (Total=6) (Total=8) liver tumor (total=2) (total=3) 480 P07724 Serum precursor 5 8 0 1 481 Q8VCM7 Fibrinogen gamma chain precursor 4 8 0 1 482 P16858 Glyceraldehyde-3-phosphate dehydrogenase 5 7 0 1 483 Q6IWE3 Glyceraldehyde-3-phosphate-dehydrogenase 0 1 0 1 484 Q99K87 Serine hydroxymethyl transferase 2 0 0 0 1 485 P50446 Keratin, type II cytoskeletal 6A 0 0 0 1 Q0VDR7 Keratin 6B 0 0 0 1 Q8BGZ7 10 days neonate skin cDNA, RIKEN 0 0 0 1 Q922U2 Keratin, type II cytoskeletal 5 (Cytokeratin-5) 0 0 0 1 Q9Z331 Keratin, type II cytoskeletal 6B 0 0 0 1 486 P02535 Keratin, type I cytoskeletal 10 0 0 0 1 Q8BVU3 0 day neonate head cDNA, RIKEN full-length 0 0 0 1 487 P38647 Stress-70 protein, mitochondrial precursor (75 0 0 0 1 488 Q31119 E beta 2 precursor (Fragment) 0 0 0 1 Q3U060 Activated spleen cDNA, RIKEN full-length 0 0 0 1 489 Q3TTY5 10 days neonate skin cDNA, RIKEN 0 0 0 1 490 Q6EJB6 U3 small nucleolar RNA-associated protein 0 0 0 1 491 Q99KG7 Hermansky-Pudlak syndrome 4 protein 0 0 0 1 492 Q9QY56 Transcription factor phi AP3 0 0 0 1 493 Q8K2E4 CDNA sequence BC031593 0 0 0 1 494 Q3UVR3 Tau-tubulin kinase 2 0 0 0 1 495 Q61414 Keratin, type I cytoskeletal 15 0 0 0 1 Q61781 Keratin, type I cytoskeletal 14 0 0 0 1 496 Q8VCW2 Keratin 25 0 0 0 1 497 Q9EQD6 Keratin intermediate filament 16a 0 0 0 1 Q9Z320 C29 0 0 0 1 *A peptide set is defined as a group of peptides matched by the Mascot software to a particular protein. In some cases, the same peptide set can be matched to more than one protein. †Numbers indicate in how many of the 6 healthy in vivo biotinylated, the 8 metastases-bearing in vivo biotinylated, the 2 healthy unperfused control or the 3 metastases-bearing unperfused control mice, resepectively, the protein was identified in healthy liver or liver tumor specimens.

Supplementary Data to Rybak et al. Page 18 of 21 Supplementary Table 2. Selection of proteins found in both metastasis and normal liver, for which more peptides were identified in metastasis specimens.

*No. of different peptides identified in Acc. # Protein Name Only Only Liver & Total Metastasis Liver Metastasis P11276 Fibronectin precursor 38 1 11 50 Q99K47 Fibrinogen, alpha polypeptide 20 1 21 Q8K0E8 Fibrinogen beta chain precursor 18 1 19 Q921I1 Serotransferrin precursor 17 5 22 Q00896 Alpha-1-antitrypsin 1-3 precursor 15 2 17 †P07724 Serum albumin precursor 8 1 9 18 †Q8VCM7 Fibrinogen gamma chain precursor 8 3 11 P11499 Heat shock protein HSP 90-beta 7 3 10 P58252 Elongation factor 2 6 2 2 10 P07901 Heat shock protein HSP 90-alpha 6 3 9 P07759 Serine protease inhibitor A3K precursor 6 2 8 P68372 Tubulin beta-2C chain 6 1 7 Q05793 Perlecan 9 1 10 P11087 Collagen alpha-1(I) chain precursor 5 5 10 Q04857 Collagen alpha-1(VI) chain precursor 4 2 6 P07309 Transthyretin precursor 4 1 5 P63017 Heat shock cognate 71 kDa protein 3 1 4 P09055 Fibronectin receptor subunit beta 3 1 4 Q01149 Collagen alpha-2(I) chain precursor 2 1 2 5 Q9Z0I9 Collagen alpha3(VI) precursor 2 1 2 5 P08122 Collagen alpha-2(IV) chain precursor 2 2 4 Q80WC8 Hist2h4 protein 2 2 4 P62631 Elongation factor 1-alpha 2 2 1 3 P60710 Actin, cytoplasmic 1 1 3 4 O55107 Basigin 1 1 2 *Numbers indicate how many different peptides of the protein were identified only in metastasis specimens, only in liver specimens or in both types of specimens, respectively. †For these proteins each 1 peptide was also identified in 1 metastasis negative control sample from an unbiotinylated mouse, presumably reflecting the high abundance of these proteins in metastasis tissue.

Supplementary Data to Rybak et al. Page 19 of 21 Supplementary Table 3. Fibronectin peptides identified in normal liver and/or metastasis.

Seq. Position *Liver *Metastasis Sequence Start End (Total = 6) (Total = 8) HYQINQQWER 59 68 6 VGDTYERPK 109 117 4 HALQSASAGSGSFTDVR 274 290 7 IGDQWDK 480 486 1 TFYQIGDSWEK 568 578 1 WKEATIPGHLNSYTIK 654 669 2 EATIPGHLNSYTIK 656 669 1 GLTPGVIYEGQLISIQQYGHR 670 690 7 WSRPQAPITGYR 830 841 2 3 SDNVPPPTDLQFVELTDVK 903 921 3 VTIMWTPPDSVVSGYR 922 937 8 VEVLPVSLPGEHGQR 938 952 8 NTFAEITGLSPGVTYLFK 958 975 7 VFAVHQGR 976 983 7 TVLVTWTPPR 1011 1020 2 8 QYNVGPLASK 1040 1049 4 NLQPGSEYTVTLVAVK 1054 1069 6 ATGVFTTLQPLR 1077 1088 1 8 LGVRPSQGGEAPR 1116 1128 7 VVTPLSPPTNLHLEANPDTGVLTVSWER 1169 1196 3 STTPDITGYR 1197 1206 7 VTWAPPPSIELTNLLVR 1375 1391 2 7 TGLDSPTGFDSSDITANSFTVHWVAPR 1446 1472 4 APITGYIIR 1473 1481 1 8 HHAEHSVGRPR 1482 1492 1 EESPPLIGQQATVSDIPR 1525 1542 8 ITYGETGGNSPVQEFTVPGSK 1570 1590 2 8 SPVQEFTVPGSK 1579 1590 6 STATINNIKPGADYTITLYAVTGR 1591 1614 5 GDSPASSKPVSINYK 1615 1629 4 TEIDKPSQMQVTDVQDNSISVR 1630 1651 8 WLPSTSPVTGYR 1652 1663 7 TASPDQTEMTIEGLQPTVEYVVSVYAQNR 1679 1707 7 †NGESQPLVQTAVTTIPAPTNLK 1708 1819 3 ‡NGESQPLVQTAVTNIDRPK 1708 1726 1 ‡IAWESPQGQVSR 1740 1751 8 ‡VTYSSPEDGIR 1754 1764 1 FSQVTPTSFTAQWIAPSVQLTGYR 1820 1843 1 5 YEVSVYALK 1878 1886 2 TKTETITGFQVDAIPANGQTPVQR 1926 1949 1 2 TETITGFQVDAIPANGQTPVQR 1928 1949 3 SYTITGLQPGTDYK 1957 1970 7 IHLYTLNDNAR 1971 1981 7 SSPVIIDASTAIDAPSNLR 1982 2000 3 8 FLTTTPNSLLVSWQAPR 2001 2017 4 5 ITGYIIK 2020 2026 5 YEKPGSPPR 2027 2035 6 §PYLPNVDEEVQIGHVPR 2165 2181 7 GVTYNIIVEALQNQR 2255 2269 4 7 RPGAAEPSPDGTTGHTYNQYTQR 2425 2447 2 *Numbers indicate in how many of the 6 healthy in vivo biotinylated mice or the 8 metastases-bearing in vivo biotinylated mice the peptide was identified in the corresponding tissue samples. All peptides are listed here which had been annotated by the Mascot software to the fibronectin database entries P11276, Q3UHL6 or Q3TCF1. †This pepetide covers a fibronectin sequence portion before AND after the EDA domain, indicating the presence of an (EDA–)-fibronectin isoform. ‡These peptides match to the sequence of the EDA domain (Seq. positions 1721 – 1810). §This peptide matches to the sequence of the IIICS stretch (Seq. positions 2082 – 2201).

Supplementary Data to Rybak et al. Page 20 of 21 Supplemental Figure 1. Expression of the extra-domain A of fibronectin (EDA) in human lung cancer. EDA is abundantly expressed in all important subtypes of lung cancer - the most common cancer in the world*. *References: Beadsmoore and Screaton, European Journal of Radiology 45 (2003) 8-17; Pirozynski, Resppyiratory Medicine 100 (2006) 2073-2084; Skarin (ed.), The Atlas of Diagnostic Oncology, Mosby-Wolfe (1996) 63-100. Percentage values refer to the number of cases of the specified subtype per 100 cases of any lung cancer. Lung cancer has been the most common cancer in the world since 1985. The annual number of new cases was estimated to be 12.4% of all new cancer cases and was estimated at 1.04 million in 1990 and 1.35 million in 2002.

Classification of lung cancer Immunohistochemical detection of and contribution of subtypes to EDA in tissue sections from lung all lung cancer cases (%)* tumors of the different subtypes

Small Cell Carcinoma (~15-25%)*

Squamous Cell Carcinoma (~30%)*

Adeno- Carcinoma (~30-50%)*

Non-Small Cell Lung Cancer Bronchio- (~75-85%)* alveolar Carcinoma (~2-10%)*

Large Cell Carcinoma (~10%)*

Supplementary Data to Rybak et al. Page 21 of 21