Defining the elastic fiber interactome Supplemental Information Defining elastic fiber interactions by molecular fishing: an affinity purification and mass spectrometry approach Stuart A. Cain*, Amanda McGovern, Elaine Small, Lyle J. Ward, Clair Baldock, Adrian Shuttleworth and Cay M. Kielty Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, United Kingdom. *Corresponding author Supplemental Fig. 1 CPL - + - + - + DTT 250 150 100 75 50 37 Supplemental Fig.1 4-12% bis-Tris gel showing purified calsyntenin-1 (C), proepithelin (P) and LOX (L), with (+) and without (-) reduction with DTT. 1 Defining the elastic fiber interactome SUPPLEMENTAL TABLE 1A Peptides detected no-bait protein controls No-bait protein (bead only) controls were carried out for all four cell compartments Samples were processed as described in the Experimental Procedures, with the exception that no bait protein was added. Scaffold was used to validate protein and peptide identification. Only proteins with >2 unique peptide sequences were counted, and each peptide had greater than 95.0% probability as specified by the Peptide Prophet algorithm (38). Shown is the number of peptides detected,with the number of unique peptide sequence number in brackets. Also shown is the experiment number for which the peptides were detected. Experiments were numbered in the sequence they were conducted and are labelled with the prefix 'ex'. SwissProt Entry ARPE Matrix ARPE Media HDF Matrix HDF Media Protein Protein Identitiy Name ex11 ex169 ex173 ex167 ex171 ex12 ex157 ex158 ex165 ex155 ex163 ex8 Total Fibronectin FINC_HUMAN 2 (2) 2 (2) Histone H3.3 H33_HUMAN 3 (2) 3 (2) Histone H4 H4_HUMAN 12 (7) 12 (7) Keratin, type I cytoskeletal 9 K1C9_HUMAN 9 (8) 9 (8) Keratin, type II cytoskeletal 1 K2C1_HUMAN 7 (7) 7 (7) Lymphoid-restricted membrane protein LRMP_HUMAN 2 (2) 2 (2) Protocadherin-19 PCD19_HUMAN 2(2) 2 (2) Vimentin VIME_HUMAN 14 (13) 14 (13) Grand Total 33 (26) 000002 (2)0016 (15) 0 0 51 (43) SUPPLEMENTAL TABLE 1B Peptides detected cell culture compartment controls Controls were carried out for all four cell compartments Samples were processed as described in the Experimental Procedures, with the exception that no bait protein was added and the samples did not undergo affinity capture LC . Scaffold was used to validate protein and peptide identification. Only proteins with >2 unique peptide sequences were counted, and each peptide had greater than 95.0% probability as specified by the Peptide Prophet algorithm (38). Shown is the number of peptides detected (indicated Pep. No), with the number of unique peptide sequence number in brackets . Also shown is the % coverage for each protein. Protein Identity SwissProt Entry ARPE Matrix ARPE Media HDF Matrix HDF Media Name Pep. No. % Cov Pep. No. % Cov Pep. No. % Cov Pep. No. % Cov Serum albumin ALBU_BOVIN 16 (6) 8.6 118 (20) 29 45 (10) 17 97 (14) 21 Actin, cytoplasmic ACTB_HUMAN 63 (12) 36 15 (5) 18 Vimentin VIME_HUMAN 13 (9) 22 43 (20) 44 Alpha-2-HS-glycoprotein FETUA_BOVIN 2 (2) 4.7 7 (4) 18 10 (4) 11 Serum albumin ALBU_HUMAN 11 (2) 3.1 Keratin, type II cytoskeletal 1 K2C1_HUMAN 6 (4) 12 14 (7) 14 Alpha-1-antiproteinase A1AT_BOVIN 4 (3) 8.4 Keratin, type I cytoskeletal 18 K1C18_HUMAN 13 (8) 18 Keratin, type I cytoskeletal 10 K1C10_HUMAN 6 (3) 6.7 6 (3) 4.7 Histone H4 H4_HUMAN 9 (5) 48 Keratin, type II cytoskeletal 2 epidermal K22E_HUMAN 3 (3) 3.6 Serotransferrin TRFE_BOVIN 5 (4) 4.7 Keratin, type II cytoskeletal 8 K2C8_HUMAN 4 (3) 6.4 Keratin, type II cytoskeletal 7 K2C7_HUMAN 4 (4) 5.1 Heterogeneous nuclear ribonucleoproteins A2/B1 ROA2_BOVIN 3 (2) 12 Supplemental Table 1 2 Defining the elastic fiber interactome SUPPLEMENTAL TABLE 2 Total number of peptides and unique peptide sequences for each protein detected after Scaffold validation Scaffold was used to validate protein and peptide identification. Only proteins with >2 unique peptide sequences were counted and each peptide had greater than 95.0% probability as specified by the Peptide Prophet algorithm (38). The proteins were then ranked by total number of peptides detected. The cell culture compartments in which the identified peptides were detected are also given. Also shown is the number of unique peptide sequence in brackets, and the total % coverage of the unique peptide sequences (calculated as described in Experimental Procedures). Peptides detected from bait proteins were included (shown in bold). The global % ubiquity was calculated for each detected protein using BEPro3. Proteins with a % ubiquity greater than 40% were treated as non-specific contaminants (indicated *). The sequences of the peptides identified for each protein are shown in Supplemental Table 4. Protein Identity SwissProt Entry ARPE Matrix ARPE Media HDF Matrix HDF Media Protein Peptide Total % Ubiquity Name Pep. No. % Cov Pep. No. % Cov Pep. No. % Cov Pep. No. % Cov Pep. No. % Cov Fibrillin-1 FBN1_HUMAN 394 (76) 35.6 760 (115) 55.8 334 (58) 26.6 649 (118) 56.5 2137 (145) 65.8 51.4 Keratin type II cytoskeletal 1 * K2C1_HUMAN 167 (34) 42.9 130 (25) 38.7 154 (32) 41.1 181 (33) 43.6 632 (42) 49.7 86.9 Plasminogen activator inhibitor 1 PAI1_HUMAN 33 (15) 49.3 320 (21) 65.4 2 (2) 6.0 54 (18) 60.4 409 (22) 65.4 38.0 Perlecan PGBM_HUMAN 117 (71) 22.1 256 (96) 31.5 23 (20) 5.5 2 (2) 0.7 398 (113) 36.5 18.6 Keratin type I cytoskeletal 10 * K1C10_HUMAN 96 (32) 54.5 37 (20) 45.7 63 (21) 38.3 123 (31) 52.8 319 (40) 63.9 48.0 Keratin type I cytoskeletal 9 * K1C9_HUMAN 46 (16) 32.3 45 (15) 39.2 63 (14) 30.0 49 (21) 37.9 203 (25) 47.4 50.2 BigH3 BGH3_HUMAN 7 (6) 9.4 123 (23) 47.0 2 (2) 6.0 132 (23) 47.0 7.3 Histone H4. H4_HUMAN 66 (7) 52.4 18 (5) 50.5 32 (7) 52.4 12 (4) 42.7 128 (7) 52.4 14.6 Fibronectin FINC_HUMAN 2 (2) 0.7 57 (37) 23.6 26 (24) 14.8 19 (16) 10.2 104 (49) 30.6 17.8 Stanniocalcin-2 STC2_HUMAN 8 (6) 27.5 44 (6) 27.5 6 (6) 27.5 45 (6) 24.8 103 (7) 30.1 38.2 Thrombospondin-1 TSP1_HUMAN 93 (28) 33.2 5 (4) 6.0 98 (28) 33.2 2.0 Proepithelin GRN_HUMAN 21 (11) 21.8 46 (15) 32.2 5 (4) 9.6 17 (9) 16.5 89 (19) 34.2 8.2 Keratin type II * K22E_HUMAN 15 (18) 20.0 6 (9) 15.8 25 (22) 30.1 41 (27) 35.0 87 (36) 45.0 48.6 Sulfhydryl oxidase 1 QSOX1_HUMAN 67 (28) 47.8 5 (4) 8.3 72 (29) 50.3 0.4 Serum albumin ALBU_HUMAN 10 (7) 11.2 15 (5) 8.7 18 (8) 12.0 22 (8) 13.1 65 (12) 18.2 22.2 Collagen alpha-1(XVIII) chain COIA1_HUMAN 26 (10) 7.4 36 (13) 11.3 62 (15) 13.0 3.4 Lamin-A/C LMNA_HUMAN 17 (8) 14.6 42 (17) 31.6 59 (19) 34.5 8.1 Calsyntenin-1 CSTN1_HUMAN 29 (16) 19.9 16 (14) 18.1 45 (19) 24.1 2.9 Agrin AGRIN_HUMAN 42 (21) 12.8 42 (21) 12.8 6.4 Collagen alpha-1(I) chain CO1A1_HUMAN 5 (4) 4.6 36 (21) 24.1 41 (22) 25.3 11.4 Insulin-like growth factor-binding protein 7 IBP7_HUMAN 2 (2) 10.6 38 (11) 51.8 40 (11) 51.8 0.4 Keratin type I cytoskeletal 14 K1C14_HUMAN 2 (10) 10.0 2 (3) 7.4 15 (16) 26.1 18 (14) 24.2 37 (25) 38.1 8.7 Collagen alpha-1(IV) chain CO4A1_HUMAN 30 (8) 7.5 5 (4) 3.5 35 (8) 7.5 0.3 Insulin-like growth factor-binding protein 4 IBP4_HUMAN 2 (2) 11.2 30 (7) 31.0 32 (7) 31.0 0.4 MAGP-1 MFAP2_HUMAN 2 (2) 9.8 13 (5) 25.1 17 (6) 35.5 32 (6) 35.5 4.1 Collagen alpha-2(IV) chain CO4A2_HUMAN 31 (13) 16.1 0.0 31 (13) 16.1 0.4 Calsyntenin-2 CSTN2_HUMAN 30 (16) 21.4 30 (16) 21.4 2.3 Collagen alpha-2(I) chain CO1A2_HUMAN 28 (18) 23.4 28 (18) 23.4 3.3 Annexin A2 ANXA2_HUMAN 2 (2) 5.6 24 (15) 50.7 26 (15) 50.7 4.2 Keratin type II cytoskeletal 6 K2C6A_HUMAN 5 (4) 14.4 15 (12) 31.4 6 (4) 19.5 26 (20) 43.1 7.5 Insulin-like growth factor-binding protein 3 IBP3_HUMAN 21 (7) 24.4 4 (3) 13.1 25 (7) 24.4 5.4 TGF beta-2 TGFB2_HUMAN 3 (3) 8.5 22 (10) 30.9 25 (10) 30.9 0.6 Histone H2B H2B1M_HUMAN 18 (5) 52.4 4 (3) 27.8 2 (2) 15.9 24 (6) 52.4 2.4 Keratin type II cytoskeletal 5 K2C5_HUMAN 3 (3) 12.5 3 (3) 14.6 16 (14) 26.1 22 (14) 33.2 7.4 Retinoic acid receptor responder protein 2 RARR2_HUMAN 22 (5) 39.3 22 (5) 39.3 1.7 Myosin-9 MYH9_HUMAN 16 (12) 8.8 2 (2) 1.4 3 (3) 1.9 21 (13) 9.5 0.4 Protein NOV homolog NOV_HUMAN 21 (9) 36.1 21 (9) 36.1 0.4 Extracellular matrix protein 1 ECM1_HUMAN 20 (11) 29.1 20 (11) 29.1 0.3 Histone H2A type 2-A H2A2A_HUMAN 11 (4) 59.2 6 (4) 35.4 2 (2) 12.3 19 (5) 67.7 0.4 Collagen alpha-1(XII) chain COCA1_HUMAN 18 (15) 6.4 18 (15) 6.4 0.4 Titin TITIN_HUMAN 3 (3) 0.2 11 (11) 0.7 2 (2) 0.1 16 (16) 0.9 0.4 Keratin type I cytoskeletal K1C16_HUMAN 15 (10) 33.8 15 (10) 33.8 7.5 Supplemental Table 2 3 Defining the elastic fiber interactome Protein Identity SwissProt Entry ARPE Matrix ARPE Media HDF Matrix HDF Media Protein Peptide Total % Ubiquity Name Pep.