Supporting Information for Proteomics DOI 10.1002/prca.200780101 Paul Cutler, Emma L. Akuffo, Wanda M. Bodnar, Deborah M. Briggs, John B. Davis, Christine M. Debouck, Steven M. Fox, Rachel A. Gibson, Darren A. Gormley, Joanna D. Holbrook, A. Jacqueline Hunter, Emma E. Kinsey, Rabinder Prinjha, Jill C. Richardson, Allen D. Roses, Marjorie A. Smith, Nikos Tsokanas, David R. Will, Wen Wu, John W. Yates and Israel S. Gloger Proteomic identification and early validation of complement 1 inhibitor and pigment epithelium-derived factor: Two novel biomarkers of Alzheimer’s disease in human plasma ª 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com Supplementary Table 1: Complete list of proteins identified from spots derived from 2D gel analysis of human plasma. Each protein was observed to be in a spot showing altered expression between Alzheimer’s disease and matched control by statistical methods as described in the Methods section. Each protein is identified by the gene description and the HUGO gene symbol. The number of “changing” spots in which this protein was observed is also given. HUGO Human Gene Number of Gene Description Symbol Spots alpha-1-B glycoprotein; A1BG 5 alpha-2-macroglobulin A2M 7 afamin; AFM 1 angiotensinogen (serpin peptidase inhibitor, clade A, member 8) AGT 4 alpha-2-HS-glycoprotein AHSG 3 albumin ALB 90 alpha-1-microglobulin/bikunin precursor; AMBP 1 annexin A1 ANXA1 1 amyloid P component, serum APCS 3 apolipoprotein A-I APOA1 14 apolipoprotein A-IV APOA4 2 apolipoprotein E APOE 2 apolipoprotein H (beta-2-glycoprotein I) APOH 3 apolipoprotein L, 1 APOL1 1 alpha-2-glycoprotein 1, zinc AZGP1 3 complement component 1, r subcomponent C1R 5 complement component 3 C3 13 complement component 4A C4A 10 complement complement 4B C4B 10 complement component 6 C6 2 complement component 9; C9 1 complement component B CFB 1 complement factor H CFH 2 caspase 14, apoptosis-related cysteine peptidase CASP14 1 clusterin (apolipoprotein J) CLU 7 collagen, type VI, alpha 3 COL6A3_V1 6 ceruloplasmin (ferroxidase) CP 6 cystatin SA CST2 1 coagulation factor X F10 2 coagulation factor XIII, B polypeptide F13B 2 epidermal growth factor receptor EGFR 2 ficolin (collagen/fibrinogen domain containing) 3 FCN3 1 fibrinogen alpha chain FGA 24 fibrinogen beta chain FGB 10 fibrinogen gamma chain FGG 16 group-specific component (vitamin D binding protein) GC 8 glutathione peroxidase 3 GPX3 1 gelsolin GSN 8 complement factor H HF1 2 histone 1, H2bb HIST1H2BB 1 haptoglobin HP 4 haptoglobin-related protein HPR 7 hemopexin HPX 14 isocitrate dehydrogenase 3 (NAD+) alpha; IDH3A 1 I factor (complement) IF 1 insulin-like growth factor binding protein, acid labile subunit; IGFALS 1 immunoglobulin heavy constant alpha 1 IGHA1 33 immunoglobulin heavy constant gamma 1 (G1m marker); IGHG1 19 immunoglobulin heavy variable 3-11 IGHV3-11 1 immunoglobulin heavy variable 4-4 IGHV4-4 12 immunoglobulin kappa constant IGKC 1 immunoglobulin lambda joining 3 IGLJ3 1 inter-alpha (globulin) inhibitor H1 ITIH1 1 inter-alpha (globulin) inhibitor H2 ITIH2 2 inter-alpha (globulin) inhibitor H4 (plasma Kallikrein-sensitive glycoprotein) ITIH4 8 kininogen 1 KNG 9 leucine-rich alpha-2-glycoprotein 1 LRG 2 lumican LUM 1 neurofilament 3 (150kDa medium) NEF3 1 orosomucoid 2 ORM2 1 protease, serine, 1 (trypsin 1) PRSS1 6 PTX1 protein; PTX1 1 retinol binding protein 4, plasma RBP4 2 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 SERPINA1 40 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 SERPINA3 10 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 6 SERPINA6 2 serpin peptidase inhibitor, clade C (antithrombin), member 1 SERPINC1 12 serpin peptidase inhibitor, clade D (heparin cofactor), member 1 SERPIND1 1 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor) member 1 SERPINF1 1 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor) member 2 SERPINF2 4 serpin peptidase inhibitor, clade G (C1 inhibitor), member 1, (angioedema, hereditary) SERPING1 13 transferrin TF 26 C-type lectin domain family 3, member B; TNA 3 transthyretin (prealbumin, amyloidosis type I) TTR 3 vitronectin (serum spreading factor, somatomedin B, complement S-protein) VTN 1 Supplementary Table 2: Complete list of proteins for which peptides were identified, derived from the non-gel based LC/MS/MS analysis of human plasma. Each protein was observed to show altered expression between Alzheimer’s disease and matched control by statistical methods as described in the Methods section. Each protein is identified by the gene description and the HUGO gene symbol. The number of “changing” peptides observed for each protein is also given. HUGO Human Gene Number of Gene Description Symbol peptides albumin ALB 7 alpha-1-microglobulin/bikunin precursor AMBP 1 apolipoprotein A-I APOA1 1 apolipoprotein B (including Ag(x) antigen) APOB 1 asp (abnormal spindle)-like, microcephaly protein ASPM 2 B-factor, properdin BF 2 complement component 3 C3 7 C-reactive protein, pentraxin-related CRP 1 coagulation factor II (thrombin) F2 2 group-specific component (vitamin D binding protein) GC 2 gelsolin GSN 3 hemopexin HPX 1 immunoglobulin kappa variable 1-9 IGKV1-9 1 inter-alpha (globulin) inhibitor H2 ITIH2 1 similar to Zinc-alpha-2-glycoprotein precursor (Zn-alpha-2-glycoprotein) (Zn-alpha-2-GP) LOC401393 1 lumican LUM 3 pregnancy-zone protein PZP 2 serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2 SERPINF2 2 transferrin TF 3 vitronectin (serum spreading factor, somatomedin B, complement S-protein) VTN 1 Supplementary Table 3: Complete list of proteins identified from spots derived from 2D gel analysis of mouse plasma. Each protein was observed to be in a spot showing altered expression between the transgenic mouse model of amyloidosis and wild type by statistical methods as described in the Methods section. Each protein is identified by the gene description and the HUGO gene symbol is given for the corresponding human orthologue. The number of “changing” spots in which this protein was observed is also given. HUGO Human Gene Symbol of orthologue Number of Gene Description (where known) Spots adiponectin ADIPOQ 2 afamin AFM 1 alpha-1-antitrypsin SERPINA1 126 alpha-2-antiplasmin SERPINF2 1 alpha-2-globulin 4 alpha-2-macroglobulin A2M 20 angiotensinogen AGT 1 antigen-like precursor (apoptosis inhibitory 6) CD5L 1 antithrombin-III SERPINC1 22 apolipoprotein E APOE 1 apolipoprotein AI APOA1 9 apolipoprotein AIV APOA4 9 apolipoprotein CIII APOC3 1 arsenical pump-driving ATPase ASNA1 1 C4B-binding protein C4BPA 1 clusterin (apo-J). CLU 1 complement 6 C6 1 complement C1 C1S 1 complement C1r C1R 1 complement C3 C3 7 complement C8 C8A 4 complement factor H CFH 1 complement factor I CFI 1 esterase 1 1 factor X F10 6 fetua_alpha-2-HS-glycoprotein AHSG 11 fetuin-B FETUB 8 fibrinogen beta FGB 12 fibrinogen gamma FGG 9 fibrinogen, alpha polypeptide FGA 3 fibronectin FN1 3 gamma actin-like protein ACTG1 1 gelsolin GSN 5 glutathione peroxidase GPX3 2 H-2 class I histocompatibility antigen, Q10 alpha chain 2 haptoglobin HP 5 heat shock 70 kDa protein 4 HSPA4 1 haemoglobin HBB 1 haemoglobin alpha chain HBA2 1 hemopexin HPX 14 histidine-rich glycoprotein HRG 1 histone H2B 1 hypothetical protein psyr03003113 1 inter alpha-trypsin inhibitor. heavy chain 4 ITIH4 4 kallikrein KLKB1 1 keratin KRT1 1 keratin type II KRT81 1 keratin. type II cytoskeletal 6G KRT71 1 kininogen KNG1 6 known chromosome CP 1 Krt2-5 protein. (keratin 5) KRT5 1 leukemia inhibitory factor receptor LIFR 4 liver carboxylesterase N 2 major urinary protein 1 11 p73 alpha protein TP73 1 paraoxonase/arylesterase 1 (pon1) PON1 2 PPBP - pro-platelet basic protein (chemokine (c- x-c motif) ligand 7) CXCL7 1 prothrombin F2 2 Q7UPPPA_RHOBA hypothetical protein; histidine-rich glycoprotein 1 serine proteinase inhibitor a3c 1 serotransferrin TF 23 serpin A3K (contraspin) 24 serpin A3K SERPINA10 1 serum albumin ALB 25 similar to alpha-2u-globulin v-mouse. 2 similar to shroom-related protein; f-actin-binding protein SHROOM3 1 SPI2 proteinase inhibitor SERPINA3 1 thymus chemokine 1 (chemokine subfamily b cys-x-cys) PPBP 2 transthyretin TTR 3 tropomyosin beta chain TPM2 1 type II keratin KRT77 1 type II keratin kb36. KRT73 1 vitamin D-binding protein GC 6 vitronectin VTN 2 zinc-alpha-2-glycoprotein AZGP1 1 Supplementary Table 4: Proteins identified from spots or peptides derived as described in Supplmentary Tables 1 and 2 for which follow up assay confirmation was performed. Sequences identified by mass spectrometry are given for each protein. Human Plasma by 2DE HUGO Number Human of Spots Gene Gene Description Symbol alpha-2-macroglobulin A2M 7 AAQVTIQSSGTFSSK ALLAYAFALAGNQDK LLLQQVSLPELPGEYSMK + Oxidation (M) MVSGFIPLKPTVK + Oxidation (M-64) NEDSLVFVQTDK NEDSLVFVQTDK + Deamidation (NQ) QFSFPLSSEPFQGSYK QGIPFFGQVR SASNMAIVDVK + Oxidation (M) SSGSLLNNAIK SSSNEEVMFLTVQVK + Oxidation (M) VGFYESDVMGR + Oxidation (M) VSVQLEASPAFLAVPVEK LPPNVVEESAR FEVQVTVPK angiotensinogen AGT 4 AAMVGMLANFLGFR + 2 Oxidation (M) ADSQAQLLLSTVVGVFTAPGLHLK ALQDQLVLVAAK DPTFIPAPIQAK QPFVQGLALYTPVVLPR + Deamidation (NQ) SLDFTELDVAAEK VGEVLNSIFFELEADER VLSALQAVQGLLVAQGR apolipoprotein A-I APOA1 14 AKPALEDLR ATEHLSTLSEK VSFLSALEEYTK QGLLPVLESFK DLATVYVDVLK VQPYLDDFQK DYVSQFEGSALGK LSPLGEEMR + Oxidation (M) AELQEGAR apolipoprotein E APOE 2