Supporting Information for Proteomics DOI 10.1002/pmic.200600056 Barbara Celegato, Daniele Capitanio, Mario Pescatori, Chiara Romualdi, Beniamina Pacchioni, Stefano Cagnin, Agnese Vigan, Luca Colantoni, Shajna Begum, Enzo Ricci, Robin Wait, Gerolamo Lanfranchi and Cecilia Gelfi Parallel protein and transcript profiles of FSHD patient muscles correlate to the D4Z4 arrangement and reveal a common impairment of slow to fast fibre differentiation and a general deregulation of MyoD-dependent genes ª 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Table A. List of genes discriminating among the two classes of patients with the same range of size deletion (EcoRI fragment > 21Kb), but differing by age at biopsy. NA: not available value. Table B. Mass spectrometry data of up and down regulated proteins of unclusterised patients, as shown in Figure 3. Table C. Mass spectrometry data of up and down regulated proteins of patients after gene transcripts cluster analysis. Table D. Common altered genes in FSHD muscle compared to a deltoideous control muscle are listed according to their biological process. Table E. Functionally classified transcripts with an increasing profile according to KpnI repeats number at D4Z4 locus. Single patient ratios and intra-class averaged ratios are reported. Table F. Functionally classified transcripts with a decreasing profile according to KpnI repeats number at D4Z4 locus. Single patient ratios and intra-class averaged ratios are reported. Table G. Ratios values of selected transcripts altered in FSHD muscle, suggesting an enrichment in slow-oxidative fibers in the most severely affected patients. Table H. MyoD target genes with an altered expression in FSHD muscle. Figure A. Hierarchical cluster analysis of the transcriptional expression profiles of 11 FSHD patients. Profiles were obtained using the Human Muscle Array 2.0 platform, which contains 4.801 muscle cDNA probes and are expressed as FSHD deltoid/control. Pearson correlation was used as distance measure. Statistical support for the nodes of the dendrogram is also shown in figure 1A and it is expressed as a fold number percent of a given node over the resampling trials. We chose the bootstrapping genes resampling procedure to build experiment dendrogram and 500 iterations. Patients are clearly divided into 4 groups, according to their EcoRI fragment length. Furthermore, patients with fragment longer than 20kb and age at biopsy >50 years (mean 57.7 +/- 10) are separated from younger (mean 25.7 +/- 5) patients with a comparable fragment size. Below, an enlargement of the global dendrogram showing a selection of the genes discriminating among the classes of patients. Figure B. Percent of differentially expressed genes in each class of FSHD patients, grouped by their biological function. Note that some functional categories are specifically affected in one class of patients. Figure C. Correlation between protein and transcript expression levels of myosin heavy chains (MYH1, MYH7, MYH2) averaged among patients of the same class. Table A Human Muscle Array 2.0 Expression ratios (FSHD/control) Averaged expression ratios Gene name and biological process Chr pos Archive ID Group A Group > 50 years Group A Group > 50 years 4 - 23 Kb 3 - 26 Kb 6 - 21 Kb 1 - 27 Kb 5 - 23 Kb 2 - 27 Kb Frgm > 21Kb Oxidative phosphorilation 2-001A09 cytochrome c oxidase subunit VIa, pol 2 (COX6A2) 16 -1.18 -1.18 1.09 1.2 1.18 1.14 -1.08 1.18 2-001G05 cytochrome c oxidase subunit VIIb (COX7B) X -1.04 -1.2 -1.01 1.24 1.1 1.11 -1.08 1.15 2-003E10 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1 (NDUFC1) 4 -1.11 -1.19 1.06 1.03 -1.05 1.25 -1.07 1.08 Protein biosynthesis 2-001D07 ribosomal protein S18 (RPS18) 6 -1.12 -1.23 1.14 -1 1.02 1.22 -1.05 1.08 2-001G01 ribosomal protein S8 (RPS8) 1 -1.08 -1.14 1.15 1.15 1.11 1.11 -1.02 1.12 2-018H06 mitochondrial ribosomal protein L46 (MRPL46) 15 -1.22 -1.22 -1.11 1.06 1.06 1.1 -1.18 1.07 2-018H02 aspartyl aminopeptidase (DNPEP) 2 -1.14 -1.14 -1.07 1.01 1.18 1.14 -1.12 1.11 Proteolysis 2-017D05 ubiquitin protein ligase E3A (UBE3A) 15 -1.33 -1.33 1.05 1.09 1.15 1.08 -1.18 1.1 2-029A02 cathepsin B (CTSB) 8 2.01 2.25 2.1 -2 -1.76 -1.94 2.12 -1.89 2-033C05 KIAA0317 gene product 14 2.35 1.89 2.11 -1.9 -1.42 -1.64 2.12 -1.63 Muscle contraction 2-008A02 creatine kinase, muscle (CKM) 19 -1.25 -1.16 -1.06 1.04 1.18 1.11 -1.15 1.11 2-021E11 tropomyosin 2 (beta) 9 2.35 2.19 1.9 -1.72 -1.4 -1.39 2.15 -1.49 2-021C11 tropomyosin 2 (beta) 9 2.29 1.95 1.82 -1.48 -1.56 -1.45 2.02 -1.49 2-001A05 titin-cap (telethonin) (TCAP) 17 2.28 1.88 1.84 -1.93 -1.33 -1.79 2 -1.64 2-010H06 troponin C2, fast (TNNC2) 20 -1.24 -1.2 -1.05 1.01 1.2 1.16 -1.16 1.12 2-022B02 troponin I, skeletal, fast (TNNI2) 11 2.7 1.95 1.64 -2.21 -1.22 -1.46 2.09 -1.53 2-024D08 troponin T1, skeletal, slow (TNNT1) 19 3.55 2.51 2.3 -2.23 -1.38 -2.18 2.79 -1.84 2-024F02 troponin T1, skeletal, slow (TNNT1) 19 3.04 2.99 1.82 -2.22 -1.37 -2.03 2.62 -1.79 2-023F09 troponin T1, skeletal, slow (TNNT1) 19 3.2 3.07 2.31 -2.09 -1.19 -2.02 2.86 -1.66 2-021E03 troponin T1, skeletal, slow (TNNT1) 19 3.35 2.35 1.88 -1.91 -1.44 -2.16 2.53 -1.79 2-023H12 troponin T1, skeletal, slow (TNNT1) 19 3.11 3.12 1.67 -1.85 -1.13 -1.68 2.64 -1.49 2-001A12 troponin T1, skeletal, slow (TNNT1) 19 2.43 2.17 1.72 -1.2 -1.35 -1.6 2.11 -1.37 2-020A10 troponin T3, skeletal, fast (TNNT3) 11 4.86 5.51 4.03 -2.56 -2.16 -1.62 4.8 -2.04 2-001H06 troponin T3, skeletal, fast (TNNT3) 11 4.6 5.46 3.84 -2.11 -1.49 -1.37 4.63 -1.6 Cell cycle control 2-008G02 zinc finger, HIT domain containing 1 7 -1.09 -1.12 -1.09 1.06 1.15 1.12 -1.1 1.11 2-007F05 protein phosphatase 2A, regulatory subunit B (PR 53) 9 -1.13 -1.2 1 1.11 1.13 1.18 -1.1 1.14 BL-007F08 retinoblastoma binding protein 7 (RBBP7) X -1.11 -1.16 1.01 1.11 1.21 1.23 -1.08 1.19 Apoptosis BL-001B06 BH3 interacting domain death agonist (BID) 22 -1.14 -1.17 1.03 1.03 NA 1.17 -1.09 1.1 2-029A02 cathepsin B (CTSB) 8 2.01 2.25 2.1 -2 -1.76 -1.94 2.12 -1.8 Regulation of transcription 2-011H11 transcription factor LZIP mRNA (CREB3) 9 -1.13 -1.15 -1.1 1.03 1.22 1.19 -1.12 1.15 2-029C02 myocyte enhancer factor 2C (MEF2C) 5 -1.35 -1.36 -1.45 1.06 1.03 1.18 -1.39 1.09 Extracellular matrix 2-025E09 laminin, alpha 4 (LAMA4) 6 4.76 4.51 3.36 -1.77 -1.76 -1.5 4.21 -1.67 2-016C10 laminin, beta 2 (laminin S) (LAMB2) 3 -1.22 -1.22 -1.01 1.14 1.12 1.16 -1.14 1.14 Other 2-009A04 lamin B1 (LMNB1) 5 -1.3 -1.15 1.08 1.06 1.03 1.12 -1.1 1.07 2-015F09 voltage-dependent anion channel 2 (VDAC2) 10 -1.13 -1.23 1.04 1.19 1.26 1.18 -1.09 1.21 2-018H10 PDZ and LIM domain 2 (mystique) 8 -1.18 -1.31 -1.12 1.15 1.21 1.17 -1.2 1.18 2-003B09 monoglyceride lipase 3 -1.16 -1.26 -1.01 1.08 1.28 1.2 -1.14 1.19 2-016G12 B-cell associated protein (REA) 12 -1.01 -1.27 -1.01 1.21 1.08 1.18 -1.08 1.16 2-016B11 coatomer protein complex, subunit epsilon (COPE) 19 -1.27 -1.22 -1.01 1.2 1.24 1.22 -1.15 1.22 BL-002H11 arachidonate 5-lipoxygenase-activating protein (ALOX5AP) 13 -1.14 -1.2 -1.08 1.12 1.04 1.18 -1.14 1.11 BL-002F03 sorting nexin 17 2 -1.17 -1.15 -1.04 -1.01 1.17 1.1 -1.12 1.09 BL-003H07 formin-like (FMNL) 17 -1.09 -1.17 -1.12 1.06 1.16 1.16 -1.12 1.12 2-029G06 SNARE associated protein snapin 1 -1.19 -1.11 -1.07 -1.04 1.1 1.11 -1.12 1.06 2-012D10 SEC13-like 1 3 -1.21 -1.41 -1.02 1.1 1.18 1.22 -1.19 1.16 Unknown 2-017E07 haloacid dehalogenase-like hydrolase domain containing 1A (GS1) X -1.09 -1.16 1.1 1.16 1.18 1.03 -1.04 1.12 2-005A01 aurora-A kinase interacting protein (AKIP) 1 -1.06 -1.24 1.1 1.22 1.1 1.12 -1.05 1.15 BL-002B09 hypothetical protein LOC51234 15 -1.1 -1.27 1.06 -1.03 -1 1.21 -1.09 1.06 2-008F10 unknown seqeunce from clone RP11-545E24 1 -1.21 -1.38 -1.02 1.15 1.18 1.2 -1.18 1.18 2-017C01 unknown seqeunce from clone CTD-2545M3 19 -1.33 -1.49 -1.04 1.02 1.06 1.19 -1.26 1.09 2-013D10 hypothetical protein FLJ10769 (FLJ10769) 13 -1.19 -1.37 -1.01 1.08 1.12 1.12 -1.17 1.11 2-001H10 ankyrin repeat domain 23 2 -1.31 -1.29 -1.12 -1.01 -1 -1.02 -1.23 -1.01 2-033G02 unknown sequence from PAC clone RP4-745K6 17 -1.15 -1.24 1.17 1.19 1.25 1.08 -1.05 1.17 2-017B04 chromosome 5 open reading frame 13 5 -1.24 -1.3 1.08 1.02 1.08 1.04 -1.13 1.05 2-020D03 SCIRP10-related protein 1 5.2 6.19 4.14 -1.73 -1.84 -1.57 5.17 -1.71 BL-002B05 zinc finger, ZZ domain containing 3 (ZZZ3) 1 -1.11 -1.2 -1.11 1.04 1.14 1.15 -1.14 1.11 2-012H02 chromosome 20 open reading frame 111 20 -1.13 -1.28 -1.02 1.11 1.14 1.18 -1.13 1.14 2-011G09 chromosome 2 open reading frame 24 2 NA -1.4 1.12 1.11 1.11 1.19 -1.09 1.14 2-005C06 hypothetical gene supported by BC055092 8 2.34 2.85 1.8 -1.55 -1.44 NA 2.33 -1.49 2-005F01 selenoprotein O 22 -1.05 -1.19 -1.05 1.15 1.14 1.15 -1.09 1.15 Table B FSHD Upregulated proteins: Swissprot/ MW (Da) MW (Da) TrEMBL pI using pI using calculated Spot Spot AC ExPASy calculated ExPASy from 2D- volume volume log2 spot N° Number Protein p-value tool from 2D-gel tool gel Control FSHD (FSHD/Control) Contraction/structur al proteins: 201 P02675 Fibrinogen beta chain precursor 0.006 8.54 8.50 55 928.15 52 673 0.043 0.073 0.76 99 O75083 WD repeat prot, 1 0.018 6.17 6.99 66 193.52 69 178 0.003 0.004 0.42 Anaerobic metabolism: 191 P00352 Aldehyde DH 1A1 0.006 6.29 6.94 54 730.65 53 304 0.039 0.097 1.31 248 P06733 Alpha enolase 0.008 6.99 7.07 47 037.77 46 415 0.159 0.219 0.46 331 P17174 Aspartate aminotransferase, cytoplasmic 0.004 6.57 7.64 46 116.31 39 931 0.059 0.111 0.91 323 P17174 Aspartate aminotransferase, cytoplasmic 0.004 6.57 7.70 46 116.31 40 573 0.09 0.15 0.74 271 P13929 Beta enolase