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Identification of Disease- and Therapy-Associated Proteome Changes in the Sera of Patients with Myelodysplastic Syndromes and del(5q) Changwei Chen PhD1, David T. Bowen MD2, Aristoteles A. N. Giagounidis MD, PhD3, Brigitte Schlegelberger MD4, Sabine Haase MD3 and Eric G. Wright PhD1 1 Centre for Oncology and Molecular Medicine, Division of Medical Science, College of Medicine, Dentistry & Nursing, Ninewells Hospital & Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK 2 Department of Haematology, St. James’s Institute of Oncology, Bexley Wing, Leeds LS9 7TF, UK 3 Clinical Research Unit, St. Johannes Hospital, Medizinische Klinik II, An der Abtei 7- 11, 47166 Duisburg, Germany 4 Institute of Cell and Molecular Pathology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany Running title: Proteomic analysis of serum proteome of myelodysplastic syndromes Corresponding author: Dr. Changwei Chen, Centre for Oncology and Molecular Medicine, Division of Medical Science, College of Medicine, Dentistry & Nursing, Ninewells Hospital & Medical School , University of Dundee, Dundee DD1 9SY, Scotland, UK; Tel: +44 (0)1382 632663; Fax: +44 (0)1382 633952; Email: [email protected] Supplementary Experimental Procedures: Protein purification and identification Identification of peaks at m/z 2791, m/z 3274, m/z 3956, m/z 3972, m/z 4282 and m/z 4298 One millilitre of MDS serum was fractionated with ProteinChip spin columns (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) according to the manufacturer’s instructions. Serum fractions were collected and profiled with IMAC30-Cu2+ arrays. The resulting mass spectra showed that these peaks were mainly present in the flowthrough fraction. To further enrich these proteins/peptides, a protocol based on reverse phase chromatography was employed. The flowthrough fraction was mixed with a sample buffer (2% TFA in 20% ACN) at 3:1 and applied to a C18 minicolumn (Amersham Biosciences, Little Chalfont, England) which had been activated and equilibrated according to the manufacturer’s instructions. After washing the column with a solution containing 0.5% TFA and 5% ACN, the bound proteins were sequentially eluted with 20%, 30%, 40%, 50%, 60% and 70% ACN. The eluates were analyzed and two fractions, i.e. eluates with 60% and 70% ACN containing the proteins/peptides were concentrated in a SpeedVac evaporator (Savant) and then processed for liquid chromatography–tandem mass spectrometry (LC-MS-MS). Briefly the LC-MS-MS was performed with an Ultimate 3000 nano-HPLC system (Dionex) connected to a LTQ Orbitrap ion trap mass spectrometer (Thermo Fisher Scientific). Samples were loaded to a 75 m15 cm PepMap C18 reverse phase column (Dionex) that was equilibrated with 30% solvent B (90% ACN in 0.08% formic acid) and 60% solvent A (0.01% formic acid in Milli-Q 2 H2O) and run at 300 nl/min. Separated peptides were eluted with a gradient of 30–90% solvent B over 21 min. The ion trap was set to detect positively charged ions using a spray voltage of 2 kV and an automated data dependent MS-MS analysis performed on the top 5 most abundant ions from each MS scan before another full MS scan was performed. Peptides were analyzed once then excluded for 90 seconds. A Mascot generic data file was generated from the MS-MS data using RAW2MSM1 prior to database searching with the Mascot search engine2 against the International Protein Index (IPI) human database3. The searching parameters were: enzyme: none; fixed modification: carbamidomethyl (C); variable modifications: acetyl (N-term), oxidation (M); mass value: monoisotopic; protein mass: unrestricted; peptide mass tolerance: ± 10 ppm; max missed cleavages: 2. Peptides above the Mascot significance threshold were considered as positive assignments. As shown in Supplementary Table 2, the peptides at m/z 3274 and m/z 3972 were identified as fragments of inter-alpha (globulin) inhibitor H4. We noticed that the peptides at m/z 4282 and m/z 4298 have the same sequence. As these two peptides have a mass difference of ca.16 Da, the peptide at m/z 4298 may represent an oxidized form of the peptide at m/z 4282. The attempts to obtain the identities of two other peptides, i.e. m/z 2791 and m/z 3956 by MS analysis were unsuccessful. Recently a paper4 reports the sequence of a same peptide at m/z 3956 which is exactly the same as that of the peak at m/z 3972 in our study. Another paper5reports the sequence of a peak at m/z 3953 which is largely the same except a few amino acid residues as that of the peptide at m/z 3972 in our study. These data indicate that the peptide at m/z 3956 may be a truncated or 3 modified form of the peptide at m/z 3972, which is supported by the array data showing that these two peaks are closely neighbouring to each other. Identification of peaks at m/z 7765, m/z 13737, m/z 13810, m/z 15089, m/z 15826 and m/z 79186 Serum samples were fractionated as described previously and the resulting fractions containing these proteins were determined by IMAC30-Cu2+ (m/z 7765 and m/z 79186) or Q10 profiling (m/z 13737, m/z 13810, m/z 15089 and m/z 15826). Proteins were then separated in a centrifugal filtering device with a 30 kDa cut-off membrane (Millipore, Watford, UK). The filtrate and the retenate containing proteins with molecular weight <30 kDa and >30 kDa respectively were dialyzed and concentrated in centrifugal filter devices with a 3 kDa cut-off membrane (Millipore, Watford, UK). Proteins with molecular weight <30 kDa were further separated by electrophoresis on 10–20% tricine gels (Invitrogen, Paisley, UK). To purify the protein at 79186 Da, the retenant was passed through an affinity column to remove albumin and IgG. The protein was finally purified by electrophoresis on a 10% glycine gel. After electrophoresis, all the gels were stained with colloidal Coomassie blue (Invitrogen). Protein bands corresponding to the individual proteins were excised from the gels and the proteins were in-gel digested with trypsin. The tryptic digests were analyzed with LC-MS-MS. Briefly the MS analysis was performed with a QSTAR XL mass spectrometer (Applied Biosystems) connected to an LC Packings ultimate nano-HPLC system. The resulting MS/MS data were searched against the IPI Human database using Mascot searching engine. The searching 4 parameters were: enzyme: trypsin; fixed modification: carbamidomethyl (C); variable modifications: acetyl (N-term), oxidation (M), pyro-glu (N-term Q), sulphone (M); mass value: monoisotopic; protein mass: unrestricted; peptide mass tolerance: ± 2 Da; fragment mass tolerance: ± 0.8 Da; max missed cleavages: 2. Peptides above the Mascot significance threshold were considered as positive assignments. Database searching identified multiple proteins from the 13 kDa bands, 3 of which i.e. serum amyloid A protein precursor (IPI00552578), APO C3 13 kDa protein (IPI00657670), and transthyretin precursor (IPI00022432) have a mass close to 13 kDa. Based on the published data on serum profiles obtained by other investigators, 68we suggested that the peaks at m/z 13810 and m/z 13737 would represent the variants of transthyretin. To test this, we prepared the serum samples depleted of transthyretin and analyzed them with Q10 array profiling. The two peaks, i.e. m/z 13737 and 13810 along with another peak at m/z 13851 disappeared from the depleted serum. These peaks were detected in the eluted sample (Supplementary Figure 1a). This assay demonstrated that these two peaks were derived from transthyretin. As the mass of the unmodified form of transthyretin is ca.13760 Da, these two peaks may represent modified forms of transthyretin. The exact modifications that these two peaks represent need to be clarified. Four proteins including transthyretin (IPI00022432), haemoglobin subunits (IPI00654755), δ (IPI00473011) and (IPI00410714) were identified from the 15 kDa gel bands. Based on the published data, 911 we speculated that these two peaks would represent the haemoglobin (m/z 15089) and (m/z 15826) chains. Thus an immunodepletion assay was carried out to remove haemoglobin from the serum samples. 5 These two peaks disappeared from the depleted serum, and reappeared in the eluted sample (Supplementary Figure 1b). The assay confirms that these two peaks indeed represent haemoglobin and chain, respectively. The identification of m/z 7765 by MS analysis was unsuccessful. As several papers report the identification of a few similar protein peaks as platelet factor 4 (PF-4), 5, 12, 13we employed an immunodepletion assay to test whether it would be PF-4. The assay showed that the m/z 7765 peak was absent in the depleted serum, and was detected in the eluted fraction (Supplementary Figure 1c), which indicates that the peak was indeed derived from PF-4. 6 References 1 Olsen JV, de Godoy L M, Li G, Macek B, Mortensen P, Pesch R et al. Mol Cell Proteomics 2005; 4: 2010–2021. 2 Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999; 20: 3551–3567. 3 Kersey PJ, Duarte J, Williams A, Karavidopoulou Y, Birney E, Apweiler R. The International Protein Index: an integrated database for proteomics experiments. Proteomics 2004; 4: 1985–1988. 4 Song J, Patel M, Rosenzweig CN, Chan-Li Y, Sokoll LJ, Fung ET et al. Quantification of fragments of human serum inter-alpha-trypsin inhibitor heavy chain 4 by a surface-enhanced laser desorption/ionization-based immunoassay.Clin. Chem. 2006; 52: 1045–1053. 5 Cho WC, Yip TT, Ngan RK, YipTT, Podust VN, Yip C et al. ProteinChip array profiling for identification of disease- and chemotherapy-associated biomarkers of nasopharyngeal carcinoma. Clin Chem 2007; 53: 241–250. 6 Escher N, Kaatz M, Melle C, Hipler C, Ziemer M, Driesch D et al. Posttranslational modifications of transthyretin are serum markers in patients with mycosis fungoides. Neoplasia 2007; 9: 254–259. 7 Liu L, Liu J, Dai S, Wang X, Wu S, Wang J et al. Reduced transthyretin expression in sera of lung cancer. Cancer Sci 2007; 98: 1617–1624. 7 8 Miguet L, Bogumil R, Decloquement P, Herbrecht R, Potier N, Mauvieux L et al. Discovery and identification of potential biomarkers in a prospective study of chronic lymphoid malignancies using SELDI-TOF-MS. J Proteome Res 2006; 5: 2258–2269. 9 Koomen JM, Shih LN, Coombes KR, Li D, Xiao LC, Fidler IJ et al. Plasma protein profiling for diagnosis of pancreatic cancer reveals the presence of host response proteins. Clin Cancer Res 2005; 11: 1110–1118. 10 Kozak KR, Amneus MW, Pusey SM, Su F, Luong MN, Luong SA et al. Identification of biomarkers for ovarian cancer using strong anion-exchange ProteinChips: potential use in diagnosis and prognosis. Proc. Natl. Acad. Sci. U S A. 2003; 100: 12343–12348. 11 Woong-Shick A, Sung-Pil P, Su-Mi B, Joon-Mo L, Sung-Eun N, Gye-Hyun N et al. Identification of hemoglobin-alpha and -beta subunits as potential serum biomarkers for the diagnosis and prognosis of ovarian cancer. Cancer Sci 2005; 96: 197–201. 12 Vermeulen R, Lan Q, Zhang L, Gunn L, McCarthy D, Woodbury RL et al. Decreased levels of CXC-chemokines in serum of benzene-exposed workers identified by arraybased proteomics. Proc. Natl. Acad. Sci. U S A. 2005; 102: 17041–17046. 13 Aivado M, Spentzos D, Germing U, Alterovitz G, Meng XY, Grall F et al. Serum proteome profiling detects myelodysplastic syndromes and identifies CXC chemokine ligands 4 and 7 as markers for advanced disease. Proc Natl Acad Sci USA 2007; 104: 1307–1312. 8