MALDI-TOF 1, # 1 1 2 Quantitative Analysis of Cashmere and Other Animal Hair Fibers in Textiles Using MALDI-TOF Mass Spectrometry Shinichi Ohashi 1,#, Yuka Demura 1, Motoaki Sano 1, and Yoichiro Yoshioka 2 1 Genome Biotechnology Laboratory, Kanazawa Institute of Technology, 3-1 Yatsukaho, Hakusan, Ishikawa 924-0838, Japan 2 Boken Quality Evaluation Institute, 1-8-15 Ue-Machi, Chuo-Ku, Osaka 540-0005, Japan Abstract : Techniques for analyzing the composition of animal hair fibers in textiles are very important to guarantee the quality of animal hair yarns and fabrics. We have recently developed new methods based on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI- TOF) mass spectroscopy in order to identify the animal species from the hair fibers. In this study, we assigned the animal species-specific peaks by tandem mass (MS/MS) analysis. We prepared cashmere-wool and cashmere-yak standard samples and observed very good linearity between the composition and the peak area ratio. The quantitative method was validated using nine blind-test samples containing known contents of cashmere, wool, and yak. The method was also validated by measuring various samples containing cashmere and other animal hair fibers, natural fibers and chemical fibers and by comparing the results with those obtained by optical microscopy. (Received 14 February, 2014 ; Accepted 6 March, 2014) # corresponding author 1 2 DNA 1-3 4 DNA 2 DNA DNA 5 I SDS-PAGE MALDI- TOF m/e 2450-2750 6 7
MALDI-TOF CBB MALDI-TOF xcise Proteome Systems CHCA MALDI-TOF AXIMA-CFRplus Matrix Science Mascot NCBI MSDB CCMI 12 DTT SDS IAA Bio-Rad 6 SDS-PAGE 45000 nano-lc KY CHCA MALDI TOF/TOF 4800 plus AB Sciex MS/MS NCBI Sigma-Aldrich HPLC HPLC Promega α- -4- CHCA Sigma-Aldrich MM400 25 mg 1.5 ml 50 mmol/l DTT 4% SDS 0.1 M ph 7.8 0.5 ml 95 15000 rpm 5 100 mmol/l IAA 25 30 25 mmol/l DTT10μl 15000 rpm 5 DTT SDS SDS-PAGE e 90 mm 83 mm 1mm AE-6513P CBB SDS-PAGE SDS DTT SDS-PAGE MALDI-TOF m/e 2450-2750 6 MALDI-TOF MS/MS 6 SDS-PAGE 45000 LC MALDI-TOF MS/MS Table 1 m/e=2691 33A 307 329
m/e=2664 33B 307 329 m/e=2504 keratin type I microfibrillar 47.6 kdalike 309 331 23 1 4 33A 33B N- NCBI BLAST 33A Table 2 keratin33a m/e=2450-2750 7 hair acidic keratin 1 keratin type I cuticular Ha 4-like Table 1 MS/ MS Table 1 Results of MS/MS analysis of animal-specific peaks. Table 2 Amino acid sequence of animal species-specific region in keratin type I.
MALDI-TOF MALDI 8,9 6 SDS-PAGE 45000 I MALDI-TOF 80% 50% 20% Fig. 1 Cashmere Peak % PAC 100/ PAc+PAw Cashmere Peak % PAC 100/ PAc+PAY PAC : PAW : PAY : Fig. 2 Calibration curve for cashmere/wool mixture. Fig. 3 Calibration curve for cashmere/yak mixture. Fig. 1 Mass spectra of cashmere/wool mixture. Fig. 2 Fig. 3 1 1 6 SDS-PAGE
Fig. 4 Fig. 5 Fig. 4 7 Fig. 5 9 1 CCMI Blind Test 9 6 2 2 Table 3 Table 3 Fig. 4 Fig. 5 Reproducibility and repeatability of calibration curve for cashmere/wool mixture. Reproducibility and repeatability of calibration curve for cashmere/yak mixture. Table 3 Peptide analysis of blind test samples*. 1-01 1-09 9 3.4 7 100% Table 4 Table 5 14 5% 2-02 2-06 15 Table 5
Table 4 Validation of peptide method (1). Table 5 Validation of peptide method (2). 3 4% 3-06 3-13 3-15
SDS-PAGE MALDI-TOF m/e=2450-2750 33A 33 I 23 m/e=2450-2750 MALDI-TOF blind test DNA 2,10 Karl Spilhaus MS/MS 1. S. Subramanian, T. Karthik, N. N. Vijayaraaghavan, J. Biotechnol., 116, 153 (2005). 2. K. Kerkhoff, G. Cescutti, L. Kruse, J. Muessig, Text. Res. J., 79, 69 (2009). 3. W. Ji, L. Bai, M. X. Yang, Forensic Sci. Int., 208, 139 (2011). 4. http : / / www. meti. go. jp / information / downloadfiles / c110204d02j.pdf 5. C. Tonetti, C. Vineis, A. Aluigi, C. Tonin, Text. Res. J., 82, 766 (2012). 6. S. Ohashi, Y. Demura, M. Sano, Sen I Gakkaishi, 68, 276 (2012). 7. S. Ohashi, Y. Demura, M. Sano, Y. Yoshioka, Sen I Gakkaishi, inpress. 8. HA Ebhardt, ZZ Xu, AW Fung, RP Fahlman, Anal. Chem., 81, 1937 (2009). 9. KM Park, YJ Bae, SH Ahn, MS Kim, Anal. Chem., 84, 10332 (2012). 10. C.Vineis, C. Tonetti, S. Paolella, P.D. Pozzo, S. Sforza, Text. Res. J., December 12, 2013 0040517513512394.