PROTEOMIC METHOD FOR THE IDENTIFICATION AND QUANTIFICATION OF ANIMAL HAIR FIBRES
C. Tonetti, S. Paolella, D.O. Sanchez Ramirez, R.A. Carletto, Francesca Truffa Giachet, Giulia Dalla Fontana, C. Vineis, A. Varesano, S. Sforza
CNR-ISMAC Istituto per lo Studio delle Macromolecole – Consiglio Nazione delle Ricerche, Biella ([email protected])
Convegno Nazionale AICTC – 27 ottobre 2017 - Biella IDENTIFICATION OF ANIMAL HAIR FIBRES
The market of high quality textiles requires suitable analytical methods for the determination of their fiber composition to guarantee that no falsification occurs, especially when cheaper fibers, like wool and yak, are blended with expensive fibers, like cashmere.
YAK
WOOL CASHMERE
Traditional methods currently used for fibres identification are light microscopy (LM), and scanning electron microscopy (SEM). IDENTIFICATION OF ANIMAL HAIR FIBRES BY LIGHT MICROSCOPY The identification is based on external and internal morphology of the fibres.
External features Internal features Profile of the fibre Pigmentation (shape, intensity, distribution) Mean Fibre Diameter Medullation (shape, intensity, distribution) Scale Ratio Scale Shape
WOOL CASHMERE YAK ALPACA VICUNA
GUANACO IDENTIFICATION OF ANIMAL HAIR FIBRES BY SCANNING ELECTRON MICROSCOPY The identification is based on the surface morphological characteristics of the fibres.
WOOL CASHMERE YAK VICUNA Mean Fibre Diameter Scale Ratio Scale Shape Scale Height (Thickness) ALPACA GUANACO Scale Angle IDENTIFICATION OF ANIMAL HAIR FIBRES BY MICROSCOPIC TECHNIQUES
Animal hair fibres, despite their different genetic origin, exhibit great similarities in chemical properties and sometimes in morphological architecture. LM analysis can play an important role for fibre identification and characterization due to the possibility to investigate the internal fibres structure. SEM analysis play a fundamental role in the quantitative analysis of wool and other specialty animal fibres based on the criterion of the height of the cuticle scale edge.
The correct identification depends on the skill of the operator and often is affected by chemical treatments to which the fibers have been submitted during textile processing PROTEOMIC METHOD: HYSTORY
A new objective method has been developed for the identification of animal hair fibres, in particular wool, cashmere and yak.
The research has been developed jointly by:
- Institute for Macromolecular Studies CNR-ISMAC Biella that is the repository of a huge knowledge about animal fibres;
- Department of Food Science of University of Parma that developed this method for the analysis and identification of food product of animal origin where is currently used. PROTEOMIC METHOD: HYSTORY 2011-2012: The first part of the research and development of the method was financed and assisted by CCMI (Cashmere and Camel hair Manufacturers Institute). The results of this program have been published on an important scientific journal: S. Paolella, M. Bencivenni, F. Lambertini, B. Prandi, A. Faccini, C. Tonetti, C. Vineis, S. Sforza, "Identification and quantification of different species in animal fibres by LC/ESI-MS analysis of keratin-derived proteolytic peptides", Journal of Mass Spectrometry Vo l . 48, N° 8, 2013 and they have been presented in two oral communications: ─ S. Sforza, B. Prandi, S. Paolella, M. Bencivenni, C. Vineis, C. Tonetti, P.D. Pozzo, G. Galaverna, A. Dossena, “MS-based peptidomics for authentication of food and non-food commodities”, 4th EuCheMS Chemistry Congress, August 26-30, 2012, Prague ─ A. Dossena, S. Sforza, B. Prandi, S. Paolella, M. Bencivenni, C. Vineis, C. Tonetti, A. Aluigi, G. Galaverna, “MS-based peptidomics as molecular characterization of food and non-food commodities”, Convegno Nazionale “MASSA 2012”, July 1-5, 2012, Palermo PROTEOMIC METHOD: HYSTORY
2013-2014: A consortium of 12 Italian leading textile industries have financed CNR-ISMAC Biella in the project to acquire the equipment and set-up the procedures to make this method of analysis available to all textile industries and to consumers.
SPONSOR
• Lanificio Ermenegildo Zegna & Figli SpA • Biella Manifatture Tessili SpA • Zegna Baruffa Lane Borgosesia SpA. • E. Thomas SpA • Filatura di Trivero SpA • Fratelli Piacenza SpA • Lanificio Cariaggi SpA • Lanificio F.lli Cerruti SpA • Loro Piana SpA • Pettinatura di Verrone SrL • Successori Reda SpA • Vitale Barberis Canonico SpA PROTEOMIC METHOD: HYSTORY
The results are reported on two papers: ─ C. Vineis, C. Tonetti, S. Paolella, P.D. Pozzo, S. Sforza, "UPLC/ESI-MS method to identify wool, cashmere and yak fibres" Textile Research Journal, Vo l . 84, N° 9, 2014 ─ Claudia Vineis, Cinzia Tonetti, Diego Omar Sanchez Ramirez, Riccardo Andrea Carletto & Alessio Varesano, “Validation of UPLC/ESI-MS method used for the identification and quantification of wool, cashmere and yak fibres”, The Journal of The Textile Institute, 2017 and they have been presented in two oral communications: ─ C. Vineis, S. Sforza, “UPLC/ESI-MS method: an innovative method of identification and quantification of different animal fibres”, 82° IWTO Congress, June 12-14, 2013, Biella. ─ C. Vineis, S. Sforza, “UPLC/ESI-MS method to identify wool, cashmere and yak fibres”, CCMI International Fiber Identification Symposium, February 19-20, 2014, Biella. PROTEOMIC METHOD: HYSTORY
Internal standard ISMAC-BI-2014-LB01: Quantitative analysis of animal hair fibres by UPLC/ESI- MS (wool, cashmere, yak), 2014
ISO Standard 20418-1: Textiles – Qualitative and quantitative proteomic analysis of some animal hair fibers – Part 1: Peptide detection using LC-ESI-MS with protein reduction (under approval)
2014-2016: The continuation of the research was financed by Compagnia of San Paolo within the project MATEx Aims of the work: Identification of specific markers for Camelids Evaluation of the influence of chemical and physical treatments on the proteomic analysis THE PRINCIPLES OF THE METHOD
Molecular markers of species
Molecules which can uniquely linked to a specific species, surviving along the production chain, allowing to trace the species in the final product.
Molecular marker for cashmere Absent in other species
Molecular marker for yak Absent in other species
Cashmere and yak simultaneously present THE PRINCIPLES OF THE METHOD
The most common ...but also other molecules can be molecular marker is DNA... used for this purpose
Protein sequences, and peptides derived from it, are genetically determined, and can be as good molecular markers for species as DNA sequences.
Peptides can be naturally present in the commodity or can be generated by enzymatic digestion of the protein fraction.
Advantages of peptide detection, rather than DNA detection:
- Proteins, hence peptides, are more abundant than DNA - Peptides are easier to analyze by liquid chromatography coupled to mass spectrometry in a very specific way, also in complex mixtures - Their quantitative determination is more straightforward and more accurate THE PRINCIPLES OF THE METHOD
Animal fibers consist principally of keratin, a high-sulfur content protein.
Keratin sequences might be slightly different in the different species, because in some specific parts there are aminoacidic variants
Cashmere …….Cys-Gln-Leu-Asn-Gln-Val-Gln……
Wool …….Cys-Gln-Leu-Ser-Gln-Val-Gln……
Yak …….Cys-Gln-Leu-Ala-Gln-Val-Gln……
Peptides containing these variants have different MW and different sequences among the species, thus might be differentially detected by Mass Spectrometry, acting as Species-Specific Molecular Markers PROCEDURE
Extraction of keratin from fibers with buffer solution (tris-HCl, thiourea, urea, DTT, pH = 8.5) for 48 h at 50°C
Enzymatic digestion wool with trypsin
yak cashmere LC-ESI-MS analysis
Specific marker-peptides identification MARKER IDENTIFICATION
Markers were identified for the specie for which they are specific, implying that for other species they are absent. Wool-Cashmere markers can be found in both wool and cashmere, but they are absent in yak. Markers were characterized by their MW, Rt and characteristic ions and their aminoacidic sequence. The markers were coupled according to their molecular weight in order to calculate the percentage of each fiber. MARKER IDENTIFICATION
The Yak markers The Cashmere markers
Yak 1 Yak 2
Sheep-Goat 1 Sheep-Goat 2
Cashmere 2 Cashmere 1 MARKER IDENTIFICATION
Wool markers
Sheep 1 Sheep-Goat 1 Sheep 2
Sheep-Goat 2 VALIDATION OF THE METHOD VALIDATION OF THE METHOD
The method was validated also for quantitative analysis. For this purpose calibration curves were set up with known standards. Wool/Cashmere Calibration
% wool measured by UPLC/ESI-MS vs % wool % cashmere measured by UPLC/ESI-MS vs actual, in wool/cashmere blends at known % cashmere actual, in wool/cashmere blends composition at known composition VALIDATION OF THE METHOD We carried out the quantitative analysis of many blind samples in different shapes: • fibres • slivers • yarns • fabrics • raw materials and with different treatments: • dyed • depigmented • bleached • finished VALIDATION OF THE METHOD
Finally reproducibility tests were carried out using a sample with unknown composition. The sample has been analysed six times in order to assess repeatability of the whole method (from the protein extraction to the LC/MS analysis).
Standard deviation: ±1.4 VALIDATION OF THE METHOD: CONCLUSIONS
The method showed 100% sensitivity (no false negatives) and 100% specificity (no false positives) on the samples tested for validation
In samples with cashmere/wool, cashmere/yak and wool/yak mixtures the LC/MS is able to have performances at the same level of SEM or slightly better
The limit of detection was estimated at 3%
The proteomic method was not influenced by different treatments to which the fibers have been subjected or by the presence of other fibers different from animal hairs (cotton, polyamide, silk…)
The proteomic method showed good repeatability, reproducibility and accuracy IDENTIFICATION OF MARKERS FOR CAMELIDS
The research was divided in two steps: 1. identification of specific markers to discriminate Camelidae (camel and South American camelids) from Bovidae (wool, cashmere and yak) 2. i dentification of specific markers to discriminate camelids from each others.
Step 1: Camelidae vs Bovidae
5 specific markers (C1-C5) for a generic Camelidae 2 specific markers (Bov1-Bov2) for a generic Bovidae 5 specific markers for individual Bovidae IDENTIFICATION OF MARKERS FOR CAMELIDS IDENTIFICATION OF MARKERS FOR CAMELIDS
The markers were coupled according to their molecular weight and retention time. Each couple has been validated for qualitative and quantitative analysis.
3 specific peptides-marker for a generic Camelidae (C1, C4, C5) can been used also for quantitative analysis limit of detection = 3% IDENTIFICATION OF MARKERS FOR CAMELIDS
Step 2: Camelidae vs Camelidae
8 specific markers to discriminate camel from South American camelids (alpaca, llama, vicuna and guanaco) APPLICATION OF THE PROTEOMIC METHOD: EXAMPLE
Declared Composition Proteomic method PA 13 % PA 13 % Alpaca 43 % Camelidae 46 % Wool 42 % Wool 38 % Cashmere 2 % Cashmere 3 %
Declared Composition Proteomic method PA 7 % PA 7 % Alpaca 23 % Camelidae 29 % Wool 44 % Wool 39 % Cashmere 3 % Cashmere 2 % Silk 23 % Silk 23 % Fabrics provided by customer company customer by provided Fabrics FUTURE WORKS
To test the repeatability and the reproducibility of the proteomic method using blends Camelidae/Bovidae and Camelidae/Camelidae
To validate qualitatively and quantitatively the specific markers for camel blended with South American camelids
To study the influence of different textile treatments (finishing and dying) on the proteomic method for the analysis of fibers from camelids
To characterized the aminoacid sequences of new peptide-markers of Camelidae and Bovidae Thank you for the attention