Research Reports

ホームHOMEResearch and DevelopmentAbout Nippi Research Institute of BiomatrixResearch Reports
Report No. 007 Species identification methods of leather and glue using collagen-derived marker peptides
Report No. Species identification methods of leather and glue using collagen-derived marker peptides
007

Overview

Leather is produced from animal skin through a tanning*1 process using chrome or vegetable agents and has long been used for various products, including clothing, footwear, bags, and gloves. In Japan, the animal source of leather products derived from cattle, horse, pig, sheep, goat, and deer must be labeled by supplier to guarantee their quality and value by law (Household Goods Quality Labeling Act). In addition, all crocodilian species, including crocodile, alligator, and caiman, are under the protection of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Detailed information such as the species and country of origin must be clearly specified for international trade of crocodilian leather. Identification of the animal source of leather is generally performed by morphological observation using electron microscopy based on the cross-sectional fiber structure of collagen and pore morphology on the leather surface, both of which are characteristic for certain animals. However, this method requires extensive operator experience to correctly identify the animal source, and discrimination between animal species with similar morphological characteristics, such as between sheep and goat and among crocodilian species, is difficult. Therefore, development of a simple and objective method to identify the animal source of leather has been desired.
We have developed various analytical methods of collagen by mass spectrometry (Report No. 004). Utilizing these technologies, we established a novel analytical method to accurately identify the animal species of leather based on interspecies differences in the amino acid sequence of type I collagen, which is the main component of leather. Species identification is achieved by detecting peptide*2 fragments from type I collagen digested with trypsin*3
Furthermore, we applied this analytical method to cultural properties. Animal glue, which is extracted from bone and skin through heating in boiling water, is mainly composed of heat-denatured collagen (gelatin). Artists and craftsmen have long used animal glue as adhesives for paintings and murals and as binding media for pigments. The animals used as raw materials for glue vary depending on the era and region. In addition, specific animal species of glue is sometimes used depending on the purpose due to differences in the physical properties among the animal origin. Species identification of animal glue used in artworks and historical materials (paintings, murals, sculptures, etc.) can provide various information, such as the context of the era and artistic materials/techniques.
This report introduces (1) species identification of leather, (2) semi-nondestructive certification of leather products, and (3) species identification of glue.
*1: The “tanning” is a treatment process to produce leather by cross-linking of collagen, which is a major protein in animal skin. This process provides stability against moisture, heat, and microbial degradation. Today, about 90% of leathers in the world are produced by chrome tanning.
*2: The “peptide” is composed of several to dozens of amino acids, and here refers to a fragment of type I collagen digested with trypsin.
*3: The “trypsin” is one of the digestive enzymes and degrades proteins to generate peptide fragments with recognition of basic amino acids (lysine and arginine).

(1) Species identification of leather (References 1 and 2)

In order to develop a method for determination of the animal origin of leather using mass spectrometry, we first established sample preparation procedures, including dechroming treatment with calcium hydroxide and subsequent digestion with trypsin, which allowed efficient generation of type I collagen-derived peptides from chrome-tanned leather in a short time (approximately 7 hours in total). We then selected 12 collagen marker peptides (6 each for mammals and reptiles) from among many tryptic peptides generated from leathers of 10 target animals (cattle, horse, pig, sheep, goat, deer, crocodile, alligator, caiman, and lizard) (Table 1). Mass spectrometric detection of these marker peptides achieved highly sensitive and accurate determination of the animal source of leather. For example, marker peptides M3, M4, and M6 are detected for sheep, while M2, M4, and M6 are detected for goat (Figure 1). We believe that this simple and rapid method is useful for routine measurements for certification of leather products.


Table 1. Detection patterns of type I collagen-derived marker peptides for species identification of leather





Figure 1. Marker peptides detected for leather from sheep and goat.

(2) Semi-nondestructive certification of leather products (Reference 2)

Morphological observation using electron microscopy, which is the conventional method for identifying the animal source of leather, requires destruction of samples, making it difficult to analyze leather products in practice. However, we enabled determination of the animal species with minimal destruction by subjecting a very small amount of leather samples collected by filing to the identification method established in Chapter (1). Commercial leather watch straps labeled as crocodile, alligator, caiman, and crocodile-embossed calf were used for testing. Samples were discreetly taken from an inconspicuous area on the undersides of the leather products. The small amount of collected leather powder (approximately 100 µg) was analyzed, and all marker peptide detection patterns were confirmed to be consistent with the labeled animal species (Table 1 and Figure 2). This method, with minimal damage to leather, is expected to be applicable in a variety of fields, such as analysis of expensive leather products and historical materials.



Figure 2. Marker peptides detected for commercial leather watch straps.

(3) Species identification of glue (References 3–5)*4

Species identification of glue used in artworks and historical materials has been increasingly performed using mass spectrometry with detection of collagen-derived peptides. However, it is difficult to unambiguously determine the animal origin of glue using conventional methods when multiple glues of different origins are used, such as due to blending with other glues to alter its properties and contamination with other glues during manufactureing process. To overcome this problem, we applied our methodology used for leahter to establish a novel species identification method of glue applicable to samples with multiple animal origins. The target animal species were set to eight species used for animal glue (cattle, horse, pig, sheep, goat, deer, rabbit, and sturgeon). We newly selected 12 type I collagen-derived marker peptides to give different detection patterns even between samples containing two species (Table 2). Using this method, we analyzed a small amount of sample taken from the tacking margin of the canvas of a painting (The Harvest by Camille Pissarro, 1882) that is part of the collection of The National Museum of Western Art (Figure 3) and showed that glues derived from two animals (cattle and sheep) were used (Figure 4). This method, which requires only minute amounts of sample, would be helpful for research of valuable artworks and historical materials.

*4: This study was conducted in collaboration with Miho Takashima at the National Museum of Western Art and Taniguchi Yoko at University of Tsukuba, and was supported by JSPS KAKENHI Grant No. 16K01187 and 19K01133.
Table 2. Detection patterns of type I collagen-derived marker peptides for species identification of animal glue






Figure 3. The Harvest (Camille Pissarro, 1882, tempera on canvas, collection of The National Museum of Western Art, Tokyo, Japan, ex-Matsukata Collection).



Figure 4. Marker peptides detected for canvas of The Harvest.

References

1. Kumazawa Y, Taga Y, Iwai K, Koyama Y. A rapid and simple LC-MS method using collagen marker peptides for identification of the animal source of leather. J Agric Food Chem. 64, 6051–6057 (2016)
2. Kumazawa Y, Hattori S, Taga Y. Semi-nondestructive certification of crocodilian leather by LC−MS detection of collagen marker peptides. Anal Chem. 91, 1796-1800 (2019)
3. Kumazawa Y, Taga Y, Takashima M, Hattori S. A novel LC–MS method using collagen marker peptides for species identification of glue applicable to samples with multiple animal origins. Herit Sci. 6, 43 (2018)
4. https://www.yomiuri.co.jp/culture/dentou/20230611-OYT1T50100/
5. https://www.yomiuri.co.jp/culture/dentou/20230618-OYT1T50077/
Back