In September 2018, the animal hair identification method using MALDI-TOF to detect species-specific peptides was released as a standard (ISO) in Japan. In addition, it will also be designated as the Japanese Industrial Standards (JIS). Even with only one hair, this identification method can be used for analysis. Therefore, it can not only prevent the counterfeiting of cashmere, but also be used for the identification of various types of hair such as feathers and human hair, as well as for the inspection of foreign objects mixed in food. This method was developed by the Institute of Genomic Biotechnology at Kanazawa Institute of Technology based on the Shimadzu MALDI-TOF system.

The identification and analysis technology of animal hair plays an important role in various fields. Animal hair is used to make various fiber products such as jackets, tops, sweaters, etc. When making such fiber products, it is the obligation to label the types of fibers and the mixing rate when using multiple fibers according to the Household Product Quality Labeling Law. In addition, when hair is mixed into food, determining the type of hair is crucial for identifying the mixing route.

Excluding moisture, about 90% of hair is composed of protein. Figure 1 shows the structure of animal hair.

Figure 1 Pattern diagram of animal hair

Hair not only prevents external impacts, but also plays a role in insulation, which is closely related to the structure of hair. The protein in hair contains a large amount of steroids, which bind to form multiple cross-linked structures between molecules. The chemical analysis of hair requires dissolution, using a substance called a reducing agent to break disulfide bonds. In addition, the obtained protein extract contains a variety of proteins, salts and other substances, so it needs to be purified by polyacrylamide gel electrophoresis (SDS-PAGE). The MALDI-TOF identification process for animal hair fibers is shown in Figure 2:

Figure 2 MALDI-TOF MS animal hair analysis method

Processed peptides were analyzed using MALDI-TOF MS, and crystal samples on metal target plates were detected using MALDI-TOF MS. Adjust the detection conditions such as laser power to ensure good resolution in the field of m/z 2450-2750 for observing animal species specific peaks.

Perform MALDI-TOF MS detection on the sample obtained by dissecting the SDS-PAGE protein bands. Find specific peak values representing different animal species.

(a)Band1 KeratinⅡ (b) Band2 KeratinⅠ

Figure 3 MALDI-TOF MS detection spectrum

By using MALDI-TOF MS technology, the peak difference interval m/z 2450-2750 and specific peak between cashmere, wool, and yak hair were identified. As shown in Figure 4.

Figure 4 Animal species specific peaks in MALDI-TOF MS

Table 1 Amino acid sequences of animal specific peptides * 1

*The peak highlighted in black is the main peak

MS/MS analysis was performed on the specific peaks of these animal species, and the results of amino acid arrangement are speculated to be shown in Table 1 (2). A peptide composed of 23 amino acids, with 1 amino acid difference between cashmere and wool, and 4 amino acids difference between cashmere and yak hair.

Many fiber products undergo chemical treatments such as bleaching, decolorization, and dyeing during processing, but the specific peak position of animal hair does not change due to these treatments. Therefore, it is clear that it can be used to identify the types of animal hair in fiber products.

Analysis of one hair

When analyzing animal hair in fiber products, in order to avoid deviations in product composition, it is advisable to sample a large area of the product and average it, and then use a portion of it. Therefore, microanalysis is rarely needed. On the other hand, when analyzing hair mixed in food, it is recommended to use as few samples as possible for analysis.

In scientific investigations, DNA analysis of hair can provide a lot of information related to personal characteristics such as blood type. However, the animal hair used in fiber products mostly only has a dry part, especially for heavily stained products, making DNA analysis difficult. By using MALDI-TOF MS method to analyze proteins, even one hair can be fully detected.

According to different animal species, the peak position of specificity also varies, but animals with the same attribute tend to have peaks at the same location. Cashmere and Angora goat wool need to be identified using a microscope. Table 2 shows the peak positions (m/z) of major animal specific peaks and the amino acid arrangements estimated from the database. The main peak positions of tanuki, fox, and dog are the same, but it can be seen that the second peak of fox is 2638 and that of dog is 2641, which can be used to make judgments about them. In addition to the animals shown here, animals that can utilize genomic databases can also infer and confirm the location of specific peaks. In this way, the type of animal hair contained can be determined by only looking at the spectrum of m/z 2450-2750, which is a major feature of MALDI-TOF MS method.

Figure 6: Animal specific peak values obtained from MALDI-TOF MS analysis of one hair

Table 2 Amino acid arrangement and peak position (m/z) of various animal hair specific peptides

*According to NCBI database（ https://www.ncbi.nlm.nih.gov/ ）Presumption

reference material:

Shimadzu App News No. 65 and Literature

1) Daiki Shinichi, Yuka Idemura, Motoaki Sano SEN’I GAKKAISHI、68（10）、276 （2012）．

2) Daiki Shinichi, Yuka Idemura, Motoaki Sano, Yoichiro Yoshioka SEN’I GAKKAISHI，70 (6)、114 (2014)．

3)ISO20418-2 Textiles ̶ Qualitative and quantitative proteomic analysis of some animal hair fibres ̶ Part 2: Peptide detection using MALDI-TOF MS．