Qualitative Identification of Cashmere and Yak Fibers by Protein

Article pubs.acs.org/IECR

Qualitative Identification of Cashmere and Yak Fibers by Protein Fingerprint Analysis Using Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry Youngmi Kim,†,‡ Taewoo Kim,† and Hyung-Min Choi*,‡ †

FITI Testing & Research Institute, 17-5 Jayang-4-Dong, Gwangjin-Gu, Seoul, 143-841 Republic of Korea Department of Organic Materials and Fiber Engineering, Soongsil University, 511 Sangdo-Dong, Dongjak-Gu, Seoul, 156-743 Republic of Korea

‡

S Supporting Information *

ABSTRACT: Protein fingerprint analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was investigated for qualitative identification and differentiation of cashmere and yak fibers. Mass spectra of a total of 25 cashmere and 7 yak fibers were obtained and analyzed. Three ion peaks that are specific only to cashmere fiber were identified as m/z 2036, 2634, and 3266. On the other hand, two ion peaks at m/z 2503 and 2519 can be used as fingerprint ions for yak fiber. This test method can be effectively used in differentiating cashmere fiber from yak fibers in the item with a fraudulent label. We hoped that the use of this identification method could reduce the fraudulent labeling problem in the luxury fiber market. and experience of the analyst for reliable fiber analyses. In addition, diverse scale patterns can be observed even within one species of the fiber, and juvenile animals often lack differences in morphological patterns.11 The results of the annual interlab study done by the Cashmere and Camel Hair Manufacturers Institute (CCMI) revealed that less than 20% of testing laboratories successfully passed the differentiation test for animal fiber mixtures.12 The limitation of AATCC TM 20A therefore describes that “their accuracy depends to a considerable extent upon the ability of the analyst to identify the individual fibers”. For the past decade, various test methods have been examined to improve the creditability of animal fiber differentiation methods. These methods are mainly categorized into four groups: (1) chemical methods such as extraction and analysis of protein fractions by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE),4 cortical cell separation,13 and external and internal lipid analyses,4 (2) instrumental methods including near-IR;14 (3) improved microscopic analysis by use of CCMI or image analysis,15,16 wavelet texture analysis,17 and an artificial neural network;18 (4) molecular biological methods such as DNA,2,19 polymerase chain reaction−restriction fragment length polymorphism,5 and immunological methods generating monoclonal antibodies.1,6 Each method claimed to be suitable for replacing the microscopic method. Most reliable test methods are, however, based on molecular biological methods such as DNA. However, this method still has a problem; it is not suitable for chemical-treated, dyed, or microbial-attacked fibers, which could damage the DNA of the cashmere fibers.20,21 Furthermore, these molecular biological methods generally are only applicable for soluble proteins, which

1. INTRODUCTION Cashmere, obtained from the domestic goat Capra Hircus Laniger, is designated as specialty or luxury fiber because of its scarcity, high economic value, softness, and luster.1−3 The price of dehaired cashmere often reaches USD 100−150/kg, which is more than 10 times higher than regular wool tops. There are sufficient amounts of fraudulent cashmere in the market because of such a high price.2,3 The fraudulent cashmere fibers have been generally produced by admixing cashmere with other cheaper fibers such as superfine, chemically treated, and enzyme-treated wool (Optim wool).1−6 However, a recent trend indicates that the most common fraudulent cashmere is now prepared by mixing cashmere with yak fibers or sole yak fibers.2,4,12 Under a microscope, these yak fibers have an appearance very similar to that of brown cashmere because of the presence of natural pigment,4 and hence except for very few experts, even regular testing laboratories often fail to distinguish cashmere from yak fibers. This further intensifies the fraudulent use of yak in lieu of cashmere. Such a problem, therefore, calls for a reliable test method to differentiate cashmere from other animal fibers to repress adulteration with cheaper fibers.5 Animal fiber identification is traditionally carried out by an expert analyst using light microscopy (LM) or scanning electron microscopy (SEM).7−10 The most widely used standardized test procedures from ASTM TM D629-08,7 IWTO TM 58-00,8 AATCC TM 20A-2012,9 and ISO 1775110 are based on identification by either LM or SEM or both. The LM test method provides information on the internal and external structures of the fibers, showing details of the shape, cuticle morphology, internal pigmentation, and medullation. On the other hand, the SEM method gives the fine structure of the cuticle cells at high resolution.1 However, the microscopic test method, regardless of standards, is often subjective because it requires a high degree of skill © XXXX American Chemical Society

Received: January 29, 2013 Revised: March 18, 2013 Accepted: March 25, 2013

A

dx.doi.org/10.1021/ie4003415 | Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Industrial & Engineering Chemistry Research

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may not be appropriate for keratin molecules.11 Therefore, new, reliable method has yet to be developed to differentiate cashmere from other cheaper fibers. A recent study indicates that the species identification of animal fiber method (SIAM) is suitable for differentiating species of avian and mammalian fibers. It is a peptide mass fingerprinting method and does not require soluble proteins, fat, nucleic acids, or visible morphological patterns.1 In this method, a mixture of species-specific and species-unspecific digested peptides is obtained by using a high content of trypsin and a reducing agent such as 2-mercaptoethanol. The digested peptides were analyzed by matrix-assisted laser desorption/ionization time-offlight (MALDI-TOF) mass spectrometry, and species-specific peptide masses of certified animal species were identified and used as standards for unknown samples.20,21 The previous study examined and identified many different hair fibers, with emphasis on the furs of domestic dog,20 or historic fibers.21 In addition, MALDI-TOF mass spectrometry has also been employed for other types of proteins such as enzymes.22,23 Even though some testing organizations such as SGS and Gene-Facts Scientific claimed to use MALDI-TOF mass spectrometry in the identification of cashmere and yak, as far as we know, no scientific paper or detailed analyses have been published so far. Therefore, in the present study, we demonstrate a simple, rapid, yet reliable method to qualitatively identify and differentiate cashmere and yak fibers by using a protein fingerprint method with MALDI-TOF mass spectrometry. Yak fiber was included in the study because it was the most common fiber in lieu of cashmere in the commercial market. We hope that such a method can be widely used for identification of cashmere among testing organizations or industry to prevent, or at least minimize, fraudulent labels.

Table 1. Types of Animal Fibers Tested by the SIAM Methods fiber type

label

species

cashmere (25)

A-1 A-2 A-3 A-4 A-5 A-6 B-1 B-2 B-3 B-4 B-5 B-6 C-1 C-2 C-3 C-4 C-5 C-6 D-1 D-2 D-3 D-4 D-5 D-6 E-1 G-5 G-6 H-1 H-2 H-3 H-4 H-5

Chinese Alashan type white Chinese Albas type white Chinese Liaoning type white Chinese brown Chinese light brown Chinese light gray Chinese bleached from white Chinese decolored from brown Chinese decolored from gray Chinese white slipe type Chinese gray slipe type Chinese brown slipe type Chinese infant type Mongolian white Mongolian light gray Mongolian brown Iranian white Iranian dark brown Afghan dark brown Afghan light brown Afghan light gray Afghan fawn Kyrgyz brown Turkish brown Australian white Chinese dark brown Chinese semibleached Chinese gray Chinese brown Chinese decolored Chinese bleached Chinese half-bleached

yak (7)

2. EXPERIMENTAL SECTION 2.1. Materials. As shown in Table 1, a total of 25 cashmere and 7 yak fibers were obtained from a fiber library of CCMI (known as the CCMI Fiber Box 2011 CCMI), Boston, MA. These fibers were used as reference specimens for the SIAM method to compare mass-to-charge (m/z) ion peaks from MALDI-TOF mass spectrometry. All other fiber samples were obtained from commercial cloths with 100% cashmere, or cashmere blends. Trypsin from hog pancreas with an activity of 1500 units/mg was purchased from Sigma-Aldrich. The following chemicals were used: 2-mercaptoethanol and acetonitrile from Merck, trifluoroacetic acid and ammonium bicarbonate from SigmaAldrich, and α-cyano-4-hydroxycinnamic acid (HCCA) and peptide calibration standard II from Bruker. All of the chemicals were reagent grade and were used without any further separation. Water purified by a Millipore water purification system (Bedford, MA) was used throughout the study. 2.2. Sample Preparation. About 4 mg of the fiber sample was placed in an Eppendorf safe-lock tube (1.5 mL size), and 100 μL of 5% 2-mercaptoethanol (25 mmol/L) was added to each sample. The sample was incubated in an Eppendorf comfort thermomixer for 30 min at 95 °C with a speed of 650 rpm. The sample tube was then cooled in an ice bath for 20 min. The trypsin solution was separately prepared by adding 2.5 mg of trypsin in 1 mL of a 25 mM aqueous ammonium bicarbonate solution. The 100 μL trypsin solution was added to the sample tube, which was again incubated for 2 h at 37 °C in a thermomixer. A total of 5 μL of the incubated solution was transferred into a fresh Eppendorf tube and mixed with 50 μL of

the matrix solution. The matrix solution was daily prepared by making a saturated HCCA solution in 50% acetonitrile/1% trifluoroacetic acid along with 5 min of ultrasonication followed by 5 min of centrifugation at 13000 rpm (Eppendorf Centrifuge, 5804R). A total of 1 μL of each sample−matrix mixture was spotted in the MALDI-TOF target plate. 2.3. Mass Spectrometry Analysis. A Bruker Autoflex speed LRF time-of-flight mass spectrometer (Bruker-Daltonic, Bremen, Germany) was used throughout the study. The sample target was MTP 384 target ground steel TF, and the laser used was a Smartbeam-II laser [355 nm, energy 100 μJ/pulse (max 500 μJ)]. This laser was equipped with a focusing lens to control the laser beam size and an attenuator to adjust the power of the laser. Analysis was in a positive mode with an ion source voltage of 19 kV and a reflector voltage of 21 kV. After analysis, the Flash detector, 2-GHz digitizer (BrukerDaltonic, Bremen, Germany) was used as a data acquisition system. The data acquisition software was in an autoexecuted mode with 70−80% laser. The resolution was >8000, and the mass range was collected from 1000 to 4000 Da. For one stored spectrum, at first 2000 shots were summed but then only allowed a maximum of 200 shots per raster position. Finally, the peak was processed using Flexanalysis (Bruker-Daltonic, Bremen, Germany) software with MS windows using an SNAP algorithm. The signal-to-noise threshold was greater than three, and the quality factor threshold was greater than 50. B



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Figure 1. Mass spectrum of the representative cashmere (A1: Chinese Alashan type white) fiber at a m/z ratio between 1000 and 4000 Da.

Figure 2. Mass spectrum of the representative yak (G5: Chinese dark brown) fiber at a m/z ratio between 1000 and 4000 Da.

yak (G5: Chinese dark brown) fibers (Figures 1 and 2) according to their mass-to-charge ratios (m/z) between 1000 and 4000 Da obtained by MALDI-TOF mass spectrometry. We analyzed some important ion peaks in mass spectra such as ratios at m/z 1109, 1151, 1486, 1521, 2036, 2086, 2503, 2519, 2634, and 3266. These ion peaks were produced from a pool of cleavage peptides, which did not originate from a single protein but from a complex mixture of several proteins.20,21 Therefore, no useful information was obtained from the ions with a mass of

For cross-validation of commercial samples, the ISO test method with a light microscope (Olympus BX 51) was used for identification of the cashmere and yak fibers.10

3. RESULTS AND DISCUSSION 3.1. General Analyses of Mass Spectra. Mass spectra of a total of 25 cashmere and 7 yak fibers are listed in the Supporting Information. Here we only display mass spectra of the representative cashmere (A1: Chinese Alashan type white) and C



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Figure 3. Peak intensities of the ion peak at m/z 1109 for various yak (seven) and cashmere (25) fibers.



less than 1000 Da, and the most suitable mass range of peptide detection was from 1000 to 3400 Da for identification of cashmere and yak.11 As shown in Figures 3 and 4, the trends of the peaks at m/z 1109 and 1151 were very similar; i.e., both cashmere and yak fibers revealed these peaks even though the peak intensities tended to be somewhat higher in cashmere than in yak. The

tendency for low peak intensity for the yak type was more prominent at m/z 1151. Especially, H3 yak fiber, Chinese decolored, was extremely low in intensity in both m/z 1109 and 1151. The low intensity peak of H3 also appeared at m/z 1521 and 2086 (both are not shown). Similarly, two decolored cashmere types, B2 and B3, also tended to lower peak intensities in most of the m/z ratios. It was expected that the trypsin process D



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other types of species including cashmere and yak in all four m/z ratios at 1109, 1151, 1486, and 1521 (not shown). 3.2. Fingerprint Peaks for Cashmere. We tried to find specific peaks only appearing in mass spectra of cashmere, so that they could be used as fingerprint peaks. In the case of m/z 1486, the intensities were high in all of the cashmere fibers, whereas

in this study generally digested only the outer protein layers of the samples. Therefore, the chemical treatment such as decoloration tended to modify characteristics of a certain detected peak. The same phenomenon was also observed in the previous study.11 On the other hand, among various species, cashmere B5 and B6 showed generally greater intensities than E



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Obviously, a more specific peak is required for use as a fingerprint peak for differentiation between cashmere and yak fibers. Contrarily, three ion peaks at m/z 2036, 2634, and 3266 could be employed as fingerprint peaks for identification of the cashmere fibers. The ion peak at m/z 2036 was only presented in the cashmere fiber, while no yak fiber showed in this peak (Figure 6). The ion peak at m/z 2634 was also very specific only to the cashmere fiber, as shown in Figure 7. No yak fiber showed in the peak at m/z 2634. The last candidate peak for identification of cashmere was at m/z 3266 (Figure 8). This peak was also clearly specific to the cashmere fiber because no yak fiber showed in the peak at this position while all of the cashmere fibers showed the peak at this position. Therefore, it was concluded that three fingerprint peaks at m/z 2036, 2634, and 3266 could be used for positive identification of cashmere fibers. In addition, both decolored cashmere fibers, B2 and B3, showed the lowest peak intensities at m/z 2036 and 3266, probably for the same reason as discussed above. It should be noted, however, that no detailed analyses were given to the intensities of the fingerprint peaks because the study was mainly for qualitative purposes. 3.3. Fingerprint Peaks for Yak. The two ion peaks at m/z 2503 and 2519 could be specifically used as fingerprint peaks for identification of yak fibers. As shown in Figures 9 and 10, yak fibers showed both peaks at m/z 2503 and 2519, whereas no cashmere fiber showed in these two peaks. Among yak fibers, H2

Figure 11. Specimen from men’s overcoat labeled as 100% cashmere.

those in yak were very low (Figure 5). For example, yak fibers showed low intensities as less than 2500 at m/z 1486 except H1 having 11437 intensity. The intensity of H1 was still less than half that of B2, Chinese decolored from brown, which exhibited the lowest intensity among all cashmere fibers. The low peak intensity at m/z 1486 could be used as a characteristic peak for indication of the yak fiber during a comparison against the cashmere fiber. Although it was not shown here, the ion peak at m/z 1486 generally appeared with very high intensity in regular wool fiber. Therefore, the ion peak at m/z 1485 may be used only between cashmere and yak but not with other animal fibers. F



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Figure 12. Mass spectra of the fiber specimen obtained from a commercial men’s overcoat: (a) warp yarn; (b) weft yarn.

3.4. Commercial Sample Analyses. To further confirm the efficacy of using the SIAM method in identification and differentiation of cashmere and yak, we applied this process to a commercial sample. A black-dyed specimen in a commercial men’s overcoat was purchased from a department store (Figure 11). This sample was originally labeled as a 100% cashmere product. As shown in Figure 12a, MALDI-TOF mass spectrometry revealed that the m/z ratio of this specific specimen was matched with cashmere only in warp yarn at m/z 2036, 2634,

(Chinese brown) demonstrated the highest intensity with 20764 for m/z 2503 and 7166 for m/z 2519. Like other peaks described above, the decolored yak, H3, exhibited again the lowest peak intensity in yak fibers as 408 for m/z 2503 and 120 for m/z 2519. Although the intensity of the ion peak in H3 was much lower compared to those of other yak fibers, it was sufficient for species recognition as a yak fiber. Therefore, the presence of two ion peaks at m/z 2503 and 2519 was a positive identification of yak fibers. G



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complex amino acid sequence analyses. Furthermore, quantitative analysis of animal fibers by the SIAM method is also theoretically possible. The study is currently underway for quantitative identification for cashmere and yak fibers to pass the validity test and for examination of other fibers in the CCMI fiber library.

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ASSOCIATED CONTENT

S Supporting Information *

Mass spectra for 25 cashmere and 7 yak fibers obtained by MALDI-TOF mass spectrometry. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 13. Fiber identification results from commercial specimens in 2012. These samples were all labeled as 100% cashmere: 53% cashmere; 41% mixture of cashmere and yak; 6% mixture of cashmere and wool.

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and 3266. On the other hand, the ion peaks of the weft yarn were identical with fingerprint ion peaks for yak reference at m/z 2503 and 2519. This substantiated that the MALDI-TOF mass spectrometry could be applicable to identification of commercial samples to detect fraudulent labeling. We further tested 32 commercial samples labeled as 100% cashmere submitted by one company in 2012. The results indicated that almost half of the samples (41%) were made of solely yak fibers or a mixture of cashmere and yak fibers, while 6% of the samples were actually a mixture of cashmere and wool, as illustrated in Figure 13. This result by MALDI-TOF analysis was also cross-validated by LM testing, as shown in Figure 14.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +822 8200626. Fax: +822 8178346. E-mail: [email protected]. kr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank to Dr. Youngmin Jeon in FITI for his helpful discussion.

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4. CONCLUSION We demonstrated that the protein fingerprint method using MALDI-TOF mass spectrometry can be specifically used in the identification and differentiation of cashmere and yak fibers. Three fingerprint ion peaks for cashmere were identified: m/z ratios at 2036, 2634, and 3266. However, two ion peaks at m/z 2503 and 2519 were very specific for yak fibers. This test method can be effectively used in qualitatively differentiating cashmere fiber from yak fibers in the item with fraudulent label without

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Figure 14. Fiber identification results from commercial specimens using LM (400×). H



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Qualitative Identification of Cashmere and Yak Fibers by Protein

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