Abstract
The demand for leather goods has grown globally in recent years. Industry revenue is forecast to reach $91.2 billion by 2018. There is an ongoing labelling problem in the leather items market, in that it is currently impossible to identify the species that a given piece of leather is derived from. To address this issue, we developed a rapid and simple method for the specific identification of leather derived from cattle, horses, pigs, sheep, goats, and deer by analysing peptides produced by the trypsin-digestion of proteins contained in leather goods using liquid chromatography/mass spectrometry. We determined species-specific amino acid sequences by liquid chromatography/tandem mass spectrometry analysis using the Mascot software program and demonstrated that collagen α-1(I), collagen α-2(I), and collagen α-1(III) from the dermal layer of the skin are particularly useful in species identification.
Keywords: quality control, labelling, product tracing, collagen
INTRODUCTION
In recent years, the demand for leather goods such as handbags, clothing, gloves, and household items has grown. Although the rising cost of raw materials has impaired profit margins, the leather goods market is forecast to continue to expand.1) To provide transparency for maintaining the quality and value of leather goods in the global supply chain, it has become neccessary to verify the specific animal origin of such products. Especially in Japan, leather materials must be labelled with their species of origin, according to the Household Goods Quality Labeling Act, when the leather items are wholly or partly made of synthetic leather, cowhide, horse leather, pigskin, sheepskin, goatskin, or deerskin.2) In addition to Japanese cases, almost all countries now have labelling requirements under textile acts. In the textile industry, there is a need to identify products that are labelled as pure cashmere but are actually made from cashmere blended with cheaper animal fibres. To meet this need, in addition to official conventional microscopic methods such as those included in ISO 17751 and JIS L 1030-2, some novel testing methods for the identification and quantification of such products using mass spectrometry have been developed.3–7) However, there is currently no officially approved method for identifying the animal species from which leather goods are derived, and the few previous studies that have been conducted in this area were mainly related to archaeology.8) Currently, many testing laboratories have been examining a vareity of approaches for identifying the animal species used in leather goods: (1) External observation (thickness, size, color and pattern, the line of the fur, gloss, touch feeling); (2) Microscopic images (hair follicles array, cross sections, leather grain, medulla); and (3) DNA analysis. However, external observations and microscopic images are subjective and time-consuming due to their reliance on an operator. In addition, DNA sometimes cannot be detected in leather specimens due to degradation that occurs during stages of the production process such as tanning and dyeing.9) Therefore, to aid the smooth distribution of leather items and protect consumers, alternative reliable identification methods would be highly desirable.
In this study, we analysed trypsin-digested peptides derived from proteins contained in leather goods using the widely-used LC/MS approach and confirmed the presence of species-specific amino acid sequences. Furthermore, we validated these target peptides with different LC/MS instruments and developed a simple and reliable method that enables the differential identification of the animal species from which high production volume leathers are derived among cattle, horses, pigs, sheep, goats, and deer, using typical m/z values.
EXPERIMENTAL
Leather samples
The samples used in this study consisted of commercial leather goods that were chrome-tanned, which is the tanning process used for 80–90% of all leather goods around the world, and with vegetable. Forty-nine samples from 6 species were used (Table 1). The samples were washed with petroleum ether (first grade, Wako, Osaka, Japan), and then air-dried prior to analysis.
Table 1. Samples from 6 types of animal leather.
| No. | Sample | Tannning | Finishing | Colour |
|---|---|---|---|---|
| C1 | Cattle | Chrome | Anilin | Brown |
| C2 | Cattle | Chrome | Pigment | Brown |
| C3 | Cattle | Vegetable | Camel | |
| C4 | Cattle | Chrome | Black | |
| C5 | Cattle | Chrome | White | |
| C6 | Cattle | Chrome | Brown | |
| C7 | Cattle | Chrome | Drumdye | Green |
| C8 | Cattle | Vegetable | Glazing | Black |
| C9 | Cattle | Vegetable | Brown | |
| C10 | Cattle | Chrome | Drumdye | White |
| H1 | Horse | Chrome | Black | |
| H2 | Horse | Chrome | Brown | |
| H3 | Horse | Chrome | Brown | |
| H4 | Horse | Chrome | Anilin | Beige |
| H5 | Horse | Chrome | Anilin | Khaki |
| H6 | Horse | Chrome | Camel | |
| H7 | Horse | Chrome | Metal | Pink |
| P1 | Pig | Chrome | Green | |
| P2 | Pig | Chrome | White | |
| P3 | Pig | Chrome | Blue | |
| P4 | Pig | Chrome | Camel | |
| P5 | Pig | Chrome | Black | |
| P6 | Pig | Chrome | Buffing | Red |
| P7 | Pig | White | Buffing | Purple |
| P8 | Pig | White | Buffing | Orange |
| S1 | Sheep | Chrome | Black | |
| S2 | Sheep | Chrome | Full grain | Black |
| S3 | Sheep | Chrome | Red | |
| S4 | Sheep | Chrome | Cream | |
| S5 | Sheep | Chrome | White | |
| S6 | Sheep | Chrome | Smooth | Brown |
| S7 | Sheep | Chrome | Pigment | Brown |
| S8 | Sheep | Chrome | Buffing | Black |
| G1 | Goat | Chrome | Black | |
| G2 | Goat | Chrome | Black | |
| G3 | Goat | Vegetable | Camel | |
| G4 | Goat | Chrome | Semi anilin | Olive |
| G5 | Goat | Chrome | Shrinking | Red |
| G6 | Goat | Chrome | Metal | Grey |
| G7 | Goat | Chrome | Metal | Beige |
| G8 | Goat | Chrome | Titling | Bronze |
| D1 | Deer | Chrome | White | |
| D2 | Deer | Chrome | Silicon | Beige |
| D3 | Deer | Chrome | Yellow | |
| D4 | Deer | Chrome | Blue | |
| D5 | Deer | Chrome | Brown | |
| D6 | Deer | Chrome | Pink | |
| D7 | Deer | Chrome | Red | |
| D8 | Deer | Chrome | Camel |
Sample preparation
The samples were placed into liquid nitrogen for 10 min and then crushed into a powder using a sample crusher (Multi-beads Shocker®, Yasui Kikai, Osaka, Japan) at 3,000 rpm for 30 s.
Powdered leather samples, 0.5 mg, were weighed directly into 1.5 mL Safe-Lock™ tubes (Eppendorf, Hamburg, Germany). Five hundred microlitres of 25 mM NH4HCO3 was added, and the resulting suspension vortexed at full speed for 1 min to mix. The samples were then incubated at 60°C with gentle shaking at 250 rpm for 1 h. After cooling to room temperature for 10 min, 20 μL of a 0.4 μg/μL sequencing grade trypsin solution (Promega, Madison, WI, USA) was added and the samples were incubated at 37°C overnight. The supernatant was filtered through a centrifugal filter unit (Agilent Technologies, Santa Clara, CA, USA) with a pore size of 0.22 μm at 4,900×g for 10 min, and the solution was frozen and evaporated using a freeze dryer (FDU-1200, EYELA, Tokyo, Japan). The following morning, the resulting peptide mixture was dissolved in 100 μL of 0.1% (v/v) formic acid, followed by centrifugal filtration under the same conditions as above.
LC/MS/MS analysis
To explore and identify species-specific peptides, LC/MS/MS analysis was performed on an LC/MS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan) interfaced with a nano reverse-phase LC system (Shimadzu). LC separation was performed using a PicoFrit™ BetaBasic® C18 column (New Objective, Woburn, MA, USA) at a flow rate of 300 nL/min. Two microlitres of each sample were injected into the instrument. Peptides were eluted using gradients of 5–45% acetonitrile containing 0.1% (v/v) formic acid and sprayed directly into the mass spectrometer. The positive ion mode was utilised as the electrospray ionisation (ESI) source in this operation. The capillary temperature and electrospray voltage were set at 200°C and 2.5 kV, respectively. MS/MS data were acquired in the data-dependent mode using the LCMS Solution software (Shimadzu) and were converted to a single text file containing the observed precursor peptide m/z, fragment ion m/z, and intensity values using Mascot Distiller (Matrix Science, London, UK). The file was analysed using the Mascot (Matrix Science) MS/MS Ion Search function to search and assign the obtained peptides to the NCBI non-redundant database. The search parameters were set as follows: Database, NCBInr; Taxonomy, all; Enzyme, trypsin; Peptide m/z tolerance, ±0.05 Da; and MS/MS m/z tolerance, ±0.05 Da.
LC/MS analysis
To validate the presence of species-specific peptides, mass spectrometry was performed using an ESI QqQ mass spectrometer system (G6460, Agilent Technologies). The system was operated using the following conditions in the positive ion mode: capillary voltage, 3.5 kV; nozzle voltage, 2.0 kV; nebuliser pressure, 50 psi; drying gas temperature, 300°C; sheath gas temperature, 350°C; fragmenter voltage, 150 V. Two microlitres of the peptide samples were injected onto a C18 reverse phase column (150 mm length and 2.1 mm inner diameter, CERI, Tokyo, Japan) that was maintained at 40°C using a 30 min linear gradient from 2–60% acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.2 mL/min.
RESULTS AND DISCUSSION
Identification of species-specific peptides for each animal leather
Trypsin-digested samples from 6 animal species were analysed and species-specific peptides were identified using the Mascot software to perform error-tolerant searches. To identify peptides containing hydroxyproline and hydroxylysine, searching for amino acid sequence information was conducted by removing a protein-translational modification and allowing for, not only the oxidation of methionine, but also of proline and lysine. According to this approach, the detected peptides matched collagen α-1(I), collagen α-2(I), and collagen α-1(III). These peptides are mainly derived from the dermal layer of skin, which makes up the major portion of most leather items. Collagen is the most abundant protein produced by mammals, and types I and III collagen are formed in animal skin in a higher proportion relative to other types of collagen. Type I collagen is a heterotrimer consisting of two α-1 chains and one α-2 chain. Collagen α-1(III) is a fibrillar collagen that is frequently associated with type I collagen.10) Although the abundance and balance of type I and III collagen may vary in animals with age or injury,11) we consistently detected species-specific peptides, the amino acid sequences for which are shown in Table 2. While creating leather items from raw skin requires processing by both chemical and physical means, the collagen peptides that enable species identification were consistently detected in all samples. Collagen-derived peptides show a high degree of amino acid sequence conservation among animal species due to their important roles in providing mechanical stability and elasticity to skin. Therefore, species discrimination could be largely accomplished based on small differences in the amino acid sequences of these peptides.
Table 2. Amino acid sequences of the species-specific peptides.
| No. | AA | Amino Acid Sequence | Protein (Collagen) | Mr (Calc) | m/z | Cattle | Horse | Pig | Sheep | Goat | Deer |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 472–486 | GEPGPAGLPGPPGER+3Ox(P) | α 1(I) | 1434.6 | 718.3 | + | − | + | + | + | + |
| GEPGPTGLPGPPGER+3Ox(P) | α 1(I) | 1464.6 | 733.3 | − | + | − | − | − | − | ||
| 2 | 493–504 | GFPGADGVAGPK+Ox(P) | α 1(I) | 1087.5 | 544.7 | + | + | + | − | − | + |
| GFPGSDGVAGPK+Ox(P) | α 1(I) | 1103.5 | 552.7 | − | − | − | + | + | − | ||
| 3 | 763–795 | GLTGPIGPPGPAGAPGDKGEAGPSGPAGPTGAR+2Ox(P) | α 1(I) | 2852.4 | 951.8 | + | − | − | − | − | − |
| GLTGPIGPPGPAGAPGDKGETGPSGPAGPTGAR+2Ox(P) | α 1(I) | 2882.4 | 961.8 | − | + | + | + | + | + | ||
| 4 | 910–933 | GETGPAGRPGEVGPPGPPGPAGEK+Ox(K)+2Ox(P) | α 1(I) | 2215.0 | 739.4 | + | − | − | − | + | + |
| GETGPAGRPGEAGPPGPPGPAGEK+Ox(K)+2Ox(P) | α 1(I) | 2187.0 | 730.0 | − | − | + | − | − | − | ||
| 918–933 | AGEVGPPGPPGPAGEK+2Ox(P) | α 1(I) | 1447.6 | 724.8 | − | − | − | + | − | − | |
| 5 | 1062–1083 | SGDRGETGPAGPAGPIGPVGAR | α 1(I) | 1974.9 | 659.3 | + | − | − | + | + | + |
| SGDRGEAGPAGPAGPIGPVGAR | α 1(I) | 1944.9 | 649.3 | − | + | − | − | − | − | ||
| SGDRGETGPAGPAGPVGPVGAR | α 1(I) | 1960.9 | 654.6 | − | − | + | − | − | − | ||
| 6 | 326–340 | GIPGPVGAAGATGAR+Ox(P) | α 2(I) | 1266.6 | 634.3 | + | − | − | + | + | + |
| GIPGPAGAAGATGAR+Ox(P) | α 2(I) | 1238.6 | 620.3 | − | + | + | − | − | − | ||
| 7 | 463–484 | EGPVGLPGIDGRPGPIGPAGAR+Ox(P) | α 2(I) | 2055.0 | 686.0 | + | + | − | − | − | + |
| EGPAGLPGIDGRPGPIGPAGAR+Ox(P) | α 2(I) | 2027.0 | 676.6 | − | − | + | + | + | − | ||
| 8 | 542–571 | GEQGPAGPPGFQGLPGPAGTAGEAGKPGER+Ox(K)+2Ox(P) | α 2(I) | 2791.3 | 931.4 | + | − | − | + | + | − |
| GEQGPAGPPGFQGLPGPAGTAGEVGKPGER+Ox(K)+2Ox(P) | α 2(I) | 2819.3 | 940.7 | − | + | − | − | − | − | ||
| 9 | 572–586 | GI(L)PGEFGLPGPAGAR+2Ox(P) | α 2(I) | 1426.7 | 714.3 | + | + | − | + | + | + |
| GIPGEFGLPGPAGPR+2Ox(P) | α 2(I) | 1452.7 | 727.3 | − | − | + | − | − | − | ||
| 10 | 590–607 | GPPGESGAAGPTGPIGSR+Ox(P) | α 2(I) | 1579.7 | 790.8 | + | − | − | + | + | − |
| GPPGESGAAGPAGPIGSR+Ox(P) | α 2(I) | 1549.7 | 775.8 | − | + | + | − | − | + | ||
| 11 | 776–792 | GDGGPPGATGFPGAAGR+2Ox(P) | α 2(I) | 1472.6 | 737.3 | + | − | + | + | + | + |
| GDGGPPGVTGFPGAAGR+2Ox(P) | α 2(I) | 1500.6 | 751.3 | − | + | − | − | − | − | ||
| 12 | 881–904 | GLPGVAGSVGEPGPLGIAGPPGAR+3Ox(P) | α 2(I) | 2130.1 | 711.0 | + | − | + | + | + | + |
| 2130.1 | 1066.0 | ||||||||||
| GLPGVAGSLGEPGPLGIAGPPGAR+3Ox(P) | α 2(I) | 2144.1 | 715.7 | − | + | − | − | − | − | ||
| 2144.1 | 1073.0 | ||||||||||
| 13 | 977–994 | GEPGPAGAVGPAGAVGPR+Ox(P) | α 2(I) | 1531.7 | 766.8 | + | − | − | − | − | + |
| GEPGPVGSVGPVGAVGPR+Ox(P) | α 2(I) | 1603.8 | 802.9 | − | + | − | − | − | − | ||
| GEPGPAGSVGPAGAVGPR+Ox(P) | α 2(I) | 1547.7 | 774.8 | − | − | + | − | − | − | ||
| GEPGPVGAVGPAGAVGPR+Ox(P) | α 2(I) | 1559.8 | 780.9 | − | − | − | + | + | − | ||
| 14 | 1066–1078 | IGQPGAVGPAGIR+Ox(P) | α 2(I) | 1207.6 | 604.8 | + | − | − | − | − | − |
| SGQPGTVGPAGVR | α 2(I) | 1181.6 | 591.8 | − | + | − | − | − | − | ||
| TGQPGAVGPAGIR | α 2(I) | 1179.6 | 590.8 | − | − | + | + | + | + | ||
| 15 | 396–413 | GEMGPAGIPGAPGLI(L)GAR+Ox(M)+Ox(P) | α 1(III) | 1651.8 | 826.9 | + | + | − | + | + | + |
| GEMGPAGIPGAPGLMGAR+2Ox(M)+Ox(P) | α 1(III) | 1701.7 | 851.8 | − | − | + | − | − | − | ||
| 16 | 933–956 | GAPGPQGPPGAPGPLGIAGLTGAR+2Ox(P) | α 1(III) | 2097.0 | 700.0 | + | − | − | + | + | − |
| α 1(III) | 2097.0 | 1049.5 | |||||||||
| GSPGPQGPPGVPGPSGLIGITGAR+3Ox(P) | α 1(III) | 2173.1 | 725.3 | − | + | − | − | − | − | ||
| GSPGPQGPPGAPGPGGISGITGAR+3Ox(P) | α 1(III) | 2088.9 | 1045.5 | − | − | + | − | − | − |
“+” means present and “−” means absent.
Selection of peptides for identification
Our objective was to select correlated marker peptides that are detected consistently by different instruments. To accomplish this, we analysed the same leather specimens using different LC/MS instrumentation. Our results showed that peptides No. 3, 4, 5, 6, 7, 9, 10, 12, 13, 14 and 15 as illustrated in Table 2, were stably detected by 2 different types of mass spectrometers.
We concluded that it is not necessary to detect all peptides to distinguish among leather from different animals, as long as the method is able to discriminate between species-specific marker peptides. Thus, a sufficient number of species-specific peptides that have a high signal-to-noise ratio at or higher than 3 were adopted as markers to avoid false positives, because of the possibility of genetic polymorphism. In the present case, we selected peptides Nos. 3, 4, and 5 of collagen α1(I), Nos. 6, 7, 9, 10, 12, 13 and 14 of collagen α2(I) and No. 15 of collagen α1(III), as illustrated in Table 2. As shown in Fig. 1, to simplify the testing method and shorten the time required to complete an analysis, we established the following procedure: First, leather derived from cattle, horses, and pigs which is produced in high volumes, should be identified by the following fragments: m/z 951.8 and m/z 604.8 for cattle, m/z 1073.0, m/z 802.9, and m/z 591.8 for horses and m/z 654.6, m/z 774.8 and m/z 851.8 for pigs. Subsequently, leather derived from sheep, goats, and deer could be identified on the basis of the matrix of 6 types of peptides. Sheep, goats and deer have the m/z 714.3 in common, which could be useful for a positive control reference in the tests. Deer leather produces an m/z 686.0 and an m/z 766.8 fragment, while sheep and goats share a common m/z 790.8 fragment and goats and deer share a common m/z 739.4 fragment. After deer leather is identified, a typical m/z 724.8 fragment can be used to distinguish between leather from the sheep and goats. As shown in Table 3, the animal types in 49 samples were correctly identified at a positive SNR using this simple method. These marker peptides showed 100% species specificity and selectivity among all tested samples, including samples that had been strongly tanned, buff-treated, or dark-coloured, which present difficulties when conventional microscopy and DNA analysis methods are used for species identification. Only sample S5 which was obtained from a young sheep provided low intensity m/z 714.3 and m/z 790.8 (under SNR value 5) fragments on an Agilent G6360 instrument as shown in Fig. 2 .
Fig. 1. Flow chart for leather identification. The type of animal from which a given leather sample is derived can be rapidly identified by detecting specific marker peptides.
Table 3. Detected marker peptides for identifying the animal species of leather samples.
| No. | Sample | m/z 951.8 | m/z 604.8 | Result | ||
|---|---|---|---|---|---|---|
| C1 | Cattle | + | + | Cattle | ||
| C2 | Cattle | + | + | Cattle | ||
| C3 | Cattle | + | + | Cattle | ||
| C4 | Cattle | + | + | Cattle | ||
| C5 | Cattle | + | + | Cattle | ||
| C6 | Cattle | + | + | Cattle | ||
| C7 | Cattle | + | + | Cattle | ||
| C8 | Cattle | + | + | Cattle | ||
| C9 | Cattle | + | + | Cattle | ||
| C10 | Cattle | + | + | Cattle | ||
| No. | Sample | m/z 1073.0 | m/z 802.9 | m/z 591.8 | Result | |
| H1 | Horse | + | + | + | Horse | |
| H2 | Horse | + | + | + | Horse | |
| H3 | Horse | + | + | + | Horse | |
| H4 | Horse | + | + | + | Horse | |
| H5 | Horse | + | + | + | Horse | |
| H6 | Horse | + | + | + | Horse | |
| H7 | Horse | + | + | + | Horse | |
| No. | Sample | m/z 654.6 | m/z 774.8 | m/z 851.8 | Result | |
| P1 | Pig | + | + | + | Pig | |
| P2 | Pig | + | + | + | Pig | |
| P3 | Pig | + | + | + | Pig | |
| P4 | Pig | + | + | + | Pig | |
| P5 | Pig | + | + | + | Pig | |
| P6 | Pig | + | + | + | Pig | |
| P7 | Pig | + | + | + | Pig | |
| P8 | Pig | + | + | + | Pig | |
| No. | Sample | m/z 714.3 | m/z 790.8 | m/z 724.8 | Result | |
| S1 | Sheep | + | + | + | Sheep | |
| S2 | Sheep | + | + | + | Sheep | |
| S3 | Sheep | + | + | + | Sheep | |
| S4 | Sheep | + | + | + | Sheep | |
| S5 | Sheep | + | + | + | Sheep | |
| S6 | Sheep | + | + | + | Sheep | |
| S7 | Sheep | + | + | + | Sheep | |
| S8 | Sheep | + | + | + | Sheep | |
| No. | Sample | m/z 714.3 | m/z 739.4 | m/z 790.8 | Result | |
| G1 | Goat | + | + | + | Goat | |
| G2 | Goat | + | + | + | Goat | |
| G3 | Goat | + | + | + | Goat | |
| G4 | Goat | + | + | + | Goat | |
| G5 | Goat | + | + | + | Goat | |
| G6 | Goat | + | + | + | Goat | |
| G7 | Goat | + | + | + | Goat | |
| G8 | Goat | + | + | + | Goat | |
| G9 | Goat | + | + | + | Goat | |
| No. | Sample | m/z 714.3 | m/z 739.4 | m/z 686.0 | m/z 766.8 | Result |
| D1 | Deer | + | + | + | + | Deer |
| D2 | Deer | + | + | + | + | Deer |
| D3 | Deer | + | + | + | + | Deer |
| D4 | Deer | + | + | + | + | Deer |
| D5 | Deer | + | + | + | + | Deer |
| D6 | Deer | + | + | + | + | Deer |
| D7 | Deer | + | + | + | + | Deer |
| D8 | Deer | + | + | + | + | Deer |
“+” means positive (above SNR value 3).
Fig. 2. Extracted ion chromatograms of peptides from sample S5 as listed in Table 1. For (a) at m/z 714.3 and for (b) at m/z 790.8.
CONCLUSION
Although labelling requirements for leather goods have been established worldwide, there is still no robust method for identifying the animal species from which the leather is derived. Therefore, we developed a method for the differential identification of cattle, horses, pigs, sheep, goats, and deer, which are the main animal sources for most commercial leather goods. This method is applicable for use in the analysis of both tanned or treated leathers that are unsuitable for DNA analysis, since the processes involved in their production cause relatively little damage to collagen, thus permitting collagen-derived peptides to be accurately detected by LC/MS. The experiments show that species-specific peptides derived from collagen α-1(I), collagen α-2(I), and collagen α-1(III) were present in the samples. These 3 fibrillar proteins are major constituents of the dermal layer of skin, which comprises the main part of leather goods. In addition, we carefully selected marker peptides that are not affected by differences among various LC/MS instruments, in order to establish a simple and rapid identification procedure.
This objective technique for identifying the animal species of leather products provides numerous practical advantages, such as high repeatability and reproducibility. We hope that the use of this method will reduce the fraudulent labelling problems associated with the leather market and expect it to be globally standardised to complement current subjective tests.
Mass Spectrom (Tokyo) 2016; 5(1): A0046
- LC/MS
liquid chromatography/mass spectrometry
- LC/MS/MS
liquid chromatography/tandem mass spectrometry
- IT-TOF
ion trap-time of flight
- ESI
electrospray ionisation
- SNR
signal to noise ratio
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