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Semi-nondestructive certification of crocodilian leather by LC–MS detection of collagen marker peptides Yuki Kumazawa, Shunji Hattori, and Yuki Taga Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05180 • Publication Date (Web): 01 Jan 2019 Downloaded from http://pubs.acs.org on January 2, 2019
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Analytical Chemistry
Semi-nondestructive certification of crocodilian leather by LC–MS detection of collagen marker peptides Yuki Kumazawa,†,‡ Shunji Hattori†,‡ and Yuki Taga*,† †Japan ‡Nippi
Institute of Leather Research, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan Research Institute of Biomatrix, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan
ABSTRACT: Leather produced from crocodile, alligator, and caiman skin is widely used in the fashion industry. Crocodilian leather is generally more expensive than mammalian leather, and the value greatly differs even between the crocodilian species. However, inappropriate labeling of the animal source on leather products sometimes arises from accidental or fraudulent substitution, which is difficult to unambiguously detect by existing methods. In the present study, animal source identification of crocodilian leather was carried out using type I collagen-derived marker peptides generated after dechroming, heat denaturation, and trypsin digestion. Definitive discrimination between the three crocodilian species and also a related species, lizard, was achieved based on the detection patterns of selected six marker peptides, determined by LC–MS. Furthermore, powdering of the leather samples enabled a reduction in the sample amount required and allowed the elimination of the dechroming step. Approximately 100 µg of powder was taken from commercial leather watch straps by filing, resulting in only slight damage to the undersides of the straps. The animal sources of the crocodilian products and also a crocodile-embossed calf product were successfully identified using a combination of the crocodilian marker peptides and previously established mammalian marker peptides. This semi-nondestructive species identification method is not only useful for certification of leather products but also for monitoring of international trade of leather and skin.
INTRODUCTION Crocodilian skin is used in the manufacturing of exotic leather products, such as garments, handbags, wallets, shoes, and watch straps. The overall volume of world trade in skin from crocodilian species, which encompasses crocodiles, alligators, and caimans, was 1.5 million items per year in 2015.1 In general, due to its unique scale patterns, crocodilian leather is more expensive than leather produced from mammals, such as cattle, pig, and goat. Furthermore, the value of leather greatly differs even between the crocodilian species, indicating the importance of certification of the animal source. Caiman leather is inferior compared to “classic” leather produced from crocodile and alligator because the bony plates in the ventral scales of caiman cause pitted and discolored appearance.2 Species declaration is required for international trade of any type of crocodilian skin and leather under the protection of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).3 Inappropriate labeling of crocodilian species on leather products sometimes arises from accidental or fraudulent substitution, which has become a problem in commercial and international trade. Therefore, a reliable and definitive method for animal source identification is in high demand for the authentication of crocodilian leather. Several morphological features of crocodilian leather give some clues to distinguishing the animal origin. For example, sensory pits in the ventral scales are specific to crocodile leather,4,5 and umbilical scars showing distinct web-like patterns are specific to alligator leather.4,6 However, these features often disappear during the leather manufacturing process, and species discrimination becomes more difficult when trying to identify the leather source of small products, such as watch straps. Microscopic observation using scanning electron microscopy (SEM) is generally used for the identification of the animal source of mammalian leather.7-9 In addition, discrimination of crocodilian leather from mammalian leather can be easily performed by SEM
observation of the cross-sectional fiber structure of collagen and the texture and pore patterns on the leather surface. However, discriminating between crocodilian species is difficult because of their similar characteristics in SEM. PCR amplification of mitochondrial DNA is a highly sensitive technique that allows high taxonomic specificity in the tracing of the animal origin, and its applications in leather identification have been reported for mammals, such as cattle, sheep, and goat.10-12 For crocodilian species, the DNA-based approach also has been used in several studies analyzing raw tissue and blood.13-15 Furthermore, a recent study detailed the identification of saltwater crocodile in processed foods using double gene targeting short-amplicon PCR, which was shown to be resistant to thermal treatment.16 However, some concerns exist with regard to leather analysis, such as DNA degradation caused by various chemical treatments for leather manufacturing (acid, alkaline, and tanning) and inhibition of PCR amplification by co-extracted compounds.17 There is only reported study where Chinese alligator was identified for tanned hide by PCR,13 but otherwise, successful species identification of crocodilian leather has not been achieved. To the best of our knowledge, unambiguous species discrimination between the three crocodilian species (crocodile, alligator, and caiman) used in leather production has not been achieved by any method. Type I collagen, consisting of two α1 chains and a genetically distinct α2 chain, is a major protein component of skin. Cross-linking of collagen by the chemical tanning process in leather production using chrome or vegetable agents is important as it renders the leather heat stable and protects it against bacterial putrefaction.18 The primary structure features of collagen are a repeating Gly-Xaa-Yaa sequence and prolyl hydroxylation at the Yaa position forming 4-hydroxyproline (4-Hyp). Over the last decade, mass spectrometric detection of interspecies differences in the amino acid sequence of target proteins has been proven to be a highly promising method for species certification, especially in the food industry.19 The
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proteomic approach using LC–MS also has been reported for collagen-based materials, including gelatin,20,21 glue,22,23 and clothing.24 The exact sequence information of peptide fragments generated by enzymatic digestion (typically using trypsin) can be obtained via database search of acquired MS/MS spectra, which enables highly sensitive and reliable species identification. However, this approach cannot be used for crocodilian species because their collagen sequence information is almost completely absent from public databases. Only type I collagen sequences of alligator species (Alligator mississippiensis and Alligator sinensis) are available in the UniProt database. We recently developed a novel LC–MS method to achieve animal source identification of mammalian leather, even when the sequence information of the target species is publicly unavailable.25 Similar to other proteomic methods, we first performed identification of peptide fragments from type I collagen by database search of MS/MS spectra obtained for trypsin-digested leathers. The high similarity in collagen sequences among vertebrates led to the identification of many peptides from common sequence regions of different animals, even for uncharacterized species. All of the identified peptides were candidates of marker peptides with no regard for the provenance of the peptide sequence. Six marker peptides were finally selected to give species-specific detection patterns between six animals that are used in leather production (cattle, horse, pig, sheep, goat, and deer). For example, although deer type I collagen sequences (both α1(I) and α2(I) chains) are not completely known, five marker peptides were detected for this species, which indicates the wide species applicability of the method. In fact, we previously demonstrated its application in the identification of the origin of glue, which additionally included rabbit and sturgeon.26 In the present study, we used this methodology for determining the animal source of leather samples derived from three crocodilian species and also a related species, lizard. An almost nondestructive sampling procedure was established for the expensive leather, which enabled species identification of commercial watch straps without causing any noticeable damage to the products. EXPERIMENTAL SECTION Leather Samples and Watch Straps. Chrome-tanned crocodile, alligator, caiman, and lizard leathers (summarized in Table S1) were kindly gifted from Horiuchi Trading (Tokyo, Japan). Crocodile, alligator, caiman, and crocodile-embossed calf watch straps were purchased from a market (Chiba, Japan). Trypsin Digestion. The leather samples were cut into approximately 1 × 1 mm pieces or were powdered using a steel file. Ten milligrams of the samples were subjected to dechroming, heat denaturation, and trypsin digestion processes as described previously.25 In brief, the samples were slowly shaken in 0.25% calcium hydroxide at room temperature for 2 h. After washing with distilled water and heating at 80°C for 30 min in distilled water, the samples were digested with 50 µg of trypsin (Sigma-Aldrich, St. Louis, MO) in 100 mM Tris–HCl/1 mM CaCl2 (pH 7.6) at 37°C for 4 h with shaking. After acidification with formic acid, the supernatants were filtered through a 0.45 µm filter and subjected to LC–MS/MS or LC–MS analysis in multiple reaction monitoring (MRM) mode. Approximately 100 µg of powders taken from the undersides of respective watch straps by filing were similarly
digested with 0.5 µg of trypsin, but without the dechroming treatment, and subjected to MRM analysis. LC–MS/MS Analysis for Peptide Identification. The tryptic digests of the leather samples were analyzed by LC– MS/MS on a maXis II quadrupole time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled to a Shimadzu Prominence UFLC-XR system (Shimadzu, Kyoto, Japan). The sample solutions were loaded onto an Ascentis Express C18 HPLC column (2.7 µm particle size, L × I.D. 150 mm × 2.1 mm; Supelco, Bellefonte, PA) with a binary gradient of 0.1% formic acid and acetonitrile at a flow rate of 200 µL/min for 30 min as described previously.25 The MS scan and MS/MS acquisition were performed over the m/z ranges of 50–2150 with a frequency of 8 Hz. Peptide identification was performed by searching the MS/MS data converted to .mgf files against a local type I collagen database, which was modified from a previous one25,26 by adding publicly available alligator and lizard sequences, using ProteinPilot software 4.0 (AB Sciex, Foster City, CA). The MS datasets have been deposited to the ProteomeXchange consortium via the jPOST partner repository with the dataset identifier PXD011575 (review only access site, https://repository.jpostdb.org/preview/11503422035bdfdd54bf 10e, with access key 1952).27,28 MRM Analysis of Marker Peptides. Marker peptides selected from the identified peptides were monitored by LC– MS in MRM mode on a 3200 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer (AB Sciex) coupled to an Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA). The tryptic digests of the leather samples and watch straps were loaded onto an Ascentis Express C18 HPLC column (5 µm particle size, L × I.D. 150 mm × 2.1 mm; Supelco) with a binary gradient of 0.1% formic acid and acetonitrile at a flow rate of 500 µL/min for 10 min as described previously.25 The MRM transitions for marker peptides are shown in Table S2. The detection threshold for maker peptides was set to a signal-to-noise ratio of 10. RESULTS AND DISCUSSION Selection of Marker Peptides for Species Discrimination of Crocodilian Leather. We first performed LC–MS/MS identification of type I collagen-derived peptides using tryptic digests of dechromed leather samples (one each from crocodile, alligator, caiman, and lizard leathers) as reported previously.25 From the identified peptide list, we excluded peptides where any one of Pro residues at the Yaa position were not hydroxylated, and peptides generated with a missed cleavage, except at the Arg- or Lys-Hyp bond, were also omitted. The remaining peptides were then monitored by MRM analysis to check whether the marker peptide candidates were detected or not for the respective species. Two rules were set for marker peptide selection to avoid false-positive detection and misidentification: (1) more than two marker peptides must be detected for each animal and (2) there must be at least two differences in the detection patterns of marker peptides between the respective species (crocodile, alligator, caiman, and lizard). Finally, six tryptic peptides from α1(I) and α2(I) chains were selected as marker peptides C1–6 (Table 1). Four marker peptides were detected for alligator (C3, C4, C5, and C6). In addition, two or four were detected for crocodile (C3 and C5) and caiman (C2, C3, C5, and C6), respectively, both of whose type I collagen sequences are
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Analytical Chemistry publicly unknown. Distinct detection was also shown for lizard (C1 and C2). MRM chromatograms of the marker peptides for the respective species are shown in Figure 1. Detection of the marker peptides by MRM analysis enabled a straightforward judgment of the animal origin with rapid analysis time (10 min). The different and species-specific detection patterns were confirmed to be reproducible for various leathers, listed in Table S1, covering almost all the crocodilian and lizard species used in leather production.1 We previously established other six marker peptides selected from type I collagen to discriminate between different types of mammalian leather (marker peptides M1–6; Table S3).25 None of the mammalian marker peptides were detected for crocodilian and lizard leather samples (Figure 1), while the newly selected marker peptides (C1–6) were not detected for leather samples derived from mammals (cattle, horse, pig, sheep, goat, and deer; Figure S1). Therefore, the combination of all 12 marker peptides can discriminate between the animal source of leather derived from crocodile, alligator, caiman, lizard, and the six mammal species. Estimation of the Effect of Powdering on Trypsin Digestion Efficiency. In a previous study, three or four 1 × 1 mm pieces (~5 mg) were taken from raw tanned leather samples to reliably detect marker peptides for the animal source identification of mammalian leather.25 To reduce the sample amount as much as possible for the analysis of expensive crocodilian leather products, the effect of a powdering treatment using a steel file on the efficiency of trypsin digestion was investigated. We compared peptide generation from powdered leather to that from 1 × 1 mm leather pieces using chrome-tanned crocodile leather digested with trypsin after dechroming using calcium hydroxide. MRM analysis of marker peptides C3 and C5 revealed that the trypsin digestion efficiency was enhanced by approximately 2fold by the powdering procedure (Figure S2). Surprisingly, in addition, marker peptides were sufficiently detected without the need for the dechroming step, while this treatment was essential for the cut pieces, consistent with our previous observation.25 The sampling procedure with filing potentially enables animal source identification with minimum destruction. Therefore, we adopted this sampling method to analyze crocodilian leather watch straps. Identification of the Animal Source of Leather Watch Straps. Four commercial leather watch straps labeled as crocodile, alligator, caiman, and crocodile-embossed calf were chosen, which were difficult to definitively distinguish the animal origin based on their appearance alone (Figure S3). Samples were discreetly taken from the undersides of the leather straps by filing (Figure 2A). Approximately 100 µg of
powder was collected by this sampling procedure (Figure 2B), and only a slight amount of damage was caused to the undersides of the straps (Figures 2C–E and S4). MRM chromatograms for the respective watch straps are shown in Figure 3. Crocodilian marker peptides were sensitively detected for respective crocodilian samples, regardless of the small sample amount (Figure 3A–C), and the detection patterns were in agreement with the declared species for the three leather products (C3 and C5 for crocodile, C3, C4, C5, and C6 for alligator, and C2, C3, C5, and C6 for caiman). In contrast, mammalian marker peptides (M1, M2, and M4) were detected for the crocodile-embossed watch strap, indicating that the leather is of cattle origin (Figure 3D). We further analyzed a lizard watch strap and successfully identified the leather as being of lizard origin (marker peptides C1 and C2; Figure S5). These results demonstrate that the LC–MS method combined with the filing sampling procedure described in this study can be used to accurately identify the animal source of crocodilian leather products with almost no destruction to the samples. CONCLUSIONS In the present study, we developed an LC–MS method using type I collagen-derived marker peptides to determine the animal source of crocodilian leather. The use of only six marker peptides enabled discrimination of these closely related species whose type I collagen sequences are almost uncharacterized. Moreover, in combination with previously established marker peptides for mammals,25 we can distinguish between 10 species (crocodile, alligator, caiman, lizard, cattle, horse, pig, sheep, goat, and deer) commonly used in leather production. The method described in this work allows definitive identification of the animal source of leather based on the detection patterns of clear MRM peaks. The analysis requires only a small amount of leather taken by filing (~100 µg) with the conventional LC–MS, which enabled seminondestructive certification of commercial watch straps. Use of high sensitive LC–MS can further reduce the sampling amount. Other leather goods, such as garments, handbags, and wallets, could be similarly analyzed with almost no destruction by sampling from inconspicuous sites. In addition, the peptide-based approach has advantages in the analysis of damaged samples in which species identification is difficult by DNA-based and morphological methods. Despite being protected by CITES, crocodilian species are threatened by the illegal hunting and trading of their valuable skins.14 Our method could also be used for monitoring the international trade of skin and leather derived from these endangered species.
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FIGURE CAPTIONS Figure 1. MRM chromatograms of type I collagen-derived marker peptides for crocodile, alligator, caiman, and lizard leathers. Detected marker peptides are indicated in boldface. Figure 2. Sampling from a crocodile leather watch strap by filing. (A) The edge of the underside of the leather was filed to collect powder. (B) Powdered sample taken from the leather. (C) The underside of the leather before filing. (D) The underside of the leather after filing. (E) The topside of the leather after filing. The arrows indicate the sampling sites. Figure 3. MRM chromatograms of type I collagen-derived marker peptides for commercial leather watch straps. (A) Crocodile, (B) alligator, (C) caiman, and (D) crocodile-embossed calf leather watch straps. Detected marker peptides are indicated in boldface.
TABLES Table 1. Type I collagen-derived marker peptides for animal source identification of crocodilian leather Crocodilec
Alligatorc
Caimanc
Lizardc
Mammalianc,d
GEOGSOGENGAOGQVGPR
−
−
−
+
−
C2
GEOGPAGLOGPAGER
−
−
+
+
−
316–327
C3
GFOGADGISGPK
+
+
+
−
−
238–252
C4
GIOGPSGPAGAAGTR
−
+
−
−
−
253–264
C5
GLVGEOGPAGAK
+
+
+
−
−
502–519
C6
GPOGESGAVGPVGPIGSR
−
+
+
−
−
Marker peptide
Chain
Positiona
α1(I)
109–126
C1
295–309
α2(I)
Sequenceb
aThe
numbering of residues begins with the triple-helical region of the chains. bO indicates 4-Hyp. cThe presence and absence of marker peptides are denoted by + and −, respectively. dThe term “Mammalian” includes cattle, horse, pig, sheep, goat, and deer.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. MRM chromatograms of marker peptides for mammalian leathers; evaluation of peptide generation with powdering; appearance of leather watch straps; pictures of leather watch straps before/after filing; MRM chromatograms of marker peptides for a lizard leather watch strap; analyzed leathers; MRM transitions of marker peptides; mammalian marker peptides (PDF)
AUTHOR INFORMATION Corresponding Author *Nippi Research Institute of Biomatrix, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan. E-mail:
[email protected]. Tel: +81-297-71-3046. Fax: +81-297-71-3041.
Notes The authors declare no competing financial interest.
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