A Rapid and Simple LC-MS Method Using ... - ACS Publications

Subscriber access provided by La Trobe University Library

Article

A Rapid and Simple LC–MS Method Using Collagen Marker Peptides for Identification of the Animal Source of Leather Yuki Kumazawa, Yuki Taga, Kenji Iwai, and Yohichi Koyama J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02132 • Publication Date (Web): 10 Jul 2016 Downloaded from http://pubs.acs.org on July 11, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

Journal of Agricultural and Food Chemistry

A Rapid and Simple LC–MS Method Using Collagen Marker Peptides for Identification of the Animal Source of Leather

Yuki Kumazawa†, Yuki Taga*,‡, Kenji Iwai†, and Yoh-ichi Koyama†,‡

†

Japan Institute of Leather Research, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan

‡

Nippi Research Institute of Biomatrix, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan

*Corresponding author (Tel: +81-297-71-3046; Fax: +81-297-71-3041; E-mail: [email protected])

ACS Paragon Plus Environment


1

ABSTRACT

2

Identification of the animal source of leather is difficult using traditional methods, including

3

microscopic observation and PCR. In the present study, a LC–MS method was developed for

4

detecting interspecies differences in the amino acid sequence of type I collagen, which is a

5

major component of leather, among six animals (cattle, horse, pig, sheep, goat, and deer). After

6

a dechroming procedure and trypsin digestion, six tryptic peptides of type I collagen were

7

monitored by LC–MS in multiple reaction monitoring mode for the animal source identification

8

using the patterns of the presence or absence of the marker peptides. We analyzed commercial

9

leathers from various production areas using this method, and found some leathers in which the

10

commercial label disagreed with the identified animal source. Our method enabled rapid and

11

simple leather certification and could be applied to other animals whether or not their collagen

12

sequences are available in public databases.

13 14

KEYWORDS: leather, collagen, mass spectrometry, species identification, scanning electron

15

microscopy


Page 2 of 29

Page 3 of 29


16

INTRODUCTION

17

Leather is produced from animal skin by chemical tanning using chrome or vegetable agents.1

18

The tanning process provides high mechanical strength and stability against heat and moisture

19

by cross-linking of collagen, which is a major protein component in skin. Leather is widely

20

used for making bags, gloves, clothes, and other products, and the global trade value of the

21

leather and leather products industry is approximately $100 billion per year. Leather can be

22

derived from various animals, such as cattle, pig, and goat. The animal source of the leather

23

must be specified on the consumer product by the supplier. However, the animal source is

24

sometimes incorrectly stated because of accidental or fraudulent substitution. In many cases, the

25

animal source cannot be easily discriminated by visual analysis.

26

Identification of the animal source of leather is generally performed by microscopic

27

observation of characteristic morphology. Scanning electron microscopy (SEM) is used for

28

identification of the animal source based on the cross-sectional fiber structure of collagen, the

29

surface texture of the grain layer, and pore patterns on the leather surface, which are all

30

characteristic for certain animals.2-4 However, extensive operator experience is required to

31

correctly identify the animal source. Leather is produced using various finishing processes,

32

including sanding to produce nubuck, embossing, and laminating, and these processes

33

sometimes make the skin pores disappear. In addition, split leather lacks skin pores because the

34

grain layer is removed. Without these pores on the surface, it is hard to discriminate among

35

animal sources of leather, especially sheep and goat, by SEM because of their similarity of the

36

cross-sectional fiber structure of collagen.

37

A DNA-based approach using PCR has been increasingly used for identification of animal

38

sources, especially for food samples,5, 6 and has also been applied to leather from cattle, pig,



39

sheep, and goat.7-10 The major advantages of the DNA-based approach are high sensitivity and

40

high taxonomic specificity. However, there are some problems with the DNA-based method for

41

leather samples. First, DNA can be damaged and degraded by manufacturing treatments,

42

especially the tanning process.11 Second, PCR amplifications are inhibited by co-extracted

43

compounds during the DNA extraction process from leather.10, 12

44

In contrast to DNA, the primary structure of protein is highly stable in the processing of

45

leather. Type I collagen consists of two α1 chains and a genetically distinct α2 chain that form

46

a stable triple-helical structure. It is the most abundant protein in connective tissues, including

47

skin. The amino acid sequence characteristic of collagen is a repeating Gly–Xaa–Yaa sequence,

48

and the X and Y positions are frequently occupied by Pro and 4-hydroxyproline (Hyp),

49

respectively. Collagen has been used for identification of the animal source for various samples,

50

including bone,13 clothing,14 gelatin,15 and glue,16 using mass spectrometric detection of marker

51

peptides following trypsin digestion. Buckley et al.13 reported species identification of bone

52

with fingerprinting analysis of 92 collagen peptide markers from 32 different mammal species

53

using MALDI–TOF–MS. The collagen fingerprinting methodology enables unambiguous

54

discrimination of various animal species, and recent studies using the method achieved species

55

identification of ancient bone samples in which morphology- and DNA-based approaches were

56

unsuccessful.17, 18 However, this method requires fractionation steps and manual confirmation

57

of each peptide peak for species identification.

58

Multiple reaction monitoring (MRM) on a triple quadrupole MS allows highly sensitive and

59

selective detection of target analytes even without purification steps.19 We recently applied LC–

60

MS analysis in MRM mode to various collagen analyses.20-25 For example, highly sensitive

61

collagen type-specific quantitation (types I and III) was performed by MRM analysis of a


Page 4 of 29

Page 5 of 29


62

tryptic digest of collagen samples and needed only 10 min per sample for data acquisition.22 In

63

this study, we established a new analytical method using collagen marker peptides with

64

detection by LC–MS in MRM mode to identify the animal source of leather, including cattle,

65

horse, pig, sheep, goat, and deer. We designed the method to give different and species-specific

66

patterns of the presence or absence of six peptide peaks for simple and unambiguous species

67

identification. We first investigated the effect of dechroming using calcium hydroxide and

68

sulfuric acid on trypsin digestion of leather for efficient peptide generation. The dechromed and

69

trypsin-digested leathers were then submitted to MS/MS analysis for identification of marker

70

peptides. Commercial leather samples from various production areas were analyzed using the

71

selected marker peptides by the high-throughput LC–MS analysis.

72 73

MATERIALS AND METHODS

74

Dechroming and Trypsin Digestion of Leather Samples. Samples of commercial leathers

75

labeled as cattle, horse, pig, sheep, goat, and deer, which were collected and stored in our

76

laboratory, were subjected to dechroming as reported previously.26 Briefly, each leather sample

77

was cut into approximately 1 × 1 mm pieces, and three or four pieces of the sample were

78

shaken slowly in 10 mL of 0.25% calcium hydroxide for 0–24 h at room temperature. After

79

washing several times with distilled water, the leather samples were shaken slowly in 10 mL of

80

1.7% sulfuric acid for 0–4 h at room temperature. The samples were washed several times with

81

distilled water and heated in distilled water at 80 °C for 30 min to denature the collagen. After

82

removal of the distilled water, digestion with 50 µg of trypsin (Sigma-Aldrich, St. Louis, MO)

83

was performed in 200 µL of 100 mM Tris-HCl/1 mM calcium chloride (pH 7.6) at 37 °C for 4



84

h. The tryptic digest was acidified with formic acid (final 1.0%), and the supernatant was

85

filtered through a 0.45 µm filter and subjected to LC–MS/MS or MRM analysis.

86

Peptide Identification Using LC–MS/MS. The tryptic digest was analyzed by LC–MS/MS

87

using a 3200 QTRAP (AB Sciex, Foster City, CA) mass spectrometer coupled to a 1200 Series

88

HPLC system (Agilent Technologies, Palo Alto, CA). The sample solution was loaded onto an

89

Ascentis Express C18 HPLC column (2.1 mm x 150 mm i.d., 2.7 µm; Supelco, Bellefonte, PA)

90

at a flow rate of 200 µL/min, and separated by a binary gradient as follows: 98% solvent A

91

(0.1% formic acid in water) for 5 min, linear gradient of 2–50% solvent B (100% acetonitrile)

92

for 15 min, 90% solvent B for 5 min, and 98% solvent A for 5 min. The separated peptides

93

were ionized and detected in positive ion mode by information–dependent acquisition, which

94

involved selecting the two most intense precursor ions of the prior survey MS scan and then

95

subjecting them to MS/MS fragmentation, with Analyst software 1.6.2 (AB Sciex). The MS

96

scan and MS/MS acquisition were performed over m/z ranges of 400–1300 and 100–1700,

97

respectively. The collision energy was automatically determined based on the mass and charge

98

state of the precursor ions using rolling collision energy. ProteinPilot software 4.0 (AB Sciex)

99

with the Paragon™ algorithm was used for peptide identification.27 The acquired MS/MS

100

spectra were searched against a local type I collagen database, which includes type I collagen

101

sequences for various animals from public databases and a previous publication.28

102

MRM Analysis of Marker Peptides. Marker peptides were monitored by LC–MS in MRM

103

mode. The sample solution was loaded onto an Ascentis Express C18 HPLC column (2.1 mm x

104

150 mm i.d., 5 µm; Supelco) at a flow rate of 500 µL/min, and separated by a binary gradient as

105

follows: 98% solvent A (0.1% formic acid in water) for 2 min, linear gradient of 2–60% solvent

106

B (100% acetonitrile) for 4 min, 90% solvent B for 2 min, and 98% solvent A for 2 min. The


Page 6 of 29

Page 7 of 29


107

MRM transitions and collision energy settings for the marker peptides are shown in Table 1.

108

The threshold for the peptide detection was set to a signal-to-noise ratio of 10.

109

SEM Analysis. The surfaces of the leather samples were cleaned with dimethylformamide,

110

and microscopic observation of the grain surface was performed using a JCM-5700 scanning

111

electron microscope (JEOL, Tokyo, Japan). Leather samples were sliced into thin layers to

112

observe the cross-sectional fiber structure by SEM.

113

114

RESULTS AND DISCUSSION

115

Optimization of Dechroming Treatment. Almost no peptides were generated from chrome-

116

tanned leathers by trypsin digestion following heat denaturation. Thus, we dechromed the

117

leathers using an established process with calcium hydroxide and sulfuric acid.26 Peptide

118

generation was investigated for one chrome-tanned cattle leather sample using three tryptic

119

peptides, which were identified as marker peptides for animal source identification in the next

120

experiment. No peptides were detected by trypsin digestion after dechroming of the leather

121

without calcium hydroxide treatment (treatment time = 0 h) and with 1 h of sulfuric acid

122

treatment (Figure 1A). Calcium hydroxide treatment dramatically increased peptide generation

123

by trypsin, and maximum peptide generation was reached with a calcium hydroxide treatment

124

time of 2 h. In contrast, subsequent sulfuric acid treatment did not enhance peptide generation

125

(Figure 1B). Therefore, sulfuric acid treatment is not required for peptide generation from

126

leather samples using trypsin. Consequently, in the subsequent experiments, we used calcium

127

hydroxide treatment (2 h) without sulfuric acid treatment for all leather samples, although

128

dechroming treatment was not required for vegetable-tanned leather (data not shown).



129

Identification of Collagen Marker Peptides. The collagen-derived peptides generated for

130

cattle, horse, pig, sheep, goat, and deer leathers after dechroming and trypsin digestion were

131

identified by LC–MS/MS analysis. Various peptides were identified for type I collagen α1 and

132

α2 chains. MRM transitions were set for the respective collagen-derived tryptic peptides to

133

monitor the presence or absence of the peptides for the leathers from the six different animals.

134

Collagen marker peptides for animal source identification were selected in accordance with the

135

following concepts: (1) presence of more than three marker peptides for each animal and (2) at

136

least two differences in the patterns of the presence or absence of marker peptides between each

137

animal. This reduced the chance of incorrect identification of the animal source. We selected

138

three marker peptides from α1(I) and three marker peptides from α2(I) in which all Pro at the Y

139

position are hydroxylated to 4-Hyp (Figure 2 and Table 1). MRM transitions for the marker

140

peptides (Table 1) were determined from the precursor ion (Q1) and fragmented product ions

141

(Q3) in the MS/MS analysis (Figure 2).

142

The patterns of the presence or absence of the six marker peptides for leather from the six

143

animal sources are summarized in Table 1. The MRM chromatograms of the marker peptides

144

(Figure 3) showed good peak shapes for all the marker peptides, and were easy to interpret as to

145

the presence or absence of the marker peptides. We investigated the trypsin digestion conditions

146

and found that most of the peptide generation was completed within 4 h of incubation at 37 °C

147

(Figure S1 in the Supporting Information). MRM analysis of the marker peptides took only

148

10 min per sample with separation by reverse-phase chromatography. Thus, species

149

identification using this method usually can be completed within a day. Detection patterns of

150

the marker peptides were different for each of the six animal sources. For example, α1(I) [316–

151

327] GFOGADGVAGPK (O indicates 4-Hyp) referred to as P1 was observed for cattle, horse,


Page 8 of 29

Page 9 of 29


152

pig, and deer leathers, and α2(I) [978–990] TGQOGAVGPAGIR (P6) was observed for pig,

153

sheep, goat, and deer leathers. Buckley et al.29 enabled discrimination between sheep and goat

154

bones using a 33 amino acid tryptic peptide, α2(I) [757-789], which differed between the two

155

species at two positions. In addition to the above peptide, we identified the following two new

156

marker peptides containing a common sequence region for discrimination between sheep and

157

goat: α1(I) [733–756] GETGPAGROGEVGPOGPOGPAGEK (P2) and α1(I) [741–756]

158

AGEVGPOGPOGPAGEK (P3). The Ala741Hyp substitution from P3 resulted in the missed

159

cleaved P2 peptide, because the Arg-Hyp bond is resistant to trypsin.30, 31 As shown in Figure 3,

160

P2 was detected for cattle, goat, and deer leathers, whereas P3 was detected only for sheep

161

leather. The new marker peptides were detected at high intensities compared with the

162

previously identified α2(I) [757-789] peptide by MRM detection (data not shown), probably

163

because of the difference in peptide length (24 and 16 vs. 33). The selected six marker peptides

164

enabled efficient and simple identification of the animal source of leather, and even allowed

165

discrimination between sheep and goat leathers.

166

Identification of the Animal Source for Commercial Leathers. We analyzed 75

167

commercial leathers from various production areas using our method. Using the presence or

168

absence of the marker peptides, all the leather samples labeled as being of cattle, horse, and pig

169

origins were identified as the same (Table 2). For leather labeled as sheep, goat, and deer, some

170

of the detected marker peptide patterns did not match the animal source given on the label. The

171

marker peptide patterns for leather samples labeled as deer seemed to differ depending on the

172

production area. For deer samples from Japan and New Zealand, the identification results

173

matched the commercial labels with detection of P1, P2, P4, P5 and P6 markers. In contrast, for

174

deer samples from Italy and the USA, detection of P1, P2, P4, and P6 was observed, which did



175

not correspond to any of the animals in this study (Figure 4C). This could be because deer are

176

classified into two subfamilies, Cervinae and Capreolinae.32 A previous study reported amino

177

acid substitution(s) in α2(I) [757-789] between the Cervinae subfamily (fallow deer) and

178

Capreolinae subfamily (roe deer).33 From the habitat distribution of deer,32 we assume that the

179

deer leathers from Japan and New Zealand are from Cervinae and those from Italy and the USA

180

are from Capreolinae. This subfamily difference in deer could explain the lack of the P5 marker

181

for the deer samples from Italy and the USA. However, authenticated deer samples are needed

182

to confirm this hypothesis. Almost all leather samples labeled as sheep and goat were identified

183

as the same species. However, the marker peptide patterns for sheep_China-1 (P2, P4, and P6;

184

Figure 4A) and goat_China-2 (P3, P4, and P6; Figure 4B) disagreed with the labeled animal

185

source. The detection patterns for sheep_China-1 and goat_China-2 indicated that they were

186

goat and sheep leathers, respectively.

187

Microscopic Observation of Sheep and Goat Leathers. To confirm the lack of a match

188

between the label and identification results for sheep_China-1 and goat_China-2, we analyzed

189

these samples by SEM to observe the fiber structure, surface texture, and skin pore patterns

190

(Figure 5). Sheep_Indonesia-1 and goat_Indonesia-1 were identified as sheep and goat,

191

respectively, using the marker peptides (Table 2), and were used as standard samples. Collagen

192

fibers ran parallel to the grain layer in both the sheep and goat standard leathers (Figure 5A and

193

B). However, the surface of the goat leather was rough compared with the smooth surface of the

194

sheep leather, and the skin pores were grouped in rows on the grain surface of the goat leather

195

but not on the sheep leather (Figure 5E and F). In the goat_China-2 and sheep_China-1

196

samples, the fiber structures were similar to those in the sheep and goat standards (Figure 5C

197

and D). In addition, in the goat_China-2 sample, the smooth surface and the uniform skin pores


Page 10 of 29

Page 11 of 29


198

were similar to the sheep standard (Figure 5G). Therefore, this sample was likely of sheep

199

origin, and this result was consistent with the identification using the marker peptides. In

200

contrast, the surface of the sheep_China-1 leather sample was not present, probably because the

201

grain layer had been removed by splitting (Figure 5H). Consequently, we were not able to

202

confirm the animal source for the sheep_China-1 sample using SEM analysis. However, our

203

method using the tryptic marker peptides provided an indication of this sample’s origin.

204

In the present study, LC–MS in MRM mode was used to identify the animal source of leather

205

using a combination of presence or absence of six marker peptides derived from type I collagen.

206

The sample preparation took approximately 7 h, including dechroming and trypsin digestion,

207

and LC–MS analysis took 10 min per sample. The MS data were easily interpreted to identify

208

the species from the detection patterns of the six marker peptides. Although we limited the

209

target animals to cattle, horse, pig, sheep, goat, and deer, other animals could be identified

210

using this method after selection of appropriate marker peptides. This strategy for animal source

211

identification using the presence or absence of marker peptides could also enable identification

212

of animals, such as snakes and alligators that are used for leather products, even if their type I

213

collagen sequences are not available in public databases. Animal source identification by

214

microscopic observation sometimes fails when the surface and fiber structure of leather are

215

disrupted (Figure 5H), but the LC–MS analysis of tryptic marker peptides successfully

216

identified all the 75 leather samples analyzed in this study. The developed method is robust and

217

can be used for screening and routine measurements.

218 219

ABBREVIATIONS USED

220

SEM, scanning electron microscopy; Hyp, hydroxyproline; MRM, multiple reaction monitoring



221 222

SUPPORTING INFORMATION AVAILABLE

223

Figure S1: Optimization of trypsin digestion of leather. Table S1: List of identified peptides

224

for cattle leathers. Table S2: List of identified peptides for horse leathers. Table S3: List of

225

identified peptides for pig leathers. Table S4: List of identified peptides for sheep leathers.

226

Table S5: List of identified peptides for goat leathers. Table S6: List of identified peptides for

227

deer leathers. This material is available free of charge via the Internet at http://pubs.acs.org.


Page 12 of 29

Page 13 of 29


228

REFERENCES

229

1.

230

Chemistry: Cambridge, UK, 2009.

231

2.

232

leather and leather products. Adv. Nat. Appl. Sci. 2012, 6, 651-659.

233

3.

234

function (L-function): Method (1): Influence of individual and location differences for

235

goatskins. J. Soc. Leather Technol. Chem. 2013, 97, 145-148.

236

4.

237

function (L-function): Method (2): Influence of individual and location differences for

238

sheepskins. J. Soc. Leather Technol. Chem. 2013, 97, 185-188.

239

5.

240

Shinmura, Y. A quick and simple method for the identification of meat species and meat

241

products by PCR assay. Meat Sci. 1999, 51, 143-8.

242

6.

243

quantitative PCR detection method for pork, chicken, beef, mutton, and horseflesh in foods.

244

Biosci. Biotechnol. Biochem. 2007, 71, 3131-5.

Covington, A. D., Tanning chemistry: the science of leather. The Royal Society of

Mirghani, M. E. S.; Salleh, H. M.; Man, Y. B. C.; Jaswir, I. Rapid authentication of

Dohshi, S. Quantitative estimation of hair follicle patterns for leather surface using k-

Dohshi, S. Quantitative estimation of hair follicle patterns for leather surface using k-

Matsunaga, T.; Chikuni, K.; Tanabe, R.; Muroya, S.; Shibata, K.; Yamada, J.;

Tanabe, S.; Hase, M.; Yano, T.; Sato, M.; Fujimura, T.; Akiyama, H. A real-time



245

7.

Schlumbaum, A.; Campos, P. F.; Volken, S.; Volken, M.; Hafner, A.; Schibler, J.

246

Ancient DNA, a Neolithic legging from the Swiss Alps and the early history of goat. J.

247

Archaeol. Sci. 2010, 37, 1247-1251.

248

8.

249

wild (Hydrochoerus hydrochaeris and Pecari tajacu) and domestic (Sus scrofa domestica)

250

species using a novel protocol. Genet. Mol. Res. 2012, 11, 672-8.

251

9.

252

a diagnostic sheep/goat peptide sequence through DNA analysis and further exploration of its

253

taxonomic utility within the Bovidae. J. Archaeol. Sci. 2013, 40, 1421-1424.

254

10.

255

Beethovens music. Mitochondrial DNA 2016, 27, 355-9.

256

11.

257

Peacock, E. E.; Gilbert, M. T. P. The survival of PCR-amplifiable DNA in cow leather. J.

258

Archaeol. Sci. 2007, 34, 823-829.

259

12.

260

709-15.

Ojeda, G. N.; Amavet, P. S.; Rueda, E. C.; Siroski, P. A. DNA extraction from skins of

Campana, M. G.; Robinson, T.; Campos, P. F.; Tuross, N. Independent confirmation of

Merheb, M.; Vaiedelich, S.; Maniguet, T.; Hanni, C. Mitochondrial DNA, restoring

Vuissoz, A.; Worobey, M.; Odegaard, N.; Bunce, M.; Machado, C. A.; Lynnerup, N.;

Lindahl, T. Instability and decay of the primary structure of DNA. Nature 1993, 362,


Page 14 of 29

Page 15 of 29


261

13.

Buckley, M.; Collins, M.; Thomas-Oates, J.; Wilson, J. C. Species identification by

262

analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-flight mass

263

spectrometry. Rapid Commun. Mass Spectrom. 2009, 23, 3843-54.

264

14.

265

Cappellini, E. Species identification of archaeological skin objects from Danish bogs:

266

comparison between mass spectrometry-based peptide sequencing and microscopy-based

267

methods. PLoS One 2014, 9, e106875.

268

15.

269

spectrometric detection of marker peptides in tryptic digests of gelatin: A new method to

270

differentiate between bovine and porcine gelatin. Food Hydrocolloids 2009, 23, 2001-2007.

271

16.

272

Tokarski, C. Identification of animal glue species in artworks using proteomics: application to a

273

18th century gilt sample. Anal. Chem. 2011, 83, 9431-7.

274

17.

275

fragmentary bone from the Late Middle/Early Upper Palaeolithic sequence of Les Cottés,

276

France. J. Archaeol. Sci. 2015, 54, 279-286.

277

18.

278

Turvey, S. T.; Reguero, M.; Gelfo, J. N.; Kramarz, A.; Burger, J.; Thomas-Oates, J.; Ashford,

Brandt, L. O.; Schmidt, A. L.; Mannering, U.; Sarret, M.; Kelstrup, C. D.; Olsen, J. V.;

Zhang, G.; Liu, T.; Wang, Q.; Chen, L.; Lei, J.; Luo, J.; Ma, G.; Su, Z. Mass

Dallongeville, S.; Koperska, M.; Garnier, N.; Reille-Taillefert, G.; Rolando, C.;

Welker, F.; Soressi, M.; Rendu, W.; Hublin, J. J.; Collins, M. Using ZooMS to identify

Welker, F.; Collins, M. J.; Thomas, J. A.; Wadsley, M.; Brace, S.; Cappellini, E.;



279

D. A.; Ashton, P. D.; Rowsell, K.; Porter, D. M.; Kessler, B.; Fischer, R.; Baessmann, C.;

280

Kaspar, S.; Olsen, J. V.; Kiley, P.; Elliott, J. A.; Kelstrup, C. D.; Mullin, V.; Hofreiter, M.;

281

Willerslev, E.; Hublin, J. J.; Orlando, L.; Barnes, I.; MacPhee, R. D. Ancient proteins resolve

282

the evolutionary history of Darwin's South American ungulates. Nature 2015, 522, 81-4.

283

19.

284

quantitative proteomics: a tutorial. Mol. Syst. Biol. 2008, 4, 222.

285

20.

286

for analyzing collagen O-glycosylations by hydrazide chemistry. Mol. Cell. Proteomics 2012,

287

11, M111.010397.

288

21.

289

of overglycosylation of collagen in osteogenesis imperfecta using hydrazide chemistry and

290

SILAC. J. Proteome Res. 2013, 12, 2225-32.

291

22.

292

novel and versatile tool for quantitative collagen analyses using mass spectrometry. J. Proteome

293

Res. 2014, 13, 3671-8.

294

23.

295

hydroxyproline-containing peptides in blood using a protease digest of stable isotope-labeled

296

collagen. J. Agric. Food Chem. 2014, 62, 12096-102.

Lange, V.; Picotti, P.; Domon, B.; Aebersold, R. Selected reaction monitoring for

Taga, Y.; Kusubata, M.; Ogawa-Goto, K.; Hattori, S. Development of a novel method

Taga, Y.; Kusubata, M.; Ogawa-Goto, K.; Hattori, S. Site-specific quantitative analysis

Taga, Y.; Kusubata, M.; Ogawa-Goto, K.; Hattori, S. Stable isotope-labeled collagen: a

Taga, Y.; Kusubata, M.; Ogawa-Goto, K.; Hattori, S. Highly accurate quantification of


Page 16 of 29

Page 17 of 29


297

24.

Taga, Y.; Kusubata, M.; Ogawa-Goto, K.; Hattori, S. Developmental stage-dependent

298

regulation of prolyl 3-hydroxylation in tendon type I collagen. J. Biol. Chem. 2016, 291, 837-

299

47.

300

25.

301

hydroxyproline (Hyp)-Gly after oral administration of a novel gelatin hydrolysate prepared

302

using ginger protease. J. Agric. Food Chem. 2016, 64, 2962-70.

303

26.

304

from chrome leather. Sci. Rept. Fac. Agr. Kobe Univ. 1983, 15, 387-391.

305

27.

306

Hunter, C. L.; Nuwaysir, L. M.; Schaeffer, D. A. The Paragon Algorithm, a next generation

307

search engine that uses sequence temperature values and feature probabilities to identify

308

peptides from tandem mass spectra. Mol. Cell. Proteomics 2007, 6, 1638-55.

309

28.

310

survival and utility. Geochim. Cosmochim. Acta 2011, 75, 2007-2016.

311

29.

312

Distinguishing between archaeological sheep and goat bones using a single collagen peptide. J.

313

Archaeol. Sci. 2010, 37, 13-20.

Taga, Y.; Kusubata, M.; Ogawa-Goto, K.; Hattori, S. Efficient absorption of X-

Itoh, K.; Nakagawa, S.; Okayama, T. Basic studies on the dechroming of shaving dust

Shilov, I. V.; Seymour, S. L.; Patel, A. A.; Loboda, A.; Tang, W. H.; Keating, S. P.;

Buckley, M.; Larkin, N.; Collins, M. Mammoth and Mastodon collagen sequences;

Buckley, M.; Kansa, S. W.; Howard, S.; Campbell, S.; Thomas-Oates, J.; Collins, M.



314

30.

Rodriguez, J.; Gupta, N.; Smith, R. D.; Pevzner, P. A. Does trypsin cut before proline?

315

J. Proteome Res. 2008, 7, 300-5.

316

31.

317

J.; Ge, Y.; Westphall, M. S.; Coon, J. J.; Greenspan, D. S. Comprehensive mass spectrometric

318

mapping of the hydroxylated amino acid residues of the alpha1(V) collagen chain. J. Biol.

319

Chem. 2012, 287, 40598-610.

320

32.

321

Mechanicsburg, PA, 1998.

322

33.

323

remains from Domuztepe, South Eastern Turkey. Archaeol. Anthropol. Sci. 2011, 3, 271-280.

Yang, C.; Park, A. C.; Davis, N. A.; Russell, J. D.; Kim, B.; Brand, D. D.; Lawrence, M.

Geist, V., Deer of the world: their evolution, behavior, and ecology. Stackpole Books:

Buckley, M.; Kansa, S. W. Collagen fingerprinting of archaeological bone and teeth


Page 18 of 29

Page 19 of 29


324

FIGURE CAPTIONS

325

Figure 1. Effect of dechroming treatments on peptide generation from leather using trypsin. A

326

sample of chrome-tanned cattle leather was incubated with (A) 0.25% calcium hydroxide for

327

either 0, 0.5, 1, 2, 4, 8, or 24 h followed by incubation with 1.7% sulfuric acid for 1 h or (B)

328

1.7% sulfuric acid for either 0, 0.5, 1, 2, or 4 h following incubation with 0.25% calcium

329

hydroxide for 2 h. Peptide generation was estimated using collagen marker peptides (P1, P2,

330

and P4) shown in Table 1. The data are expressed as relative intensity with (A) 24 h or (B) 0 h

331

being 100%, and represent the mean ± SD of three replicates.

332

333

Figure 2. MS/MS spectra of marker peptides for species identification of leather. (A–F)

334

MS/MS spectra of P1–P6 marker peptides were obtained from cattle, goat, sheep, sheep, pig,

335

and pig leathers. The peaks in blue and red are used for Q1 and Q3 transitions, respectively, for

336

MRM analysis of the marker peptides as summarized in Table 1.

337

338

Figure 3. MRM chromatograms of marker peptides for leather from six animal species (cattle,

339

horse, pig, sheep, goat, and deer). Detected marker peptides are indicated in bold face.

340



341

Figure 4. MRM chromatograms of marker peptides in unmatched leather samples. Tryptic

342

digests of (A) sheep_China-1, (B) goat_China-2, and (C) deer_USA-1 were analyzed by LC–

343

MS in MRM mode. Detected marker peptides are indicated in bold face.

344

345

Figure 5. SEM images of (A–D) cross sections and (E–H) surfaces of leather samples.


Page 20 of 29

Page 21 of 29


TABLES Table 1. List of Marker Peptides for Animal Species Identification Chain

α1(I)

α2(I)

Positiona

Marker peptide

Cattlec

Horsec

Pigc

Sheepc

Goatc

Deerc

Q1 (m/z)d

Q3 (m/z)d

zd

Collision energyd

Time (min)d

316–327

P1

GFOGADGVAGPK

+

+

+

−

−

+

544.76

643.34

2

30.0

5.3

733–756

P2

GETGPAGROGEVGPOGPOGPAGEK

+

−

−

−

+

+

739.35

964.98

3

25.3

5.0

741–756

P3

AGEVGPOGPOGPAGEK

−

−

−

+

−

−

724.85

1035.51

2

37.9

5.0

361–374

P4

GFOGSOGNIGPAGK

+

+

−

+

+

+

644.31

826.44

2

34.3

5.2

502–519

P5

GPOGESGAAGPAGPIGSR

−

+

+

−

−

+

775.87

811.44

2

40.1

5.1

978–990

P6

TGQOGAVGPAGIR

−

−

+

+

+

+

598.82

669.40

2

32.3

5.1

Sequenceb

a

The numbering of residues begins with the triple-helical portion of the chains. For example, first residue corresponds to residue 178 of Uniprot # P02453 (bovine type I

collagen α1 chain) and residue 89 of Uniprot # P02465 (bovine type I collagen α2 chain). bO indicates 4-Hyp. cThe presence and absence of marker peptides are denoted by + and −, respectively. dMRM transitions (Q1 and Q3) for the marker peptides are shown with their charge state, collision energy, and retention time.



Page 22 of 29

Table 2. Species Identified for Leathers from Various Production Areas Species on label Cattle

Horse

Pig

Sheep

Goat

Sample

P1

P2

P3

P4

P5

P6

Identified species

Japan-1, Japan-2, Japan-3

+

+

−

+

−

−

Cattle

China-1, China-2, China-3

+

+

−

+

−

−

Cattle

India-1, India-2, India-3

+

+

−

+

−

−

Cattle

Pakistan-1, Pakistan-2, Pakistan-3

+

+

−

+

−

−

Cattle

Bangladesh-1, Bangladesh-2

+

+

−

+

−

−

Cattle

Italy-1, Italy-2, Italy-3

+

+

−

+

−

−

Cattle

USA-1, USA-2, USA-3

+

+

−

+

−

−

Cattle

Japan-1, Japan-2

+

−

−

+

+

−

Horse

Mongolia-1

+

−

−

+

+

−

Horse

France-1

+

−

−

+

+

−

Horse

Poland-1, Poland-2, Poland-3

+

−

−

+

+

−

Horse


+

−

−

−

+

+

Pig

China-1, China-2, China-3

+

−

−

−

+

+

Pig


−

−

+

+

−

+

Sheep

China-1

−

+

−

+

−

+

Goat

China-2, China-3

−

−

+

+

−

+

Sheep


−

−

+

+

−

+

Sheep


−

−

+

+

−

+

Sheep

Indonesia-1, Indonesia-2

−

−

+

+

−

+

Sheep


−

−

+

+

−

+

Sheep

China-1, China-3

−

+

−

+

−

+

Goat

China-2

−

−

+

+

−

+

Sheep


−

+

−

+

−

+

Goat


−

+

−

+

−

+

Goat

22


Page 23 of 29


Deer

Indonesia-1, Indonesia-2, Indonesia-3

−

+

−

+

−

+

Goat

Italy-1

−

+

−

+

−

+

Goat


+

+

−

+

+

+

Deer


+

+

−

+

−

+

Deer?

USA-1, USA-2, USA-3

+

+

−

+

−

+

Deer?

New Zealand-1, New Zealand-2, New Zealand-3

+

+

−

+

+

+

Deer

23



Fig. 1 83x85mm (300 x 300 DPI)


Page 24 of 29

Page 25 of 29


Fig. 2 173x123mm (300 x 300 DPI)



Fig. 3 173x109mm (300 x 300 DPI)


Page 26 of 29

Page 27 of 29


Fig. 4 83x104mm (300 x 300 DPI)



Fig. 5 177x67mm (300 x 300 DPI)


Page 28 of 29

Page 29 of 29


Graphic for table of contents 85x44mm (300 x 300 DPI)


A Rapid and Simple LC-MS Method Using ... - ACS Publications

Recommend Documents