The identification of three mammalian gelatins by liquid chromatography-high resolution mass spectrometry

Accepted Manuscript The identification of three mammalian gelatins by liquid chromatography-high resolution mass spectrometry Xiao-Mei Sha, Zi-Zi Hu, Zong-Cai Tu, Lu-Zheng Zhang, Zhi Li, Xin Li, Tao Huang, Hui Wang, Lu Zhang, Hui Xiao PII:

S0023-6438(17)30724-7

DOI:

10.1016/j.lwt.2017.10.001

Reference:

YFSTL 6564

To appear in:

LWT - Food Science and Technology

Received Date: 2 May 2017 Revised Date:

6 September 2017

Accepted Date: 1 October 2017

Please cite this article as: Sha, X.-M., Hu, Z.-Z., Tu, Z.-C., Zhang, L.-Z., Li, Z., Li, X., Huang, T., Wang, H., Zhang, L., Xiao, H., The identification of three mammalian gelatins by liquid chromatographyhigh resolution mass spectrometry, LWT - Food Science and Technology (2017), doi: 10.1016/ j.lwt.2017.10.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

The identification of three mammalian gelatins by liquid chromatography-high

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resolution mass spectrometry

3 Xiao-Mei Sha a, Zi-Zi Hu a, Zong-Cai Tu

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Wang b,c, Lu Zhang a, Hui Xiao e,*

a,b,*

, Lu-Zheng Zhang a, Zhi Li d, Xin Li a, Tao Huang b, Hui

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a

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Science, Jiangxi Normal University, Nanchang, Jiangxi 330022, China

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b

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Key Laboratory of Functional Small Organic Molecule, Ministry of Education and College of Life

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi,

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330047, China

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c

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Nanchang, Jiangxi, 330047, China

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Shandong Dong-e E-Jiao Co., Ltd., Dong'e, Shandong, 252201, China

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Regeneron Pharmaceuticals, Inc, Tarrytown, New York, 10591, USA

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Corresponding author. Tel.: 86 791 88121868; fax: 86 791 88305938.

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E-mail address: [email protected]; [email protected]

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Engineering Research Center for Biomass Conversion, Ministry of Education, Nanchang University,

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ABSTRACT High-performance liquid chromatography (HPLC) and linear-ion trap (LTQ)/Orbitrap

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high-resolution mass spectrometry were combined to systematically differentiate three mammalian

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gelatins including bovine-hide gelatin, porcine-hide gelatin and donkey-hide gelatin. Interestingly,

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hemoglobin was found only in the donkey-hide gelatin while not in bovine-hide gelatin and

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porcine-hide gelatin. In marker peptide determination for each individual gelatin, 28, 27 and 14

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distinct peaks were detected to represent the bovine-hide gelatin, porcine-hide gelatin and

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donkey-hide gelatin, respectively. In the gelatin mixtures, all gelatins can be differentiated by their

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marker peptides. However, the number of detectable marker peptides in each gelatin decreased with

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reduced concentration of target gelatin in their mixtures. Under low content of target gelatin (less than

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10%), 11, 15 and 5 marker peptides were detected in the gelatin mixture digests for bovine-hide

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gelatin,

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high-resolution mass spectrometry could be a promising technology to accurately identify

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bovine-hide gelatin, porcine-hide gelatin and donkey-hide gelatin for controlling the quality of food

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products added with gelatin.

gelatin

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donkey-hide

gelatin,

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HPLC-LTQ/Orbitrap

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KEYWORDS

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bovine-hide gelatin; porcine-hide gelatin; donkey-hide gelatin; identification; high-resolution mass

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spectrometry

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1. Introduction Gelatin which is obtained by partial hydrolysis of collagen, is a mixture including the

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components with different molecular weights (Azilawati, Hashim, Jamilah, & Amin, 2014; Nur, Che,

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Rn, Aina, & Amin, 2014). Gelatin shows multiple functional properties, such as gelling properties,

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emulsifying properties, foaming properties, film-forming properties, and so on. Therefore, gelatin is

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widely used in many industries referred to food pharmacy, cosmetic and photographic material (Sha,

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Tu, Liu, Wang, Shi, Huang, et al., 2014). In present, most of commercial gelatins are derived from

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mammalian. Among them, pig and cow are two common types of mammalian which are used to

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produce gelatin in the world (Tabarestani, Maghsoudlou, Motamedzadegan, & Mahoonak, 2010).

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Besides, belonging to the equine family, donkey is another kind of mammalian whose skin is also a

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good material for gelatin preparation. The progenitor of donkey was the small grey donkey of

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northern Africa, which was domesticated on the shores of the Mediterranean Sea before about 4000

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BC (Polidori, Pucciarelli, Ariani, Polzonetti, & Vincenzetti, 2015). The population of donkey in the

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world is about 41 million, including one half from Asia, just over one quarter found in Africa, and the

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rest from Latin America (Polidori, Ariani, Micozzi, & Vincenzetti, 2016). As an important kind of

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Traditional Chinese Medicines (TCM), donkey-hide gelatin has been widely sold in many regions,

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especially in Asian countries, such as China, Japan, Korea, Malaysia, and so on.

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Bovine spongiform encephalopathy (BSE) and foot-and-mouth disease (FMD) make more and

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more people to care for the safety of mammalian gelatins (Sha, Tu, Wang, Huang, Duan, He, et al.,

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2014). People belonging to Judaism and Islam, don’t take any pork-related food. Meanwhile, Hindus

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don’t eat cow-related products. With the intake of gelatins from certain sources, some persons may

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cause an immune response (Cheng, Wei, Xiao, Zhao, Shi, Liu, et al., 2012). On the other hand,

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ACCEPTED MANUSCRIPT donkey-hide gelatin is sold with much higher price than other gelatins. Consumers are eager to know

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if other gelatins mainly including bovine-hide gelatin and porcine-hide gelatin are added into the

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products of donkey-hide gelatin. Therefore, it will be an interesting and meaningful work to

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differentiate bovine-hide gelatin, porcine-hide gelatin and donkey-hide gelatin.

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Physicochemical methods based on HPLC (Nemati, Oveisi, Abdollahi, & Sabzevari, 2004) and

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calcium phosphate precipitation (Hidaka & Liu, 2003) cannot effectively trace the source of gelatin

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from their mixture, because of their close similarity in structure and physicochemical properties

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(Venien & Levieux, 2005). Fourier transform infrared (FTIR) spectroscopy is a rapid method to trace

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the origin of gelatin (Hashim, Man, Norakasha, Shuhaimi, Salmah, & Syahariza, 2010), but also can’t

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distinguish various gelatins from their mixture (Lee, Kim, Jo, Jung, Kwon, & Kang, 2016). ELISA

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(enzyme-linked immunosorbent assay) has been used for speciation, however, the very high

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homology between collagen sequences of mammals makes it impossible to provide species-specific

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detection for gelatin (Hashim, Man, Norakasha, Shuhaimi, Salmah, & Syahariza, 2010). The

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polymerase chain reaction (PCR) assay was reported to differentiate gelatins (Mutalib, Muin,

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Abdullah, Hassan, Wan, Sani, et al., 2015; Shabani, Mehdizadeh, Mousavi, Dezfouli, Solgi,

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Khodaverdi, et al., 2015; Tasara, Schumacher, & Stephan, 2005). However, because of hot water and

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acidic condition used frequently in manufacturing process, the original species-specific DNA may be

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denatured to make the identification difficult. Previous studies suggested that LC-MS/MS approach

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can offer more accurate and reliable gelatin speciation than PCR or ELISA-based methods (Grundy,

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Reece, Buckley, Solazzo, Dowle, Ashford, et al., 2016). Despite of high similarity, the small

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divergence in sequences between gelatin species allows mass spectrometry based technique to

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distinguish gelatin sources by their characteristic marker peptides. Differentiating bovine gelatin and

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ACCEPTED MANUSCRIPT porcine gelatin by mass spectrometry has been reported (Yilmaz, Kesmen, Baykal, Sagdic, Kulen,

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Kacar, et al., 2013; Zhang, Liu, Wang, Chen, Lei, Luo, et al., 2009). In addition, Cheng, et al. (2012)

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reported that mass spectrometry could be used to identify five gelatins including donkey-hide gelatin,

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bovine-hide gelatin, pig-hide gelatin, tortoise shell glue and deerhorn glue. However, only one

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characteristic peptide for each gelatin was identified in Cheng, et al's study. If the peptide was missed

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in the testing process including gelatin digestion and mass spectrometry determination, the

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identification of gelatins will be failed. Therefore, a systematic study to distinguish bovine-hide

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gelatin, porcine-hide gelatin and donkey-hide gelatin needs to be established.

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Gelatin contains a range of post-translational modifications that are essential for triple helix

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assembly and stability, intermolecular cross-linking and strength of fibrils and tissue function

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(Fernandes, Farnand, Traeger, Weis, & Eyre, 2011). One of these post-translational modifications is

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abundant hydroxyproline (Hyp) resulted from the posttranslational hydroxylation of some proline

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residues (Song & Mechref, 2013). The difference of mass between proline and leucine is 16 Da,

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therefore, hydroxylation of proline almost offsets the gap. Zhang, et al. (2009) also stated that because

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of the similar mass of Hyp and Leu, hydroxylation of Pro made the sequence identification much

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more difficult. However, mass spectrometer with enough resolution and mass accuracy could

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effectively distinguish the tiny mass difference (0.036 Da), and then make the identification much

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easier. Therefore, in this work we utilized Linear-ion trap (LTQ)/Orbitrap mass spectrometry which

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allows us to unambiguously distinguish gelatin sources due to its extremely high resolution and mass

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accuracy of better than 60,000 and 15 ppm, respectively.

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Precisely speaking, unique marker peptides and their modifications of bovine-hide gelatin,

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ACCEPTED MANUSCRIPT porcine-hide gelatin and donkey-hide gelatin were accurately identified in their mixtures by

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LTQ/Orbitrap mass spectrometry in this study. High-energy C-trap dissociation (HCD) was selected

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to determine the sequence and identify the modification owing to its capability to detect the low mass

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fragments, which is often not detectable due to the low mass cutoff in ion trap collision-induced

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dissociation (CID). Our results showed HPLC- LTQ/Orbitrap mass spectrometry was a strong tool to

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differentiate three gelatins when the content of each gelatin was lower than 10% in the mixtures. It

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should provide a method for quality control of the products containing gelatin in food industry.

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2. Materials and Methods

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2.1. Materials

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Donkey-hide gelatin was obtained from Shandong Dong E E Jiao Co., Ltd. (Liaocheng,

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Shandong, China). Bovine-hide gelatin (G9382), porcine-hide gelatin (G2500) and Lys-C were

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purchased from Sigma Chemical Co. (St. Louis, MO, USA). Trypsin was purchased from Promega

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Corporation (Madison, WI, USA). All other reagents used were of analytical grade.

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2.2. Donkey-hide gelatin pretreatment

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Forty mg/ml of the donkey-hide gelatin solution was prepared in the deionized water at 50 °C in

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waterbath. The gelatin solution was centrifuged at 14,000 ×g for 20 min. After cooling down, the

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supernatant was further filtered through a 0.45 µm filter. The filtrate was freeze-dried for next use.

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2.3. Sample digestion

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According to our previous report with some modifications, gelatin samples were digested as 6

ACCEPTED MANUSCRIPT following procedures (Sha, Tu, Wang, Huang, Duan, He, et al., 2014). One hundred grams of

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bovine-hide gelatin, porcine-hide gelatin and donkey-hide gelatin were all dissolved in 100 ml of the

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digestion buffer composed of 20 mM pH 8.0 Tris-HCl, 50 mM NaCl, and 50 mM CaCl2. Three kinds

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of gelatin solution were mixed as three different ratios of 1:1:1, 1:1:10 and 10:10:1 (w:w:w) for

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bovine-hide gelatin, porcine-hide gelatin and donkey-hide gelatin, respectively. 10 µl of the pure

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gelatin solution or their mixed solution with gelatin concentration of 1mg/ml were mixed with 10 µl

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of the digestion solution in a 500 µl centrifuge tube. The ProteaseMax Surfactant (Promega

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Corporation, Madison, WI, USA) was added into the solution with the concentration of 0.01% (w/v).

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1.5 µl of 100 mM dithiothreitol (DTT) was added to the tube and the whole sample was incubated at

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95 °C for 5 min, and then cooled down in the ice bath. 3 µl of the alkylation buffer (100 mM

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iodoacetamide) was added to the tube and incubated in the dark at room temperature for 20 min. 2 µl

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of 0.05 µg/µl Lys-C was added to hydrolyze the gelatin sample at 37 °C for 4 h. 2 µl of 0.1 µg/µl

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trypsin was added to further hydrolyze the gelatin at 37 °C for overnight. 2µl of 5%(w/v)

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trifluoroacetic acid (TFA) was added to quench the digestion.

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2.4. Liquid chromatography and mass spectrometry analysis

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A Shimadzu HPLC (Shimadzu, Kyoto Japan), with two LC-10AD pumps, was used to generate a

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gradient with a 50 µL/min flow rate. Solvent A was 5% acetonitrile in H2O, 0.1% formic acid (FA),

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whereas solvent B consisted of 95% acetonitrile in H2O, 0.1% FA. For analysis of proteolytic peptides,

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3 µg of digested gelatin sample was injected onto a 2.0 mm i.d. × 100 mm C18 column (Phenomenex

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Inc., Torrance, CA, USA). After desalting for 5 min with 2% B, the peptides were eluted at 50 µL/min

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with a 2-20% gradient for 40 min and 20-95% gradient for 2 min. The effluent was infused into a

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ACCEPTED MANUSCRIPT LTQ - Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, CA) for MS/MS

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analysis to identify the characteristic peptides of the gelatins. The signals detected in the precursor ion

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scanning were further submitted for a HCD fragmentation to detect those fragment ions including low

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m/z range. The normalized collision energy was set to 35% for HCD. Dynamic exclusion was enabled

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with exclusion duration of 90 s.

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2.5. Database search

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Thermo proteome discoverer (ver. 1.4.0.288) was used to generate the mgf file from mass

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spectrometric data, and then Mascot Daemon (ver. 2.4.0, Matrix Science, Boston, MA, USA) was

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used to perform the searches against the NCBI database and the in-house database including donkey,

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bovine and porcine type Ι collagen sequences which were acquired from the uniprot database and a

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published patent (Bell, Neff, Polarek, & Seeley, 2001). Carbamidomethyl (C) was set as a fixed

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modification, deamidation (N and Q) and oxidation (P, K and M) were set as variable modifications.

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Additional search settings included a maximum of 5 missed cleavages, peptide tolerance of ±25 ppm,

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and MS/MS tolerance of ±0.5 Da. The peptides with a Mascot ion score >40 were considered to be

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positive identification or extensive similarity (p <0.05) (Zhang, Tu, Wang, Huang, Shi, Sha, et al.,

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2014). According to the alignment results, the targeted peptides were those characteristic peptides

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unique to donkey-hide gelatin, bovine gelatin or porcine gelatin.

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3. Results and discussion

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3.1. Composition specificity in the donkey-hide gelatin

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The mass spectrometry results showed that the donkey-hide gelatin contained hemoglobin with 8

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Mascot score of 99 for α-subunit and 69 for β-subunit. In contrast, in both bovine and porcine gelatin

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samples, no hemoglobin was detected. Fig. 1 shows the MS/MS spectra of the four distinct peptides

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from donkey hemoglobin. The mass of the peptide (1040.5482) with m/z of 521.28142+ matched well

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with the theoretical mass (1040.5365) of the sequence

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donkey hemoglobin. Moreover, the HCD MS/MS of the peptide generated a series of y ions (y1-y8 in

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Fig. 1A), unambiguously confirming the sequence of the peptide. In Fig. 1B, the mass of the peptide

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(1476.6974) with m/z of 739.35602+ matched well with that of the Asn deamidation form (1476.6845)

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with the sequence

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the α-subunit of the donkey hemoglobin. The series of b and y ions ensured the positive identification

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of the peptide sequence (b3, y1, y3-y12 and y14) and Asn deamidation based on the mass difference

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between y11 and y12 ions, which was the corresponding mass of aspartic acid or isoaspartic acid.

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Similarly the peaks with m/z of 493.79172+ and 637.87482+ in Fig. 1C and 1D were positively

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identified as the peptides of 9AAVLALWDK17 and

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donkey hemoglobin with the error of 9.4 and 13.1 ppm, respectively.

MFLGFPTTK41 from the subunit α of the

VGG*NAGEFGAEALER32 (the asterisk indicated deamidation of Asn21) from

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LLVVYPWTQR40 from the β-subunit of the

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As results show donkey-hide gelatin contains hemoglobin while bovine-hide gelatin and

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porcine-hide gelatin do not. Despite of that it is quite interesting as donkey-hide gelatin is a common

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Chinese herb and has long been used as blood tonic sold in many countries. Given the processing

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procedures for all three gelatin preparations are similar, including fat-removing, heating, filtration,

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concentrating, gelation and drying, the detected hemoglobin from donkey-hide gelatin may be caused

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by certain interaction between donkey collagen and hemoglobin. The precise reason is currently under

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investigation in our lab.

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3.2. Theoretical unique peptides for differentiating three types of gelatin

To differentiate bovine-hide gelatin, porcine-hide gelatin and donkey-hide gelatin by mass

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spectrometry, unique peptides for each type of gelatin are listed in Table 1 for searching the detected

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characteristic peptides. As shown in Table 1, 49, 50 and 55 theoretical unique sequence fragments

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were found for bovine-hide gelatin, porcine-hide gelatin and donkey-hide gelatin, respectively.

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Although there were more amino acid residues in α1 chain (1463, 1449 and 1463 for bovine, porcine

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and donkey collagen, respectively) than that in α2 chain (1364, 1366 and 1364 for bovine, porcine and

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donkey collagen, respectively) of the collagen, sequence divergence was higher in α2 chain than α1

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chain. The number of theoretical unique peptides from α1 chain was 18, 19 and 23 for bovine-hide

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gelatin, porcine-hide gelatin and donkey-hide gelatin, respectively. While 31, 31 and 32 theoretical

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unique peptides from α2 chain were found for these three types of gelatin, respectively. We further

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executed the sequence alignment between any two types of gelatin. The similarity percentages of α1

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chain of the collagen were 95.9%, 96.6% and 95.3% for bovine-porcine, bovine-donkey and

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porcine-donkey, respectively. For α2 chain, the sequence similarity were 95.3%, 94.9% and 94.7% for

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bovine-porcine, bovine-donkey and porcine-donkey, respectively.

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3.3. Detection and identification of the unique peptides

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The sequence coverage obtained from LC/MS/MS for α1 chain was 67%, 75% and 68% for

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bovine, porcine and donkey collagen, respectively. For α2 chain, the sequence coverage of 76%, 84%

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and 69% was obtained for bovine, porcine and donkey collagen, respectively. As shown in Tables 2-4,

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28, 27 and 14 distinct peaks were detected in individual bovine-hide gelatin, porcine-hide gelatin and

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donkey-hide gelatin, respectively. 10

ACCEPTED MANUSCRIPT Fig. 2 shows three unique peptides without any modification identified by the tandem mass

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spectrometry for bovine-hide gelatin , porcine-hide gelatin and donkey-hide gelatin, respectively. The

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peaks with m/z of 780.92042+ (Fig. 2A), 773.91212+ (Fig. 2B) and 765.91422+ (Fig. 2C) were identified

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from α1 chain of bovine-hide gelatin (1066GETGPAGPAGPIGPVGAR1083), porcine-hide gelatin

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(1052GETGPAGPAGPVGPVGAR1069) and donkey-hide gelatin (1066GEAGPAGPAGPIGPVGAR1083),

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respectively. The mass of three peptides matched well with the theoretical mass with the error of 13.2,

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12.7 and 12.3 ppm, respectively. A series of y and b ions (y1-16 and b2) generated by HCD MS/MS,

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undoubtedly confirmed the sequences for these three unique peptides. The three peptides were very

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similar in sequence with only one or two amino acid residue difference. Recognizing the specific

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fragment ions was the key to distinguish the gelatin source. For instance, y16 ion was the direct

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evidence to judge whether the gelatin was from bovine or donkey (Figure 2(A), 2(C)). The unique

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sequences without any modifications were very valuable for identifying gelatin proteins because there

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was no complicated interference. As listed in Tables 2-4, the amounts of unique peaks without any

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modification were 4, 8 and 2 for bovine-hide gelatin, porcine-hide gelatin and donkey-hide gelatin,

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respectively.

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Gelatin contains a number of modifications including hydroxylation (on proline and lysine) and

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deamidation (on glutamine and asparagine). The more complicated conditions makes identification

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more difficult. It could be because of the various modification levels which were found in the

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identical sequence, shown in table 2-4. Fig. 3 shows the mass spectra of the unique peaks from the

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same sequence but with different number of modification sites of hydroxylation in donkey-hide

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gelatin. The peaks with m/z 902.45762+ and 910.45542+ were from the sequence of

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793

TGPPGPSGISGPPGPPGAAGK813 in the α2 chain of the donkey-hide gelatin with three and four 11

ACCEPTED MANUSCRIPT Pro hydroxylation sites, respectively. As shown in Fig. 3, the mass of two peptides matched well with

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the theoretical mass with the error of 11.5 and 11.7 ppm, respectively. HCD MS/MS generated a

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series of y and b ions, positively confirming the identical sequence for these two unique peptides. For

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the peak with m/z 902.45762+, Pro795, Pro805 and Pro808 were identified to be hydroxylated by

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y19-b4, y8-y9 and y5-y6 ions in Fig. 3A, respectively. As shown in Fig. 3B, four proline

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hydroxylation sites of Pro795, Pro796, Pro805 and Pro808 in the peak of 910.45542+ were confirmed

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by y18-y19, y17-y18, y8-y9 and y5-y6 ions, respectively.

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3.4. Detection of unique peptides for gelatins from their mixture

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The common condition in food adulteration for gelatin products is to mix undesired gelatins into

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targeted gelatin. Therefore, It is necessary to further examine the marker peptides for each gelatin in

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the gelatin mixtures to determine the food authenticity. Because of the high similarity in three gelatins,

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the peptides produced by three gelatins may disturb each other's identification. To verify whether

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those unique peptides were useful to trace the gelatin source, bovine-hide gelatin, porcine-hide gelatin

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and donkey-hide gelatin were mixed as the ratio of 1:1:1, 1:1:10 and 10:10:1 (w:w:w), respectively.

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The results indicated all detectable peptides could be easily recognized without strong peaks

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interference. Fig. 4. shows representative marker peptides of donkey-hide gelatin, bovine-hide gelatin

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and porcine-hide gelatin from their mixture as the ratio of 1:1:1 (w:w:w). The gelatin marker peptides

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from bovine-hide (1037.99262+), porcine-hide (1062.03002+) and donkey-hide (733.35002+), were

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clearly identified without any interference.

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Table 2 lists all the unique peptides identified for bovine-hide gelatin when mixed with

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porcine-hide gelatin and donkey-hide gelatin. When the bovine-hide gelatin content was relatively 12

ACCEPTED MANUSCRIPT high at 10:10:1 (w:w:w for bovine-hide gelatin: porcine-hide gelatin: donkey-hide gelatin), 16 unique

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peptides were identified for bovine-hide gelatin by accurate mass and tandem mass spectrometry.

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When its relative content was lowered, the number of detected unique peptides was decreased to 14

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and 11, at mixing ratio of 1:1:1 and 1:1:10 (w:w:w for bovine-hide gelatin: porcine-hide gelatin:

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donkey-hide gelatin), respectively. This suggested that the dilution factor was a very important issue

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that needed to be considered in the detection of marker peptides in the mixture.

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Similarly, as shown in Table 3, the number of marker peptides for porcine-hide gelatin was 17,

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17 and 15, when its relative contents was gradually reduced in gelatin mixtures with the mixing ratio

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of 10:10:1, 1:1:1 and 1:1:10 (w:w:w for bovine-hide gelatin: porcine-hide gelatin: donkey-hide

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gelatin), respectively. For the identification of donkey-hide gelatin, Table 4 listed various marker

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peptides in different samples. The detected marker peptides for donkey-hide gelatin were 12, 10 and 5

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in three kinds of gelatin mixture with the mixing ratio of 1:1:10, 1:1:1 and 10:10:1 (w:w:w for

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bovine-hide gelatin: porcine-hide gelatin: donkey-hide gelatin), respectively. It indicated that five

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unique peptides including m/z 733.34772+, 765.90422+, 649.33083+, 1073.06092+ and 802.92182+, were

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useful to identify donkey-hide gelatin when its content was as low as about 5% of total gelatin.

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The marker peptides which could be detected in the low concentration, were very important to

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trace the gelatin source in food industry. In addition, It is worth mentioning that all three types of

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unique gelatin marker peptides can be identified at various mixing ratios in one LC-MS/MS run, is

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essential in gelatin authentication. LC-MS/MS can not only determine that food is adulterated, but

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also can define the origin of the adulteration. It should be noted that this method is not limited to the

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three types of mammalian gelatin, it can be extended to any additional gelatins as long as the

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sequences of the gelatins are known.

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4. Conclusion Based on the identification results of characteristic peptides, hemoglobin was found only in the

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donkey-hide gelatin, but not in bovine-hide and porcine-hide gelatin. According to the results from

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the multiple sequence alignment software, 49, 50 and 55 theoretical unique sequence fragments were

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found for bovine-hide gelatin, porcine-hide gelatin and donkey-hide gelatin, respectively. In the

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individual gelatin, 28, 27 and 14 unique peaks were detected by HPLC-LTQ/Orbitrap mass

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spectrometry to represent the bovine-hide gelatin, porcine-hide gelatin and donkey-hide gelatin,

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respectively. In the mixtures of three mammalian gelatins, detectable marker peptides were easily

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recognized without strong peaks interference. However, the number of marker peptides was affected

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by the concentration of targeted gelatin. When the content of target gelatin in the mixture was less to

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10%, 11, 15 and 5 marker peptides were detected in the gelatin mixture digests for bovine-hide gelatin,

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porcine-hide gelatin and donkey-hide gelatin, respectively. With the high resolution and accuracy,

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HPLC-LTQ/Orbitrap high-resolution mass spectrometry could be a potential technology for quality

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control of various gelatin products in food industry. However, other food ingredients including sugar

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and oil, should be considered to possibly disturb the identification. Therefore, simulative products and

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real foods containing varied gelatins, need to be further researched for improving gelatin

287

identification method by liquid chromatography-high resolution mass spectrometry.

288

5. Acknowledgements

289

This study was supported by the National Natural Science Foundation of China (No. 31660487), the

290

earmarked fund for China Agriculture Research System (CARS-45), the Key Research Project of

291

Jiangxi Province (No. 20161BBF60096, 20161BBF60021), the earmarked fund for jiangxi

AC C

EP

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M AN U

SC

RI PT

272

14

ACCEPTED MANUSCRIPT 292

Agriculture Research System (No. JXARS-03), the Collaborative Innovation Center for Major

293

Ecological Security Issues of Jiangxi Province and Monitoring Implementation (No. JXS-EW-00),

294

and the Project of Education Department of Jiangxi Province (No. GJJ150303).

RI PT

295 296

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ACCEPTED MANUSCRIPT Table 1. The theoretical unique peptides for three types of gelatin unique peptides from bovine-hide gelatin

unique peptides from porcine-hide gelatin

9

α1: LLLLLAATALLTHGQEEGQEEGQEEDIPPVTCVQNGLR

46

unique peptides from donkey-hide gelatin

9

α1: LLLLLAATALLTHGQEEGQEEGQQGQEEDIPPVTCVQNGLR

49

α1:9LLLLLAATALLTHGQEEGQEEGQEEDIPAVTCIQDGLR46

α1:55PVPCQICVCDNGNVLCDDVICDELK79

α1:58PVPCQICVCDNGNVLCDDVICDEIK82

α1:51AVWKPEPCR59

α1:80DCPNAK85

α1:83NCPSAR88

α1:60VCICDNGNVLCDDVICEDTK79

171

α1: STGISVPGPMGPSGPR

α1:89VPAGECCPVCPEGEVSPTDQETTGVEGPK117

186

190

α1: GLSGPPGAPGPQGFQGPPGEPGEPGASGPMGPR α1:315PGPPGPAGAR324

α1:791GEAGPSGPAGPTGAR795

α1:373GEPGPPGPAGAAGPAGNPGADGQPGGK399

α1:835GDAGPPGPAGPAGPPGPIGNVGAPGPK861

α1:451GEPGPTGVQGPPGPAGEEGK470

α1:918PGEVGPPGPPGPAGEK933

α1:508GPAGERGSPGPAGPK522

α1:934GAPGADGPAGAPGTPGPQGIAGQR957

α1:829GGPTGPPGPIGSVGAPGPK847

α1:1026DGSPGAK1032

α1:970QGPSGPSGER979

α1:1066GETGPAGPAGPIGPVGAR1083

α1:1012DGAPGPK1018

α1:1141GPPGSAGSPGK1151

α1:1022GESGPAGPPGAPGAPGAPGPVGPAGK1047

1288

α1:

1309

α1:430GNSGEPGAPGNK441

α1:

α1:472GEPGPTGLPGPPGER486

α1:487GGPGAR492 α1:631GEQGPAGSPGFQGLPGPAGPPGESGK656 α1:697GSNGAPGNDGAK708 α1:751GADGSPGK758 α1:835GDAGPPGPAGPAGPPGPIGSVGAPGPK861

α1:1052GETGPAGPAGPVGPVGAR1069 1156

VFCNMETGETCVYPTQPSVAQK

α1:89DECCPVCPEGQVSPTDDQTTGVEGPK114

α1:127GPSGPPGR134

M AN U

α1:1170TGDAGPAGPPGPPGPPGPPGPPSGGYDLSFLPQPPQEK1207

222

SC

α1:352GEGGPQGPR360

α1:80NCPGASVPK88

RI PT

α1:86VPTDECCPVCPEGQESPTDQETTGVEGPK114

α1:882VGPPGPSGNAGPPGPPGPVGK902 1193

TGDAGPVGPPGPPGPPGPPGPPSGGFDFSFLPQPPQEK

α1:903EGGK906

α1:1274VFCNMETGETCVYPTQPSVPQK1295

α1:1322HVWYGESMTGGFQFEYGGQGSDPADVAIQLTFLR1355

α1:1308HVWYGESMTDGFQFEYGGEGSDPADVAIQLTFLR1341

α1:1014EGSPGAEGSPGR1025

α1:1435RLPIIDVAPLDVGAPDQEFGFDVGPACFL1463

α1:1391FTYSVIYDGCTSHTGAWGK1409

α1:1026DGSPGPK1032

α2:33GPSGDR38

α1:1421RLPIIDVAPLDVGAPDQEFGIDLSPVCFL1449

α1:1066GEAGPAGPAGPIGPVGAR1083

α2:98GPPGASGAPGPQGFQGPPGEPGEPGQTGPAGAR130

α2:53DGDDGIPGPPGPPGPPGPPGLGGNFAAQYDGK84

α1:1170TGDAGPVGPPGPPGPPGPPGPPSAGFDFSFLPQPPQEK1207

α2:281GEVGLPGLSGPVGPPGNPGANGLPGAK307

α2:85GVGAGPGPMGLMGPR99

α1:1288VFCNMETGETCVYPTQPQVAQK1309

α2:326GIPGPVGAAGATGAR340

α2:100GPPGAVGAPGPQGFQGPAGEPGEPGQTGPAGAR132

α1:1322HVWYGESMTDGFQFEYGGQGSDPADVAIQLTFLR1355

α2:359GEPGAVGQPGPPGPSGEEGK378

α2:235GNDGSVGPVGPAGPIGSAGPPGFPGAPGPK264

α1:1435RLPIIDVAPLDIGAPDQEFGIDIGPVCFL1463

α2:283GEVGLPGVSGPVGPPGNPGANGLPGAK309

α2:9TLLLLAVTSCLATCQSLQEATAGK32

α2:380GSTGEIGPAGPPGPPGLR397 497

504

α2: GPSGDPGK

α2:505AGEKGHAGLAGAR517

EP

TE D

α1:1319EKR1321

AC C

376

310

α2: GAAGLLGVAGAPGLPGPR

327

α1:994GPPGPVGPPGLAGPPGESGR1013

α2:98GPPGASGAPGPQGFQGPAGEPGEPGQTGPAGAR130

α2:382GPNGEVGSAGPPGPPGLR399

α2:179GHKGLDGLK187

α2:518GAPGPDGNNGAQGPPGLQGVQGGK541

α2:414AGVMGPPGSR423

α2:197GEPGAPGENGTPGQAGAR214

α2:542GEQGPAGPPGFQGLPGPAGTAGEAGK547

α2:424GPTGPAGVR432

α2:281GEVGLPGLSGPVGPPGNPGANGLTGAK307

α2:590GPPGESGAAGPTGPIGSR607

α2:451GFPGSPGNVGPAGK464

α2:380GPNGEPGSTGPAGPPGLR397

17

ACCEPTED MANUSCRIPT α2:620GEPGVVGAPGTAGPSGPSGLPGER643

α2:674GAPGAIGAPGPAGANGDR691

α2:507NGEKGHAGLAGAR519

α2:505PGDKGHAGLAGAR517

α2:793TGPPGPSGISGPPGPPGPAGK813

α2:574GIPGEFGLPGPAGPR588

α2:662GEIGNPGR669

α2:829SGETGASGPPGFVGEK844

α2:664GDVGSPGR671

α2:692GEAGAAGPAGPAGPR706

α2:845GPSGEPGTAGPPGTPGPQGLLGAPGFLGLPGSR877

α2:748GENGPVGPTGPVGAAGPAGPNGPPGPAGSR777

α2:776GDGGPPGVTGFPGAAGR792

α2:905GPPGNVGNPGVNGAPGEAGR924

α2:795IGPPGPSGISGPPGPPGPAGK815

α2:925DGNPGNDGPPGR936

α2:831TGETGASGPPGFAGEK846

α2:947GYPGNAGPVGAAGAPGPQGPVGPVGK972

α2:847GPSGEPGTAGPPGTPGPQGILGAPGFLGLPGSR879

α2:845GPSGEPGTAGPPGTPGPQGLLGAPGILGLPGSR877

α2:977GEPGPAGAVGPAGAVGPR994

α2:907GPPGAVGNPGVNGAPGEAGR926

α2:881GLPGVAGSLGEPGPLGIAGPPGAR904

α2:1022GHNGLQGLPGLAGHHGDQGAPGAVGPAGPR1051

α2:939DGQAGHK945

α2:793TGPPGPSGISGPPGPPGAAGK813 α2:829AGETGASGPPGFAGEK844

α2:1079GSQGSQGPAGPPGPPGPPGPPGPSGGGYEFGFDGDFYR1116

α2:979GEPGPAGSVGPAGAVGPR996

α2:977GEPGPVGSVGPVGAVGPR994

α2:1122SPTSLR1127

α2:1006GEKGEPGDK1014

α2:1204AQPEDIPVK1212

α2:1024GHNGLQGLPGLAGHHGDQGAPGPVGPAGPR1053

α2:1016GLPGIK1021

α2:1217NSKAKK1222

α2:1068TGQPGAVGPAGIR1080

α2:1022GHNGLQGLPGLAGQHGDQGAPGSVGPAGPR1051

α2:1223HVWVGETINGGTQFEYNVEGVTTK1246

α2:1081GSQGSQGPAGPPGPPGPPGPPGPSGGGYDFGYEGDFYR1118

α2:1052GPAGPTGPVGK1062

α2:1319TNEWQK1324

α2:1219NSKVKK1224

α2:1066SGQPGTVGPAGVR1078

α2:1337LPILDIAPLDIGGADQEIR1355

α2:1225HVWLGETINGGTQFEYNMEGVTTK1248

α2:1079GSQGSQGPAGPPGPPGPPGPPGPSGGGYDFGYDGDFYR1116

α2:1356LNIGPVCFK1364

α2:1321TNEWR1325

α2:1217SSKAKK1222

—

α2:1339LPILDIAPLDIGDADQEVSVDVGPVCFK1366

α2:1223HIWLGETINGGTQFEYNVEGVTTK1246

—

—

α2:1272NSIAYLDEETGNLK1285

—

—

—

—

—

AC C

—

974

EP

TE D

M AN U

α2:947GYPGNAGPVGAVGAPGPHGPVGPTGK972

IGQPGAVGPAGIR

949

α2:905GPPGAVGAPGVNGAPGEAGR924

α2: GYPGNPGPAGAAGAPGPQGAVGPAGK

α2:

1078

506

RI PT

α2:497GPTGEPGK504

1066

499

α2:398GSPGSR403

α2: GPTGDPGK

α2: GDIGSPGR

377 378 379 380 381

α2:465EGPAGLPGIDGR476

669

SC

662

α2:995GPSGPQGVR1003

α2:1287AVTLQGSNDVELVAEGNSR1305 α2:1306FTYTVLVDGCSR1317

—

α2:1319TNEWGK1324

—

α2:1337LPILDIALLDIGGADQEFGLDIGPVCFK1364

The marker peptides were obtained from the results of the multiple sequence alignment software. The sequence number was listed based on the actual position of the sequence. The modification sites were unknown and need to further verified. α1 and α2 indicate the sequence fragments are from α1 and α2 chains of the collagen, respectively. — represents there are no more theoretical unique peptides for bovine-hide gelatin and porcine-hide gelatin. 18

ACCEPTED MANUSCRIPT Table 2. The detected unique peptides for bovine-hide gelatin

*

*

*

*

α1: GDAG PPGPAGPAGPPGPIG NVGA PGP KGAR 907

* *

*

864

933

α1: GPRGETGPAGRPGEVG P PG PPGPAGEK 934

*

α1: GAPGADGPAGAPGTPGPQGIAG QR 934

*

957

*

957

*

957

α1: GAPGADGPAGAPGTPGP QGIAG QR 934

*

934

*

α1: GAPGADGPAGA PGTPGPQGIAG QR *

*

α1: GAPGADGPAGA PGTPGP QGIAG QR 934

*

*

*

*

957

α1: GA PGADGPAGA PGTPGP QGIAG QR

957

α1:934GAPGADGPAGA*PGTPGP*QGIAG*QRGVVGL*PG*QR966 934

*

*

*

*

*

*

*

α1: GA PGADGPAGA PGT PGP QGIAG QRGVVGL PG QR 1066

α1:

GETGPAGPAGPIGPVGAR

1083

1062

α1:

SGDRGETGPAGPAGPIGPVGAR

326

*

α2: GI PGPVGAAGATGAR 308

*

*

1083

340

*

*

α2: GAAGL PGVAGA PGL PGPRGI PGPVGAAGATGAR 359

*

*

*

620

*

*

620

*

*

*

340

378

α2: GE PGAVG Q PGP PGPSGEEGK

α2: GE PGVVGA PGTAGPSGPSGL PGER α2: GETGLRGDIGS PGR

643

669

*

*

793

* *

α2: GA PGAIGA PGPAGA NGDRGEAGPAGPAGPAGPR *

*

813

α2: TG P PGPSGISGPPGP PG PAGK 844

α2: SGETGASGPPGFVGEK 829

*

844

α2: SGETGASGP PGFVGEK 947

*

977

*

973

*

1066

*

*

972

α2: GY PGNAGPVGAAGA PGPQGPVGPVGK α2: GE PGPAGAVGPAGAVGPR *

994

α2: HG NRGE PGPAGAVGPAGAVGPR α2:

1066

α2:

IG QPGAVGPAGIR *

*

1078

IG Q PGAVGPAGIR

1029.9956

2+

1037.5011

2+

1037.9931

2+

1045.9905

2+

1078

994

864.7741

3+

842.7574

3+

1029.5187

2+

1030.0062

2+

1037.5118

2+

1038.0048

2+

1046.0017

2+

706

Error(ppm)

m/z

Error(ppm)

m/z

Error(ppm)

9.6

—

–

—

–

—

–

10.7

—

–

—

–

—

–

9.7

—

–

—

–

—

14.7 10.3 10.4 11.3 10.7

1029.9956

1045.9905

996.4866

996.4973

10.7

780.9101

2+

780.9204

2+

659.3363

3+

659.3436

3+

634.3413

2+

634.3485

2+

11.4

937.8433

3+

10.4

—

926.9237

2+

11.1

—

1068.0218

2+

1076.0193

2+

2+

927.7815

3+

923.4525

2+

738.8519

2+

746.8494

2+

1131.0691

2+

766.8944

2+

666.6682

3+

597.3355

2+

605.3329

2+

1068.0338

2+

1076.0304

2+

694.3569

2+

893.95182+ 927.7920

3+

923.4620

2+

738.8599

2+

746.8573

2+

1131.0831

2+

11.2 10.3

2+ 2+

— —

780.9115

2+

659.3342

3+

634.3417

2+

1068.0209

2+

1076.0193

2+

2.1

-0.04

1037.9910

2+

1045.9870

2+

–

—

0.69

780.9109

634.3406 —

–

—

0

2+

2+

1068.0210 1076.016

-2.49 -3.67

2+

2+

1029.5038

0.18

1029.9952

2+

-0.35

1037.4977

2+

-3.26

1037.9929

2+

-0.14

1045.9900

2+

-0.51

–

—

–

—

1.11 –

—

–

-0.9

–

—

—

-3.19

–

—

–

1.81

–

—

-1.05

-1.08

659.3362

3+

-0.14

634.3411

2+

-0.39

–

—

-1.13

1076.0166

2+

-2.5

–

—

–

—

—

–

—

–

—

11.3 10.2 10.9 10.6 12.4

766.9028

11.0

666.6754

3+

10.9

597.3418

2+

605.3397

2+

10.6 11.2

927.7811 923.4511

2+

— 746.8495

2+

2+

—

–

—

0.17

-0.91 –

— 597.3364

2+

605.3330

2+

927.7802

-1.54

–

— 766.8937

-0.45

1.58 0.11

746.8483

– – 2+

-1.39 –

— 766.8934

-1.38

2+

-1.32 –

— 597.3346

2+

605.3325

2+

-1.5 -0.8

–

1068.0206

—

3+

–

2+

9.6

3+

–

780.9092

—

-3.18

–

2+

–

-0.78

– 2+

10.1

2+

19

0.007 –

1037.9952

3+

11.1

2+

—

3+

13.2

–

—

11.1

2+

BG:PG:DG=10:10:1

m/z

985.83433+

937.8335

BG:PG:DG=1:1:10

Error(ppm)

985.82343+

893.94322+

*

829

1029.5036

2+

694.3499

α2:656GETGLRGDIGS*PGRDGAR673 674

842.7492

3+

641.3189

2+

TE D

643

*

*

864.7649

3+

926.9134

α2: GE PGVVGA PGTAGPSGPSGLPGER

656

966

641.3127

m/z

RI PT

835

2+

BG:PG:DG=1:1:1

M AN U

α1: GEAGPSGPAGPTGAR

795

EP

791

pure BG

SC

Theoretical m/z

detected unique peptides from bovine gelatin

AC C

382

– –

927.7816

3+

0.079

923.4507

2+

-1.94 –

— 746.8478

2+

–

— 766.8923

-2.04

2+

-2.83 –

— 597.3348

2+

-1.18

605.3320

2+

-1.61

ACCEPTED MANUSCRIPT *P indicates proline hydroxylation; *N and *Q indicate deamidization of Asparagine and Glutamine, respectively. DG, BG and PG denote donkey-hide gelatin, bovine-hide gelatin and porcine-hide gelatin, respectively.

EP

TE D

M AN U

SC

RI PT

— represents undetectable in this work.

AC C

383 384 385

20

ACCEPTED MANUSCRIPT

386

Table 3. The detected unique peptides for porcine-hide gelatin pure PG

970

*

*

*

α1: QGPSGPSGERGPPGP MGP PGLAG PPGESGR 1022

*

1022

*

α1: α1:

GESGPAGP PGAPGAPGAPGPVGPAGK *

1047

GESGPAGP PGA PGAPGAPGPVGPAGK GETGPAGPAGPVGPVGAR

1069

1048

α1:

SGDRGETGPAGPAGPVGPVGAR

1069

1048

α1:

SGDRGETGPAGPAGPVGPVGARGPAGPQGPR

1078

α2:85GVGAGPGPMGLMGPR99 283

*

*

*

*

α2: EGPAGL PGIDGRPGPIGPAGAR *

*

309

*

506

574

*

*

α2: GI PGEFGL PGPAGPR

588

*

α2: GI PGEFGL PG PAGPR 745

588

*

*

α2: GP KGENGPVGPTGPVGAAGPAGPNGPPG PAGSR 795

* *

*

*

777

815

α2: IG P PGPSGISGPPGP PG PAGK 831

846

α2: TGETGASGPPGFAGEK 949

*

*

974

α2: GY PGNPGPAGAAGA PGPQGAVGPAGK

*

*

1008

GHNGLQGLPGLAGHHGDQGAPGPVGPAGPR

1024

*

1024

*

*

1024

*

*

α2: α2:

*

1067

GHNGLQGL PGLAGHHGDQGAPGPVGPAGPRGPAGPSGPAGKDGR *

1067

GHNGLQGL PGLAGHHGDQGA PGPVGPAGPRGPAGPSGPAGKDGR

1068

387

1064

GHNGLQGLPGLAGHHGDQGAPGPVGPAGPRGPAGPSGPAGKDGR

1024

α2:

1053

GHNGLQGL PGLAGHHGDQGA PGPVGPAGPRGPAGPSGPAGK

*

α2:

1053

GHNGLQGL PGLAGHHGDQGA PGPVGPAGPR

1024

α2:

1053

GHNGLQGL PGLAGHHGDQGAPGPVGPAGPR

1024

α2:

TGQPGAVGPAGIR

1080

695.6050

4+

1062.0408

2+

1070.0409

2+

2+

—

11.2

676.6922

3+

637.6463

3+

727.3753

2+

735.3728

2+

955.1409

3+

929.4707

2+

731.8441

2+

1067

929.1003

3+

10.7 13.1

1062.0300

2+

1070.0247

2+

Error (ppm)

m/z

Error (ppm)

m/z

—

—

—

—

-3.38

654.6647

3+

-2.11

—

1062.0280

2+

1070.0260

2+

-0.85

-2.57

1070.0243

-2.45

12.8

695.6133

12.0

—

—

—

—

—

677.34872+

10.0

—

—

—

—

—

2+

11.7

676.6997

3+

11.0

637.6533

3+

11.0

727.3837

2+

735.3810

2+

955.1516

3+

929.4811

2+

11.2

731.8528

2+

12.0

11.5 11.1 11.2

1208.6063 676.6920

2+

3+

—

0.33

-2.06 -0.39 —

727.3719

2+

735.3710

2+

955.1401

3+

929.4710

2+

-4.76 -2.42 -0.82 0.25

654.663

1208.6060 676.6912

2+

3+

—

-1.24 -2.19

-2.36 -1.57 —

727.3741

2+

735.3704

2+

955.1404

3+

929.4699

2+

-1.75 -3.33 -0.5 -0.87

-2.72

2+

4+

3+

— 3+

1062.0267

654.6728

773.9013

Error (ppm)

2+

3+

-0.61

2+

-1.87

929.1010

773.9121

1208.6229

773.9018

0.54

—

2+

12.7

2+

BG:PG:DG=10:10:1

773.9016

2+

-0.93

654.6633

3+

-1.72 — —

1208.6065 676.6909

2+

3+

—

-1.95 -1.93 —

727.3717

2+

-5.02

735.3712

2+

-2.16

955.1397

3+

-1.27

929.4684

2+

-2.52

1103.0502

11.2

1103.0356

-1.97

1103.036

-1.63

1103.0361

774.89192+

774.90172+

12.7

774.89142+

-0.69

774.89012+

-2.27

774.89092+

-1.25

798.9056

4+

798.9138

4+

10.3

—

—

—

—

—

—

695.3538

4+

695.3640

4+

699.3525

4+

699.3608

4+

703.3512

4+

703.3602

4+

12.8

738.1715

5+

738.1810

5+

12.9

—

—

—

—

—

—

797.4034

5+

797.4141

5+

13.4

—

—

—

—

—

—

800.6024

5+

800.6115

5+

11.4

—

—

—

—

—

—

670.0024

6+

670.0098

6+

11.1

—

—

—

—

—

590.8253

2+

590.8322

2+

AC C

1024

α2:

654.6644

3+

EP

α2: HGNRGE PGPAGSVGPAGAVGPRGPSG PQGIRGEK α2:

773.9023

2+

1103.0378

α2:979GE*PGPAGSVGPAGAVGPR996 975

1070.0269

2+

929.1138

11.4

BG:PG:DG=1:1:10

TE D

*

1062.0295

2+

1208.6088

486

α2: GE PGNIGF PGPKGPTGDPGK 574

3+

m/z

M AN U

*

487

3+

677.34192+

α2: GEVGL PGVSGPVGP PGN PGANGL PGAK 465

923.78233+

929.1035

1047

1052

α1:

999

923.77183+

Error (ppm)

RI PT

α1:970QGPSGPSGERGPPGP*MGPPGLAGP*PGESGR999

m/z

BG:PG:DG=1:1:1

SC

detected unique peptides from porcine gelatin

Theoretical m/z

2+

2+

14.7 12.0

11.7

—

695.3546

4+

699.3530

4+

703.3519

4+

590.8256

*P indicates proline hydroxylation; *N and *Q indicate deamidization of Asparagine and Glutamine, respectively. 21

— 2+

2+

1.16 0.79 0.95

0.56

—

— 2+

—

—

699.3502

4+

703.3502

4+

590.8241

2+

-3.23 -1.4

-2.02

—

— 2+

-1.52

695.3534

4+

-0.43

699.3508

4+

-2.44

703.3499

4+

-1.84

590.8234

— 2+

-3.16

ACCEPTED MANUSCRIPT DG, BG and PG denote donkey-hide gelatin, bovine-hide gelatin and porcine-hide gelatin, respectively.

EP

TE D

M AN U

SC

RI PT

— represents undetectable in this work.

AC C

388 389 390

22

ACCEPTED MANUSCRIPT

391

Table 4. The detected unique peptides for donkey-hide gelatin pure DG

*

*

* *

902

α1: VGPPG PSG NAG P PGPPGPVGK 1066

α1:

GEAGPAGPAGPIGPVGAR

1083

1062

α1:

SGDRGEAGPAGPAGPIGPVGAR

776

*

*

α2: GDGGP PGVTGF PGAAGR 793

*

793

* *

*

1083

792

*

813

α2: TG PPGPSGISGP PGP PGAAGK *

*

813

α2: TG P PGPSGISGP PGP PGAAGK 829

*

844

α2: AGETGASGP PGFAGEK 881

*

*

*

α2: GL PGVAGSLGE PGPLGIAGP PGAR

904

*

*

*

972

α2: GERGY PG NAGPVGAVGA PGPHGPVGPTGK 977

*

973

*

*

973

*

*

α2: GE PGPVGSVGPVGAVGPR

994

α2: HG NRGE PGPVGSVGPVGAVGPR

994 *

1006

α2: HG NRGE PGPVGSVGPVGAVGPRGPSG PQGVRGDK

m/z

Error (ppm)

m/z

Error (ppm)

733.34952+

733.35812+

11.7

733.35052+

1.27

733.34822+

-1.8

733.34772+

-2.47

921.4551

2+

921.4649

2+

10.7

2+

1.60

921.4539

2+

-1.31

—

—

765.9048

2+

765.9142

2+

765.9059

2+

649.3328

3+

649.3408

3+

649.3323

3+

-0.81

751.3552

2+

751.3628

2+

751.3565

2+

1.72

—

–

902.4472

2+

902.4576

2+

902.4463

2+

-1.05

—

–

910.4447

2+

910.4554

2+

910.4430

2+

-1.81

—

–

724.8362

2+

724.8451

2+

724.8361

2+

-0.18

—

2+

-6.01

2+

1073.0800

2+

921.4565

12.3 12.3 10.2 11.5

649.3331

3+

751.3556

2+

902.4472

2+

11.7

—

12.2 10.6

724.8375

2+

1073.0624

2+

2.21 0.41 0.57

-0.05 – 1.76

1.41

765.9042

2+

-0.82

649.3308

3+

-3.08

-5.78

1073.062

767.72343+

13.0

767.71343+

-0.13

767.71133+

-2.84

—

661.5782

4+

661.5864

4+

12.4

—

–

—

–

—

802.9232

2+

802.9325

2+

690.6874

3+

690.6957

3+

806.1594

4+

806.1683

4+

11.6

TE D

EP

-1.58

—

–

11.0

—

–

DG, BG and PG denote donkey-hide gelatin, bovine-hide gelatin and porcine-hide gelatin, respectively.

23

802.9219

2+

12.0

*P indicates proline hydroxylation; *N indicate Asparagine deamidization. — represents undetectable in this work.

765.9065

2+

767.71353+

AC C

392 393 394 395 396 397

Error (ppm)

1073.0686

α2:947GY*PG*NAGPVGAVGA*PGPHGPVGPTGK972 944

m/z

RI PT

882

BG:PG:DG=10:10:1

Error (ppm)

SC

α1:472GE*PGPTGL*PGP*PGER486

BG:PG:DG=1:1:10

m/z

M AN U

detected unique peptides from donkey gelatin

BG:PG:DG=1:1:1

Theoretical m/z

–

1073.0609

2+

-7.15 – –

802.9203

2+

-3.64

690.6862

3+

-1.67

—

–

–

—

–

—

802.9218

2+

-1.74

ACCEPTED MANUSCRIPT 398

Captions for Figures

399 400 401 402 403 404

Fig. 1. Peptide identification of hemoglobin from donkey-hide gelatin. (A): peptide 33MFLGFPTTK41

405 406 407 408

Fig. 2. MS/MS spectra of marker peptides. (A):

from the α-subunit of donkey hemoglobin; (B): peptide β-subunit of donkey hemoglobin; (D) peptide

31

LLVVYPWTQR40 from β-the subunit of donkey

chain of bovine-hide gelatin; (B): peptide porcine-hide gelatin; (C): peptide

1066

RI PT

hemoglobin.

peptide 1066GETGPAGPAGPIGPVGAR1083 from α1

1052

GETGPAGPA GPVGPVGAR1069 from α1 chain of

GEAGPAGPAGPIGPVGAR1083 from α1 chain of donkey-hide

SC

gelatin.

793

A: the peptide hydroxylated) 793

in

* *

TG*PPGPSGISGP*PGP*PGAAGK813 (Pro795, Pro805 and Pro808 were α2

the *

chain

*

TG P PGPSGISGP PGP PGAAGK

of

813

the α2 chain of the donkey-hide gelatin.

the

donkey-hide

gelatin;

(B):

the

peptide

(Pro795, Pro796, Pro805 and Pro808 were hydroxylated) in

934

TE D

Fig. 4. Characteristic peptides from three gelatins in the mixing ratio of 1:1:1 (w:w:w). A: peptide GAPGADGPAGA*PGTPGP*QGIAG*QR957 (Pro945 was hydroxylated; Glu951 and Glu956 were

deamidated)

in

1022

*

the

α1

chain

of

GESGPAGP PGAPGAPGAPGPVGPAGK

porcine-hide gelatin; C: peptide

472

*

1047

the

bovine-hide

gelatin;

(B):

peptide

(Pro1030 was hydroxylated) in the α1 chain of the

*

GE PGPTGL PGP*PGER486 (Pro474, Pro480 and Pro483 were

EP

hydroxylated) in the α1 chain of the donkey-hide gelatin.

AC C

422

M AN U

Fig. 3. Different modification level identification by tandem mass spectrometry for marker peptides.

415 416 417 418 419 420 421

VGG*NAGEFGAEALER32 (Asn21 was

deamidated) from the α-subunit of donkey hemoglobin; (C): peptide 9AAVLALWDK17 from the

409 410 411 412 413 414

18

24

423 424 425

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 1

25

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

426 427

Fig. 2

428

26

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

429 430

Fig. 3

431

27

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

432 433

Fig.4

434 435 28

ACCEPTED MANUSCRIPT 1. HPLC-LTQ/Orbitrap MS/MS was used to identify three mammalian gelatins. 2. Hemoglobin was found only in the donkey-hide gelatin while but not in bovine and porcine gelatins. 3. 28, 27 and 14 distinct peaks were detected in pure bovine, porcine and donkey gelatins, respectively. 4. Detectable marker peptides decreased with reduced concentration of target gelatin in their mixtures.

AC C

EP

TE D

M AN U

SC

RI PT

5. 11, 15 and 5 marker peptides were used to represent above three gelatins in low content, respectively.