CN114062686A

CN114062686A - Method for identifying salmon by using specific peptide fragment group

Info

Publication number: CN114062686A
Application number: CN202111416103.2A
Authority: CN
Inventors: 张晓梅; 张鸿伟; 蒋冰雪; 林超; 赵雪; 徐杰; 古淑青
Original assignee: Qingdao Customs Technology Center
Current assignee: Qingdao Customs Technology Center
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-02-18

Abstract

The invention relates to a method for identifying salmon, which is Salmonidae (Salmonoidae) fish, by using a special peptide fragment group, wherein the method comprises the following steps: atlantic salmon (Salmo salar), Pacific salmon (Oncorhynchus keta) and rainbow trout (Oncorhynchus mykiss); the specific peptide fragment group comprises polypeptides shown in SEQ ID NO. 1-14.

Description

Method for identifying salmon by using specific peptide fragment group

Technical Field

The invention relates to the technical field of biology, in particular to a method for identifying salmon by using a specific peptide fragment group.

Background

Salmon (Salmonidae ) has delicious meat, is rich in DHA, EPA, astaxanthin and other bioactive substances, and is deeply loved by consumers. Salmon is a trade name of fishes belonging to the family Salmonidae, the class Osteichthyes, the order Salmoniformes, the genus Salmon, most commonly salmon (Atlantic salmon, Pacific salmon, silver salmon) and trout (rainbow trout, golden trout). Among them, the Atlantic salmon (Salmo salar) is considered to have the best quality and is mostly imported to high-altitude ocean cold regions such as Norway, Denmark and Chilean. Pacific salmon (Oncorhynchus keta) migrates into the waters of Heilongjiang, Turkey river and the like in China, and the Wusuli river is more. The rainbow trout (Oncorhynchus) of Pacific salmon is originally produced in the Pacific coast of North America, is introduced in 1959 in China and is one of the main freshwater cultured fishes in China. Compared with Atlantic salmon, Pacific salmon and rainbow trout have poor mouthfeel and low production cost and nutritional value, but consumers are difficult to distinguish by the appearance of fish. Driven by benefits, illegal merchants impersonate salmonids and rainbow trout impersonate salmonids with low price, and the fraudulent behaviors not only harm the credibility of aquatic product industry, but also damage the quality integrity and the interests of consumers of salmon products. Therefore, it is necessary to identify the species of salmon.

At present, the salmon species identification technology is mainly a DNA-based molecular biology technology and a proteomics technology. In the DNA detection technology mainly based on PCA, the operation steps are complicated and the time consumption is long.

Disclosure of Invention

Therefore, the invention provides a method for identifying salmon by using a specific peptide fragment group, which adopts a characteristic polypeptide method to identify different salmon species, is not influenced by protein stability and deep processing technology, and is a method capable of efficiently and accurately identifying salmon species.

The invention firstly relates to the application of a group of characteristic peptide fragments in identifying Salmonidae (Salmonoidae) fish subspecies,

the Salmonidae fish subspecies are as follows: atlantic salmon (Salmo salar), Pacific salmon (Oncorhynchus keta) and rainbow trout (Oncorhynchus mykiss);

specifically, the characteristic peptide segment is as follows:

(1) characteristic peptide stretches of the following proteins from Atlantic salmon (Salmo salar):

the peptide fragment with the sequence shown in SEQ ID NO. 1is from the protein glycogen phosphorinase, muscle form (gi | 213515556);

the peptide segment with the sequence shown in SEQ ID NO.2 is derived from a protein myostatin-binding protein C, fast-type-like (gi | 929275161);

the peptide segment with the sequence shown as SEQ ID NO.3 comes from protein sarcoplasic/endo coplasic reticulum ATPase1 isofomm X2(gi | 929274646);

the peptide segment with the sequence shown as SEQ ID NO.4 is derived from protein myosin, light polypeptide 3-1(gi | 213514656);

the peptide segment with the sequence shown as SEQ ID NO.5 comes from protein phosphoglycerate kinase (gi | 929235375);

the peptide segment with the sequence shown in SEQ ID NO.6 is derived from a protein myostatin binding protein H-like (gi | 213511568);

the peptide segment with the sequence shown as SEQ ID NO.7 is derived from protein sarcolemnin-like isofomm X2(gi | 929094737);

the peptide segment with the sequence shown as SEQ ID NO.8 is derived from protein L-lactate dehydrogenase A chain (gi | 213510970);

the peptide segment with the sequence shown in SEQ ID NO.9 is derived from a protein Myoglobin (gi | 221221136);

(2) characteristic peptide stretches of the following proteins from pacific salmon (Oncorhynchus keta):

the peptide segment with the sequence shown in SEQ ID NO.10 is derived from protein triosephosphate isomerase B-like (gi | 1695879275);

the peptide segment with the sequence shown in SEQ ID NO.11 is derived from protein myosin-2-like isofomm X2(gi | 1695910459);

(3) characteristic peptide stretches of the following proteins from rainbow trout (Oncorhynchus mykiss):

the peptide fragment with the sequence shown in SEQ ID NO.12 is derived from a protein Isocitrate dehydrogenase [ NADP ] (gi | 642108671);

the peptide segment with the sequence shown in SEQ ID NO.13 is derived from protein myosin-2-like isofomm X2(gi | 1211256714);

the peptide segment with the sequence shown in SEQ ID NO.14 is derived from protein myosin-2-like isofomm X2(gi | 1211256714);

more specifically, mass spectrometry detection mass-to-charge ratio (m/z) data of each characteristic peptide fragment is as follows:

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO. 1is 582.266;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.2 is 617.856;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.3 is 934.439;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.4 is 803.441;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.5 is 991.462;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.6 is 541.263;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.7 is 772.894;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.8 is 492.956;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.9 is 548.299;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.10 is 620.326;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.11 is 706.430;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.12 is 466.254;

the mass-to-charge ratio (m/z) of the characteristic peptide segment shown in SEQ ID NO.13 is 746.412;

the mass-to-charge ratio (m/z) of the characteristic peptide fragment shown in SEQ ID NO.14 is 591.275.

The invention also relates to a method for identifying the Salmonidae (Salmonoidae) fish subspecies by using a mass spectrometric detection method through the characteristic peptide segment, wherein the Salmonidae fish subspecies are as follows: atlantic salmon (Salmo salar), Pacific salmon (Oncorhynchus keta) and rainbow trout (Oncorhynchus mykiss), characterized in that the method comprises the steps of:

(1) sample pretreatment

1) Weighing and grindingAdding protein extract (8M urea and 50mM NH) into powdered Salmonidae fish sample to be detected₄HCO₃) Extracting protein, centrifuging at high speed and low temperature, and collecting supernatant;

2) adding DTT (1mol/L) into the protein supernatant, and reacting for 1 hour at 60 ℃;

3) adding IAA (1mol/L) into the solution cooled to room temperature, and reacting for 1 hour at room temperature in a dark place;

4) centrifuging with a 10KDa molecular weight cut-off ultrafiltration centrifugal tube, and repeatedly washing the protein on the membrane with ammonium bicarbonate solution for 3 times;

5) adding a Trypsin enzyme solution into the protein solution, and carrying out enzymolysis for 16 hours at 37 ℃;

6) centrifuging by adopting a 10KDa molecular weight cut-off ultrafiltration centrifugal tube to collect lower-layer peptide filtrate, and waiting for detection on a computer;

(2) and (3) detecting by mass spectrometry, and matching the detection result with a standard spectrogram of the characteristic peptide segment of SEQ ID NO.1-14, thereby obtaining the species information of the sample to be detected.

The mass spectrum detection comprises the following steps:

1) an AB SCIEX tripleTOF mass spectrometry system is adopted,

mobile phase A: 0.1% formic acid-water, mobile phase B: 0.1% formic acid-acetonitrile;

the flow rate is 0.25 mL/min;

elution gradient: 0-2 min, 5% B; 2-27 min, 5-20% of B; 27-37 min, 20-35% B; 37-39 min, 35-80% B; 39-42 min, 80% B; 42-46 min, 5% B;

TOF scan range: 350-1500 Da, positive ion mode, GS 1: 35, GS 2: 45, Curtain Gas: 35, ISVF: 5500, TEM: 500, DP: 100, CE: 10.

or 2) an AB SCIEX 5500 triple quadrupole detection system is adopted,

the flow rate is 0.35 mL/min;

elution gradient: 0-10 min, 10-10.5% B; 10-10.5 min, 10.5-45% B; 10.5-13 min, 45-95% B; 13-13.1 min, 95-10% B; 13.1-15 min, 10%;

electrospray ion source, positive ion reaction mode, detection mode: MRM, spray voltage: 5500V, ion transfer tube temperature: 575 deg.c; atomizing gas pressure: 60psi, auxiliary heating gas pressure: 50psi, air curtain pressure: 35 psi.

The invention also relates to application of the characteristic peptide fragment group in preparing a detection kit for identifying the Salmonidae (Salmonidae) fish subspecies, wherein the characteristic peptide fragment group is any one or any combination of SEQ ID NO.1-14, and preferably, the kit is a kit based on mass spectrometry detection.

Drawings

FIG. 1, mass spectrum of characteristic peptide fragment SEQ ID NO.1VEEVDAMDAGK of Atlantic salmon.

FIG. 2 shows the mass spectrum of characteristic peptide segment SEQ ID NO.2IVEAPGPPEVVK of Atlantic salmon.

FIG. 3, mass spectrum of Atlantic salmon characteristic peptide fragment SEQ ID NO.3 EFDDLPSFELQSDAVR.

FIG. 4, mass spectrum of Atlantic salmon characteristic peptide segment SEQ ID NO.4 AAAPAPEPEVVAAPPPLDLSTVK.

FIG. 5 is a mass spectrum of characteristic peptide segment SEQ ID NO.5VLNNMEIGNSLYDDEGAK of Atlantic salmon.

FIG. 6, mass spectrum of characteristic peptide segment SEQ ID NO.6TGDWFNILEHYAR of Atlantic salmon.

FIG. 7, Atlantic salmon characteristic peptide fragment SEQ ID NO.7 EEEVEDVLSINLR mass spectrum.

FIG. 8 is a mass spectrum diagram of Atlantic salmon characteristic peptide segment SEQ ID NO.8 LITHTVLVGEPVGSR.

FIG. 9 is a mass spectrum of the characteristic peptide fragment SEQ ID NO.9LFAEHPETLTLFPK of Atlantic salmon.

FIG. 10, mass spectrum of Pacific salmon characteristic peptide fragment SEQ ID NO. 10AHVSEAVANSVR.

FIG. 11, mass spectrum of Pacific salmon characteristic peptide fragment SEQ ID NO. 11VGGGTATLTLPLLAK.

FIG. 12, mass spectrum of rainbow trout characteristic peptide fragment SEQ ID NO. 12ATDFVVSKPGTFK.

FIG. 13, mass spectrum of rainbow trout characteristic peptide fragment SEQ ID NO. 13DAAPAVAGAPGAPISVK.

FIG. 14, mass spectrum of rainbow trout characteristic peptide segment SEQ ID NO. 14SAGEIEEYQR.

Fig. 15 is a graph showing the results of randomly selected salmon samples.

In FIGS. 1-15, the ordinate is Intensity, in cps (counts per second); the abscissa is the Retention Time in min (minutes).

Detailed Description

Example 1 screening of characteristic peptide fragments of Salmon

1. DDA acquisition and establishment of mass spectra library

Three groups of salmon samples identified 912 proteins, 13615 peptides together at 95% confidence level, and the results of this library search were used to construct a relatively quantitative mass spectrum library of SWATH. Atlantic salmon identification protein 682 bars; pacific salmon identification protein 549 strips; the rainbow trout identified protein 513 strips. Wherein, the Atlantic salmon unique protein 395 bar; 269 strips of unique proteins from pacific salmon; rainbow trout has 218 unique proteins.

2. SWATH data collection

Analyzing SWATH data by using Peakview, checking peptide segment information corresponding to unique proteins, focusing on an extracted ion flow graph of each polypeptide, comparing actual fragments with spectrogram fragments and abundance distribution of the peptide segments in a sample, and screening to obtain the peptide segments with detection specificity.

3. Screening of species characteristic peptide fragments

Based on the above candidate peptide fragments, a Blastp search was performed at NCBI, and when the amino acid sequences of the peptide fragments are 100% matched and unique, the peptide fragments are considered to be biospecific.

4. MRM method optimization and detection analysis

And (3) calculating a peptide fragment transmission ion pair by adopting skyline, and optimizing a gradient elution program according to a spectrogram result. Determining the optimal target peptide fragment collision energy, depolymerization voltage, retention time and the like of the MRM acquisition method, and establishing a laboratory daily rapid detection method, which is shown in Table 1.

TABLE 1 characteristic peptide fragment MRM Mass Spectrometry parameters

Example 2 sequence Source and alignment information for Atlantic salmon (Salmo salar) specific Polypeptides

Through mass spectrometry, the polypeptide SEQ ID NO. 1-9 has independent response in Atlantic salmon (Salmo salar), good peak shape and high intensity, and has no response in Pacific salmon (Oncorhynchus keta) and rainbow trout (Oncorhynchus mykiss). Specifically, the method comprises the following steps:

FIG. 1is a mass spectrum of polypeptide VEEVDAMDAGK in Atlantic salmon, with m/z 582.266.

FIG. 2 is a mass spectrum of polypeptide IVEAPGPPEVVK in Atlantic salmon, with m/z 617.856.

FIG. 3 is a mass spectrum of polypeptide EFDDLPSFELQSDAVR in Atlantic salmon, with m/z 934.439.

FIG. 4 is a mass spectrum of polypeptide AAAPAPAPEPEVVAAPPPLDLSTVK in Atlantic salmon, with m/z 803.441.

FIG. 5 is a mass spectrum of polypeptide VLNNMEIGNSLYDDEGAK in Atlantic salmon, with m/z 991.462.

FIG. 6 is a mass spectrum of polypeptide TGDWFNILEHYAR in Atlantic salmon, with m/z 541.263.

FIG. 7 is a mass spectrum of polypeptide EEEVEDVLSNILR in Atlantic salmon, with m/z 772.894.

FIG. 8 is a mass spectrum of polypeptide LITHVLVGEPVGSR in Atlantic salmon, with m/z 492.956.

FIG. 9 is a mass spectrum of polypeptide LFAEHPETLTLFPK in Atlantic salmon, with m/z 548.299.

Example 3 sequence Source and alignment information for Polypeptides specific for Pacific salmon (Oncorhynchus keta) and Rainbow trout (Oncorhynchus mykiss)

Through mass spectrometry analysis, the polypeptide SEQ ID NO. 10-11 has independent response in Pacific salmon (Oncorhynchus keta), good peak shape and high intensity, and has no response in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Specifically, the method comprises the following steps of,

FIG. 10 is a mass spectrum of polypeptide AHVSEAVANSVR in Pacific salmon, with m/z 620.326.

FIG. 11 is a mass spectrum of polypeptide VGGGTATLTLPLLAK in Pacific salmon, with m/z 706.430.

Through mass spectrometry analysis, the polypeptide SEQ ID NO. 12-14 has independent response in rainbow trout (Oncorhynchus mykiss), good peak shape and high intensity, and has no response in Atlantic salmon (Salmo salar) and Pacific salmon (Oncorhynchus kata). In particular, the method comprises the following steps of,

FIG. 12 is a mass spectrum of polypeptide ATDFVVSKPGTFK in rainbow trout, wherein m/z is 466.254.

FIG. 13 is a mass spectrum of polypeptide DAAPAVAGAPGAPISVK in rainbow trout, wherein m/z is 746.412.

FIG. 14 shows the mass spectrum of polypeptide SAGEIEEYQR in rainbow trout, wherein m/z is 591.275.

Example 4 Salmon sample treatment and detection procedure

1. A sample pretreatment step:

(1) taking 2g of a sample to be detected, adding 20mL of protein extracting solution (8M urea and 50mM ammonium bicarbonate) for vertical shaking, centrifuging and taking supernatant to obtain a crude protein extracting solution.

(2) mu.L of the supernatant was added with 8. mu.L of DDT (Dithiothreitol) and reacted at 60 ℃ for 1 hour.

(3) After the temperature was reduced to room temperature, 40. mu.L of in-situ IAA (Iodoacetamide) was added and the reaction was carried out for 1 hour at room temperature in the dark.

(4) Selecting a 10KDa interception ultrafiltration centrifugal tube, and centrifuging at 12000r/min for 20 min. Taking NH₄HCO₃The solution washes the membrane for 3 replicates.

(5) Adding enzyme according to the mass ratio of enzyme to substrate of 1:50, and carrying out enzymolysis for 16h at 37 ℃.

(6) Centrifuging for 20min at 12000r/min with 10kDa filter membrane, collecting lower layer peptide fragment filtrate in liquid phase vial to be tested

2. Detection on machine

(1) An AB SCIEX tripleTOF system is adopted, specifically;

mobile phase A: 0.1 percent of formic acid-water,

mobile phase B: 0.1 percent of formic acid-acetonitrile,

the flow rate was 0.25mL/min,

elution gradient: 0-2 min, 5% B; 2-27 min, 5-20% of B; 27-37 min, 20-35% B; 37-39 min, 35-80% B; 39-42 min, 80% B; 42-46 min, 5% B.

or (2) detecting with AB SCIEX 5500 triple quadrupole rod,

mobile phase A: 0.1 percent of formic acid-water,

mobile phase B: 0.1 percent of formic acid-acetonitrile,

the flow rate was 0.35 mL/min.

Elution gradient: 0-10 min, 10-10.5% B; 10-10.5 min, 10.5-45% B; 10.5-13 min, 45-95% B; 13-13.1 min, 95-10% B; 13.1-15 min, 10%.

Comparing the mass spectrum detection result with the mass spectrum spectrogram of each specific polypeptide in the embodiment 2-4, and judging that the tissue sample is a corresponding species salmon sample when the mass spectrum detection spectrogram of any one of the embodiment 2-4 appears.

Through further identification, in a randomly selected salmon sample, the mass spectrometric analysis of the sample accords with the characteristic peptide fragment mass spectrogram of the rainbow trout, no response exists in Atlantic salmon and Pacific salmon, and the result is shown in figure 15, so that the sample is the rainbow trout sample.

And (5) verifying that the identification result is consistent with the species of the sample.

Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not used to limit the protection scope of the present invention.

SEQUENCE LISTING

<110> Qingdao customs technology center

<120> a method for identifying salmon using specific peptide fragment group

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 11

<212> PRT

<213> Salmo salar

<400> 1

Val Glu Glu Val Asp Ala Met Asp Ala Gly Lys

1 5 10

<210> 2

<211> 12

<212> PRT

<213> Salmo salar

<400> 2

Ile Val Glu Ala Pro Gly Pro Pro Glu Val Val Lys

1 5 10

<210> 3

<211> 16

<212> PRT

<213> Salmo salar

<400> 3

Glu Phe Asp Asp Leu Pro Ser Phe Glu Leu Gln Ser Asp Ala Val Arg

1 5 10 15

<210> 4

<211> 25

<212> PRT

<213> Salmo salar

<400> 4

Ala Ala Ala Pro Ala Pro Ala Pro Glu Pro Glu Val Val Ala Ala Pro

1 5 10 15

Pro Pro Leu Asp Leu Ser Thr Val Lys

20 25

<210> 5

<211> 18

<212> PRT

<213> Salmo salar

<400> 5

Val Leu Asn Asn Met Glu Ile Gly Asn Ser Leu Tyr Asp Asp Glu Gly

1 5 10 15

Ala Lys

<210> 6

<211> 13

<212> PRT

<213> Salmo salar

<400> 6

Thr Gly Asp Trp Phe Asn Ile Leu Glu His Tyr Ala Arg

1 5 10

<210> 7

<211> 13

<212> PRT

<213> Salmo salar

<400> 7

Glu Glu Glu Val Glu Asp Val Leu Ser Asn Ile Leu Arg

1 5 10

<210> 8

<211> 14

<212> PRT

<213> Salmo salar

<400> 8

Leu Ile Thr His Val Leu Val Gly Glu Pro Val Gly Ser Arg

1 5 10

<210> 9

<211> 14

<212> PRT

<213> Salmo salar

<400> 9

Leu Phe Ala Glu His Pro Glu Thr Leu Thr Leu Phe Pro Lys

1 5 10

<210> 10

<211> 12

<212> PRT

<213> Oncorhynchus keta

<400> 10

Ala His Val Ser Glu Ala Val Ala Asn Ser Val Arg

1 5 10

<210> 11

<211> 15

<212> PRT

<213> Oncorhynchus keta

<400> 11

Val Gly Gly Gly Thr Ala Thr Leu Thr Leu Pro Leu Leu Ala Lys

1 5 10 15

<210> 12

<211> 13

<212> PRT

<213> Oncorhynchus mykiss

<400> 12

Ala Thr Asp Phe Val Val Ser Lys Pro Gly Thr Phe Lys

1 5 10

<210> 13

<211> 17

<212> PRT

<213> Oncorhynchus mykiss

<400> 13

Asp Ala Ala Pro Ala Val Ala Gly Ala Pro Gly Ala Pro Ile Ser Val

1 5 10 15

Lys

<210> 14

<211> 10

<212> PRT

<213> Oncorhynchus mykiss

<400> 14

Ser Ala Gly Glu Ile Glu Glu Tyr Gln Arg

1 5 10

Claims

1. The application of a group of characteristic peptide fragments in identifying Salmonidae (Salmonidae) fish subspecies is as follows: atlantic salmon (Salmo salar), Pacific salmon (Oncorhynchus keta) and rainbow trout (Oncorhynchus mykiss); the peptide is characterized in that the characteristic peptide segment is as follows:

the peptide fragment with the sequence shown in SEQ ID NO.14 is derived from the protein myosin-2-like isofomm X2(gi | 1211256714).

2. The use of claim 1, wherein the mass spectrometry detected mass-to-charge ratio (m/z) data of each characteristic peptide fragment is:

3. A method for identifying a Salmonidae (Salmonidae) fish subspecies using the signature of claim 1 or 2, said Salmonidae fish subspecies being: atlantic salmon (Salmo salar), Pacific salmon (Oncorhynchus keta) and rainbow trout (Oncorhynchus mykiss), characterized in that the method is a mass spectrometric detection method, comprising the following steps:

(1) sample pretreatment

1) Weighing a salmonidae fish sample to be detected, which is ground into powder, adding a protein extracting solution (8M urea and 50mM NH4HCO3) to extract protein, centrifuging at a high speed and a low temperature, and collecting supernatant;

4. The method of claim 3, wherein the step of mass spectrometric detection in step (2) comprises:

1) an AB SCIEX tripleTOF mass spectrometry system is adopted,

the flow rate is 0.25 mL/min;

or 2) an AB SCIEX 5500 triple quadrupole detection system is adopted,

the flow rate is 0.35 mL/min;

5. The application of a group of characteristic peptide fragments in preparing a detection kit for identifying Salmonidae (Salmonoidae) fish subspecies is disclosed, wherein the group of characteristic peptide fragments is any one or any combination of SEQ ID NO.1-14, and preferably, the kit is based on mass spectrometry detection.