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CN-122017105-A - Identification method of characteristic proteins of sea cucumber producing area based on label-free data independent acquisition mass spectrometry technology

CN122017105ACN 122017105 ACN122017105 ACN 122017105ACN-122017105-A

Abstract

The invention discloses a method for identifying characteristic proteins of sea cucumber producing areas based on a label-free data independent acquisition mass spectrum technology, and belongs to the technical field of food science and analytical chemistry. The identification method of the characteristic proteins of the sea cucumber producing area based on the label-free data independent acquisition mass spectrum technology comprises the steps of sea cucumber sample collection and protein extraction, peptide digestion and classification, construction of a sea cucumber specific data dependent acquisition spectrum library, data independent acquisition mass spectrum detection, protein identification and quantification of data based on the spectrum library, identification of differential expression proteins and identification of the sea cucumber producing area based on the differential proteins, wherein the identification depth of the obtained proteins exceeds 6,278, and the differential expression proteins and key metabolic pathways of the Liaoning sea cucumber and the Liaoning sea cucumber can be accurately identified.

Inventors

  • WU QIONG
  • DONG XIUPING
  • JIAO JIAN
  • WANG ZHEMING
  • WAN SHIWEI
  • YU XILIANG
  • Cai Houde
  • PAN YI

Assignees

  • 大连工业大学

Dates

Publication Date
20260512
Application Date
20260206

Claims (10)

  1. 1. The identification method of the characteristic protein of the sea cucumber producing area based on the label-free data independent acquisition mass spectrum technology is characterized by comprising the following steps: (1) Sample collection and protein extraction Collecting sea cucumbers in known places and unknown places, and respectively extracting proteins from body wall tissues of each sea cucumber sample and body wall tissues mixed with all sea cucumber samples; (2) Digestion and fractionation of peptide fragments Carrying out trypsin enzymolysis on the proteins extracted in the step (1) respectively by adopting a filter membrane auxiliary sample preparation method, centrifuging and collecting supernatant; grading, desalting and quantifying ultraviolet enzymolysis supernatant corresponding to protein extracted from body wall tissues mixed by all sea cucumber samples by using a high-pH reverse-phase grading kit; (3) DDA spectrum library construction Mixing all the graded and quantitative peptide fragments mixed by all the sea cucumber samples in the step (2) in an equivalent way, and carrying out DDA mass spectrometry to construct a sea cucumber DDA mass spectrometry library; (4) DIA Mass Spectrometry library construction Configuring the supernatant of each sea cucumber sample obtained in the step (2) by adopting a mass spectrum platform which is the same as that of the step (3), and carrying out mass spectrum detection by using a DIA mode to construct a DIA mass spectrum library; (5) Data analysis Performing library searching on DIA data based on the DDA spectrum library constructed in the step (3) by using professional software, so as to realize protein identification and quantification of each sample; (6) Identification of characteristic proteins of sea cucumber producing area Proteins were analyzed both known and unknown using exploratory proteomic study criteria, and proteins characteristic of sea cucumber origin were identified based on t-test P-values <0.05 and absolute fold changes >1.5 or < 0.67.
  2. 2. The method of claim 1, wherein the protein extraction of step (1) is performed by reference to an existing extraction process.
  3. 3. The method according to claim 1, wherein the filter-assisted sample preparation method in step (2) is characterized in that proteins are reduced by dithiothreitol, then the reaction mixture is transferred to a 10 kDa molecular weight cut-off centrifugal filter by iodoacetamide alkylation at room temperature in the dark, washed sequentially with UA buffer and ammonium bicarbonate, and trypsin is added after washing to carry out enzymolysis.
  4. 4. The method according to claim 1, wherein the enzymolysis reaction in the step (2) is carried out at a temperature of 35-37 ℃ for 15-18 hours.
  5. 5. The method according to claim 1, wherein the centrifugation in the step (2) is performed for 5-10 min at 10000 Xg-15000 Xg.
  6. 6. The method of claim 1, wherein the desalting in step (2) is performed using a C18 solid phase extraction column.
  7. 7. The method of claim 1, wherein the uv quantification of step (2) is performed at a wavelength of 280 nm.
  8. 8. The method of claim 1, wherein the chromatographic conditions for the DDA mass spectrometry of step (3) are reversed phase C18 column, mobile phase A of 0.1% formic acid, mobile phase B of 84% acetonitrile containing 0.1% formic acid.
  9. 9. The method according to claim 1, wherein the mass spectrum detection condition of the DDA mass spectrum analysis in the step (3) is that a positive ion mode is adopted, acquisition parameters are that a mass range of a full-scan mass spectrum is set to 350-1800 m/z, resolution is 60000, an automatic gain control target value is 1×10 6 , a maximum injection time is 50 ms, a dynamic exclusion time is set to 10.0 s to reduce repeated fragmentation, precursor ions are fragmented by high-energy collision dissociation under 30 eV standardized collision energy in a secondary spectrum acquisition process by adopting a 1.5 m/z isolation window, a secondary spectrum recording resolution is 30000, an automatic gain control target value is 1×10 5 , and a maximum injection time is 50 ms.
  10. 10. The method of claim 1, wherein the parameters of mass spectrometry in the DIA mode in step (4) are that each DIA cycle comprises a full scan and 44 consecutive isolation windows covering 350-1800 m/z, the full scan is acquired in profile mode, the resolution is 120000, the automatic gain control target value is 3 x 10 6 , the maximum injection time is 30 ms, the DIA window is fragmented at 30 eV collision energy, the secondary spectrum resolution is 30000, the automatic gain control target value is 3 x 10 6 , and the maximum injection time is automatically adjusted.

Description

Identification method of characteristic proteins of sea cucumber producing area based on label-free data independent acquisition mass spectrometry technology Technical Field The invention relates to the technical field of food science and analytical chemistry, in particular to a method for identifying characteristic proteins of sea cucumber producing areas based on a label-free data independent acquisition mass spectrum technology. Background Sea cucumber is used as a high-value ocean food, and the quality and market value of the sea cucumber are closely related to the production place. Sea cucumber produced in Liaoning China enjoys higher market premium due to the superior collagen integrity. However, the molecular basis of this geographical difference, especially at the proteomic level, is not yet clear. At present, the proteomics research aiming at food geographic traceability mostly adopts a data dependent acquisition mass spectrometry (DDA) technology or a quantitative method based on marking, which generally has the defects of poor reproducibility, limited coverage range and the like of low-abundance proteins due to random ion selection deviation, and the marking method (such as TMT, iTRAQ and the like) improves the accuracy of quantification, but has complex sample processing flow, high cost and limited channel number, and is difficult to be applied to analysis of a large-scale sample queue. Therefore, there is an urgent need in the art for an emerging mass spectrometry data acquisition mode suitable for large-queue sample analysis to overcome the randomness problem of traditional data-dependent acquisition mass spectrometry technology, so as to achieve higher reproducibility, deeper coverage depth and more accurate quantitative result, thereby providing sufficient theoretical and technical support for the origin traceability identification of ocean foods, particularly sea cucumbers, which are high-value ocean foods. Disclosure of Invention Aiming at the defects existing in the prior art, the invention aims to provide a method for identifying characteristic proteins of sea cucumber producing areas based on a label-free data independent acquisition mass spectrum technology, which is used for carrying out data acquisition and processing on sea cucumber samples based on the label-free data independent acquisition mass spectrum technology, wherein more than 6000 proteins can be identified in one analysis, the coverage depth is obviously superior to that of the existing label-free methods such as SWATH, maxQuant, the defect that a spectrogram library is seriously biased towards high-abundance proteins when a general DDA method is used for the sea cucumber samples is thoroughly overcome, and the accuracy of identifying the sea cucumber producing areas is obviously improved. In order to achieve the above object, the technical scheme provided is as follows: The invention provides a method for identifying characteristic proteins of sea cucumber producing areas based on a label-free data independent acquisition mass spectrum technology, which comprises the following steps: (1) Sample collection and protein extraction Collecting sea cucumbers in known places and unknown places, and respectively extracting proteins from body wall tissues of each sea cucumber sample and body wall tissues mixed with all sea cucumber samples; (2) Digestion and fractionation of peptide fragments Carrying out trypsin enzymolysis on the proteins extracted in the step (1) respectively by adopting a filter membrane auxiliary sample preparation method, centrifuging and collecting supernatant; grading, desalting and quantifying ultraviolet enzymolysis supernatant corresponding to protein extracted from body wall tissues mixed by all sea cucumber samples by using a high-pH reverse-phase grading kit; (3) DDA spectrum library construction Mixing all the graded and quantitative peptide fragments mixed by all the sea cucumber samples in the step (2) in an equivalent way, and carrying out DDA mass spectrometry to construct a sea cucumber DDA mass spectrometry library; (4) DIA Mass Spectrometry library construction Configuring the supernatant of each sea cucumber sample obtained in the step (2) by adopting a mass spectrum platform which is the same as that of the step (3), and carrying out mass spectrum detection by using a DIA mode to construct a DIA mass spectrum library; (5) Data analysis Performing library searching on DIA data based on the DDA spectrum library constructed in the step (3) by using professional software, so as to realize protein identification and quantification of each sample; (6) Identification of characteristic proteins of sea cucumber producing area Proteins were analyzed both known and unknown using exploratory proteomic study criteria, and proteins characteristic of sea cucumber origin were identified based on t-test P-values <0.05 and absolute fold changes >1.5 or < 0.67. In one embodiment, the protein extraction of st