Method for analyzing free plasma membrane protein extracellular domain in sample

Abstract

The invention belongs to the field of molecular diagnosis, and particularly relates to a method for analyzing a free plasma membrane protein extracellular domain in a sample. The invention provides a method for analyzing free plasma membrane protein extracellular domains in a sample. By using the method disclosed by the invention, the free plasma membrane protein extracellular domain in the sample can be accurately identified, so that a reference is provided for the activation condition of a signal channel and the searching of potential disease treatment and / or prevention targets and markers.

Description

Technical Field

[0001] The present invention belongs to the field of molecular diagnosis, and in particular, relates to a method for analyzing free extracellular domains of plasma membrane proteins in a sample. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC), also known as pancreatic cancer, is one of the most deadly cancers, with a 5-year survival rate of less than 10% and a median survival time of less than 6 months. The main reason is that most pancreatic cancers are difficult to detect early, and most are already in the middle and late stages when diagnosed, missing the best time for radical surgical resection. At the same time, it is less sensitive to comprehensive treatments such as chemotherapy and radiotherapy, and its strong metastasis is also an important factor leading to the high mortality rate of pancreatic cancer patients.

[0003] The extracellular domains (ECDs) of many membrane proteins can be cleaved from the cell membrane into the extracellular environment by proteolytic enzymes, a process called ectodomain shedding (ES). The ES of the membrane protein family may play an important role in regulating intercellular signaling in the PDAC tumor microenvironment.

[0004] However, existing methods are unable to accurately analyze the shedding of extracellular domains of plasma membrane proteins in samples, and thus are unable to further search for potential markers that can predict pancreatic cancer metastasis.

[0005] Therefore, there is a need in the art for a method for analyzing the shedding of extracellular domains of plasma membrane proteins in samples, and through this method, potential marker combinations can be found, which can assist in predicting the metastasis of pancreatic cancer and provide clinicians with further treatment and detection strategies. Summary of the invention

[0006] The applicant creatively invented a method for identifying extracellular domain shedding, that is, a method capable of analyzing free extracellular domains in a sample, and further discovered through this method that there is a significant correlation between the level of extracellular domain shedding of receptor tyrosine kinase (AXL) and the level of its ligand GAS6 and pancreatic cancer metastasis.

[0007] In view of this, in a first aspect, the present invention provides a method for analyzing free extracellular domains of plasma membrane proteins in a sample, comprising the following steps:

[0008] S1. constructing a plasma membrane protein database, wherein the database includes sequence information of the extracellular domain and intracellular domain of the plasma membrane protein;

[0009] S2, enriching the plasma membrane proteome on the cell membrane surface, collecting the cell secretory proteome (containing free plasma membrane proteins), and analyzing the unique polypeptides in the extracellular domain and intracellular domain of the plasma membrane proteins in both, wherein the unique polypeptide refers to a polypeptide that matches only the intracellular domain or extracellular domain of one protein in the proteome of one species;

[0010] S3, identifying unique polypeptides of the extracellular domain and the intracellular domain, wherein when the count of the unique polypeptides in step S2 is ≥ 2, the unique polypeptide is identified as the extracellular domain or the intracellular domain; and

[0011] S4. Analyze the free extracellular domain of the plasma membrane protein, wherein the ratio of the number of unique extracellular domain polypeptides / the number of unique intracellular domain polypeptides of the same plasma membrane protein in the plasma membrane protein group and the secretory protein group is calculated respectively, and normalized, and the normalization formula is as follows:

[0012] Among them, when Index Training When ≥2, it was defined as a free extracellular domain of plasma membrane protein.

[0013] Furthermore, constructing the database in step S1 may be to distinguish the amino acid sequence information of the extracellular domain and the intracellular domain of the protein through a public database.

[0014] Further, the enrichment in step S2 is performed by a hydrazide probe. Further, the hydrazide probe carries a biotin group. Further, the hydrazide probe can be modified to have higher solubility.

[0015] In some specific embodiments, the structural formula of the hydrazide probe is as follows:

[0016]

[0017] In some specific embodiments, the plasma membrane protein ectodomain shedding is from the ectodomain shedding in tumor cells (Index test More specifically, 22 of the 45 shed plasma membrane proteins in the secretome were expressed in at least 6 tumors by Index test ≥2 were identified and defined as plasma membrane proteins shed in tumors.

[0018] The method of the present invention can accurately identify the free extracellular domain of plasma membrane proteins in the sample, thereby providing a reference for the activation of signal pathways, and the search for potential targets and markers for disease treatment and / or prevention.

[0019] In a second aspect, through the above method, the present invention has discovered a marker combination for assisting in predicting pancreatic cancer metastasis, comprising:

[0020] The free extracellular domain of the receptor tyrosine kinase (sAXL), and

[0021] Growth arrest-specific protein 6 (GAS6).

[0022] In a third aspect, the present invention provides a composition for assisting in predicting pancreatic cancer metastasis, comprising:

[0023] First reagent: a reagent for detecting the content of free extracellular domain of receptor tyrosine kinase (sAXL); and

[0024] Second reagent: a reagent for detecting the content of growth arrest-specific protein 6 (GAS6).

[0025] Furthermore, the composition further comprises:

[0026] The third reagent: hydrazide probe.

[0027] Further, the hydrazide probe carries a biotin group. Further, the hydrazide probe can be modified to have higher solubility.

[0028] In some specific embodiments, the structural formula of the hydrazide probe is as follows:

[0029]

[0030] Furthermore, the term "reagent" refers to reagents and / or kits required for any method capable of detecting the protein content.

[0031] Furthermore, any reagent required for any method capable of detecting the absolute content of the protein.

[0032] In some specific embodiments, the method can be immunoblotting, sandwich method (ELISA), or mass spectrometry.

[0033] Furthermore, the reagent may be an antibody used in immunoblotting or sandwich assay, such as an anti-AXL extracellular domain antibody or an anti-GAS6 antibody.

[0034] Further, the reagent may be a reagent for mass spectrometry. Further, it also includes a stable isotope labeled heavy peptide, and further, a stable isotope labeled heavy peptide as shown in the table.

[0035] Further, the hydrazide probe carries a biotin group. Further, the hydrazide probe can be modified to have higher solubility.

[0036] In some specific embodiments, the structural formula of the hydrazide probe is as follows:

[0037]

[0038] In a fourth aspect, the present invention provides a model for assisting in predicting pancreatic cancer metastasis, comprising:

[0039] p=P GAS6 -P sAXL , where P GAS6is the content of GAS6, is the content of the extracellular domain of free receptor tyrosine kinase (sAXL);

[0040] Among them, when p>0, it indicates that pancreatic cancer has metastasized, and when p≤0, it indicates that pancreatic cancer has not metastasized.

[0041] Further, the p-value includes the p-value of pancreatic cancer tumor and pancreatic cancer stroma.

[0042] Furthermore, when the p values ​​of both are greater than 0, it indicates that pancreatic cancer has metastasized, and when the p values ​​of both are less than or equal to 0, it indicates that pancreatic cancer has not metastasized.

[0043] In a fifth aspect, the present invention provides a device, comprising:

[0044] at least one processor; and

[0045] a memory communicatively connected to at least one of the processors; wherein,

[0046] The memory stores instructions executable by the processor, and the instructions are used to be executed by the processor to implement any of the above methods for analyzing free extracellular domains of plasma membrane proteins in a sample or any of the above models for assisting in predicting pancreatic cancer metastasis.

[0047] In some embodiments, the device further includes at least one input device and at least one output device; in the device, the processor, memory, input device, and output device are connected via a bus.

[0048] In a sixth aspect, the present invention provides a storage medium storing computer instructions, wherein the computer instructions are used to be executed by the computer to implement any of the methods for analyzing free extracellular domains of plasma membrane proteins in a sample as described above or any of the models for assisting in predicting pancreatic cancer metastasis as described above.

[0049] In a seventh aspect, the present invention provides a device for analyzing free extracellular domains of plasma membrane proteins in a sample, comprising the following modules:

[0050] S1. constructing a plasma membrane protein database module, wherein the database includes sequence information of the extracellular domain and intracellular domain of the plasma membrane protein;

[0051] S2, enriching the plasma membrane proteome on the cell membrane surface, collecting the cell secretory proteome (containing free plasma membrane proteins), and analyzing the unique polypeptides in the extracellular domain and intracellular domain of the plasma membrane proteins in both, wherein the unique polypeptide refers to a polypeptide that matches only the intracellular domain or extracellular domain of one protein in the proteome of one species;

[0052] S3, a module for identifying unique polypeptides of the extracellular domain and the intracellular domain, wherein when the count of the unique polypeptides in step S2 is ≥ 2, the unique polypeptide is identified as the extracellular domain or the intracellular domain; and

[0053] S4. Analyze the free extracellular domain module of plasma membrane proteins, wherein the ratio of the number of unique extracellular domain polypeptides / the number of unique intracellular domain polypeptides of the same plasma membrane protein in the plasma membrane protein group and the secretory protein group is calculated respectively, and normalized, and the normalization formula is as follows:

[0054] Among them, when Index Training When ≥2, it was defined as a free extracellular domain of plasma membrane protein.

[0055] In an eighth aspect, the present invention provides use of the above-mentioned composition and model for preparing a kit for assisting in predicting pancreatic cancer metastasis.

[0056] Furthermore, the present invention provides use of the above composition and model in preparing a kit for using blood to assist in predicting pancreatic cancer metastasis.

[0057] The present invention also provides the use of the above device for searching for tumor diagnostic markers.

[0058] In a ninth aspect, the present invention provides a kit comprising the composition as described above.

[0059] Furthermore, the kit also includes the storage medium as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Schematic diagram of the method for identification of extracellular domain shedding of plasma membrane (PM) proteins;

[0061] Figure 2 Schematic diagram of the method for analyzing free extracellular domains of plasma membrane proteins in samples;

[0062] Figure 3 This is a quantitative linear graph for quantifying the peptides of AXL intracellular domain (ICD), extracellular domain (ECD) and GAS6. The ordinate is the mass spectrometry peak area of ​​the peptide, and the abscissa is the absolute amount of the peptide;

[0063] Figure 4 Figure 2 Correlation analysis between the relative levels of sAXL and GAS6 and lymph node metastasis in patients. P values ​​are derived from chi-square tests. Patients with consistent trends in the cancer cell area and the stroma area in the same tumor (sAXL>GAS6 or sAXL<GAS6 in both areas) were used for statistical analysis. DETAILED DESCRIPTION

[0064] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0065] Example 1. Identification of extracellular domain shedding of plasma membrane (PM) proteins

[0066] like Figure 1 As shown, in principle, because N-glycosylation modification occurs in the ECD of membrane proteins, full-length membrane proteins (membrane proteome) will be obtained after glycoprotein enrichment of cell samples, while the secretory proteome of cells contains the ECD of membrane proteins shed. The S-PM proteome of tissue samples enriched with glycoproteins contains full-length membrane proteins and shed ECD (if ES occurs). The inventors collected mass spectrometry data of the secretory proteome and membrane proteome of cell lines, as well as the glycosylated proteome (secretory protein and membrane proteome) of tumor tissues, divided the amino acid sequence of plasma membrane proteins into ECD and ICD, and performed database retrieval on the plasma membrane data of cells and tumor tissues, thereby identifying the ECD and ICD of plasma membrane proteins in cell and tissue data sets. The unique peptide ratio of ECD / ICD in the cell secretome and tissue S-PM proteome data sets is normalized with the ratio in the cell membrane proteome data set, and then the card value standard is set to identify the plasma membrane proteins that have shed in tumor tissues.

[0067] like Figure 2As shown in the figure, the specific data analysis process is as follows: PM proteins with extracellular domains shed in PDAC tumor tissues were identified by using the secretome and PM proteome of 8 pancreatic cell lines as training sets according to the following steps: (1) Full-length PM proteins in the PM proteomes of 8 cell lines were quantified: 1012 extracellular domains (ECDs) or intracellular domains (ICDs) were identified from 773 PM proteins. For the two technical replicates of any PM protein, both ECD and ICD strictly required at least 2 unique peptides. Strict chi-square values ​​ensured the accuracy of the ratio calculated based on the number of unique peptides of full-length PM protein ECD and ICD. (2) PM proteins shed in the secretome (S) of 8 cell lines were quantified: 806 ECDs or ICDs were identified from 667 PM proteins. For the two technical replicates of any PM protein, ECD required at least 2 unique peptides to be identified. For those PM proteins shed with quantified ECD but no paired ICD identified in the secretome, the missing values ​​of the number of unique peptides were replaced with 1 for ratio calculation. (3) Quantification of intact PM proteins and shed PM proteins in tumors: 1387 ECDs or ICDs were quantified from 1041 proteins. ECD or ICD requires at least 1 unique peptide. (4) Identification of truly shed proteins in the secretome and PM proteome. The ECD / ICD ratios of 107 PM proteins were quantified in the secretome and plasma membrane proteome. In order to identify truly shed proteins in the secretome, rather than contamination by full-length PM proteins in cell debris, the ECD / ICD ratio in the secretome of each cell line was normalized by the ECD / ICD ratio in the PM proteome to generate an index called Index. Training The larger the value, the higher the ECD shedding level of PM protein. Training ≥2, a total of 45 PM proteins with extracellular domain shedding were identified. The same normalization criteria were used to determine the PM protein shedding level in tumors, generating an index called test Overall, 22 of the 45 PM-shedding proteins in the secretome were expressed in at least six tumors by Index test ≥2 were identified and defined as PM proteins shed in tumors.

[0068] Through the method of the present invention, the applicant creatively discovered that the shedding of AXL, that is, the level of its free extracellular domain and the level of GAS6, can assist in predicting the metastasis of pancreatic cancer.

[0069] Example 2: Measurement method of sAXL and GAS6

[0070] Therefore, we further measured the levels of sAXL and GAS6 in the samples, using stable isotope-labeled heavy peptides as internal standards (see the table below) to absolutely quantify the intracellular and extracellular domains of AXL and the ligand GAS6 of AXL in the cancer cell and matrix regions of 50 tumor tissues by the targeted proteomics method "parallel reaction monitoring (PRM)". The cancer cell and matrix regions were harvested from frozen tumor tissue sections (15 μm thick) by LCM, and the collection area was 20 mm 2 . Glycoproteins were enriched by the published FISGlyco method (Huang, P., Li, H., Gao, W., Cai, Z., Tian, ​​R., (2019). A Fully Integrated Spintip-Based Approach for Sensitive and Quantitative Profiling of Region-Resolved in Vivo Brain Glycoproteome. Analytical Chemistry 91, 9181-9189.). Some improvements were made, and the hydrazide microspheres were replaced with hydrazide probes with higher labeling efficiency. After labeling, the labeled proteins were enriched by streptavidin microspheres, and non-glycopeptides were released by trypsin digestion. Stable isotope-labeled heavy peptides (600 attomoles per peptide) were added to each sample, and one-third of each sample was loaded for PRM absolute quantitative analysis. PRM-MS analysis was performed on an Orbitrap Exploris480 mass spectrometer (Thermo Fisher Scientific) and an UltiMate3000RSLCnano chromatography system (Thermo Fisher Scientific).

[0071] The peak areas of peptide fragment ions were extracted from the PRM raw files using Skyline (version 20.2.0.286). Data that met the following criteria were accepted for further analysis: the retention times of endogenous light peptides and stable isotope-labeled heavy peptides were the same; the retention time variation of all samples was within ±2 minutes; and the mass difference was within ±5 ppm. After each ion peak was manually inspected to confirm that a clear peak shape could be extracted, the peak areas of all fragment ions were summed as the peak area of ​​the peptide. The absolute amount of the light peptide was calculated by dividing the peak area of ​​the light peptide by the peak area of ​​its corresponding heavy peptide and then multiplying it by the absolute amount of the heavy peptide. Because 20 mm 2 Tissues were sectioned and one-third of the sample was loaded for nano-LC-MS / MS analysis, so the absolute amount of light peptides per square millimeter of tissue section was calculated by dividing 20 and multiplying by 3. For proteins quantified by two peptides, the average was calculated to represent the protein / domain level.

[0072] Table 1. Stable isotope labeled heavy peptides used for absolute quantification of AXL and GAS6

[0073] Gene name Domain sequence Isotope-labeled amino acids AXL Extracellular domain (ECD) TATITVLPQQPR <![CDATA[R( 13 C6 15 N4)]]> AXL Extracellular domain (ECD) APLQGTLLGYR <![CDATA[R( 13 C6 15 N4)<!-- 5 --> ]]> AXL Intracellular domain (ICD) HGDLHSFLLYSR <![CDATA[R( 13 C6 15 N4)]]> GAS6 - IAVAGDLFQPER <![CDATA[R( 13 C6 15 N4)]]> GAS6 - AVPLSVALVDYHSTK <![CDATA[K( 13 C6 15 N2)]]>

[0074] Example 3: Relationship between AXL shedding and GAS6 levels and pancreatic cancer metastasis

[0075] According to the method of Example 2, the levels of sAXL and GAS6 in 50 clinical pancreatic cancer tumor tissues were detected. The detected levels were then analyzed for metastasis. Surprisingly, when the tumor samples were classified according to the relative levels of sAXL and GAS6 in the stroma and PCC regions, the inventors found a significant correlation between AXL shedding and PDAC metastasis ( Figure 4 ),from Figure 4 It can be seen that when the sAXL level is lower than the GAS6 level, the patient is more likely to metastasize.

Claims

1. A method for analyzing free extracellular domains of plasma membrane proteins in a sample, comprising the following steps: S1. constructing a plasma membrane protein database, wherein the database includes sequence information of the extracellular domain and intracellular domain of the plasma membrane protein; S2, enriching the plasma membrane proteome on the cell membrane surface, collecting the secretory proteome of the cell, and analyzing the unique polypeptides in the extracellular domain and intracellular domain of the plasma membrane protein in the two, wherein the secretory proteome contains free plasma membrane proteins, and the unique polypeptide refers to a polypeptide that matches only the intracellular domain or extracellular domain of one protein in the proteome of one species; S3, identifying unique polypeptides of the extracellular domain and the intracellular domain, wherein when the count of the unique polypeptides in step S2 is ≥ 2, the unique polypeptide is identified as the extracellular domain or the intracellular domain; and S4. Analyze the free extracellular domain of the plasma membrane protein, wherein the ratio of the number of unique extracellular domain polypeptides / the number of unique intracellular domain polypeptides of the same plasma membrane protein in the plasma membrane protein group and the secretory protein group is calculated respectively, and normalized, and the normalization formula is as follows: Among them, when Index Training When ≥2, it was defined as a free extracellular domain of plasma membrane protein.

2. The method according to claim 1, characterized in that The enrichment in step S2 is performed by using a hydrazide probe.

3. The method according to claim 2, characterized in that The structure of the hydrazide probe is:

4. A marker combination for assisting in predicting pancreatic cancer metastasis, comprising: The free extracellular domain of the receptor tyrosine kinase (sAXL), and Growth arrest-specific protein 6 (GAS6).

5. A composition for assisting in predicting pancreatic cancer metastasis, comprising: First reagent: a reagent for detecting the content of free extracellular domain of receptor tyrosine kinase (sAXL); as well as Second reagent: a reagent for detecting the content of growth arrest-specific protein 6 (GAS6).

6. The composition according to claim 5, characterized in that The composition further comprises: The third reagent: hydrazide probe.

7. A model for assisting in predicting pancreatic cancer metastasis, comprising: p=P GAS6 -P sAXL , where P GAS6 is the content of GAS6, P sAXL is the content of free extracellular domain of receptor tyrosine kinase (sAXL); Among them, when p>0, it indicates a high risk of pancreatic cancer metastasis, and when p≤0, it indicates a low risk of pancreatic cancer metastasis.

8. A device comprising: at least one processor; as well as a memory communicatively connected to at least one of the processors; wherein, The memory stores instructions executable by the processor, wherein the instructions are used to be executed by the processor to implement the method for analyzing free extracellular domains of plasma membrane proteins in a sample according to any one of claims 1 to 3 or the model for assisting in predicting pancreatic cancer metastasis according to claim 7.

9. A storage medium storing computer instructions, wherein the computer instructions are used to be executed by the computer to implement the method for analyzing free extracellular domains of plasma membrane proteins in a sample as described in any one of claims 1 to 3 or the model for assisting in predicting pancreatic cancer metastasis as described in claim 7.

10. A device for analyzing free extracellular domains of plasma membrane proteins in a sample, comprising the following modules: S1. Construct a plasma membrane protein database module, including: The database includes extracellular domain and intracellular domain sequence information of plasma membrane proteins; S2, enriching the plasma membrane proteome on the cell membrane surface, collecting the secretory proteome of the cells, and analyzing the unique polypeptides in the extracellular domain and intracellular domain of the plasma membrane protein in the two, wherein the unique polypeptide refers to a polypeptide that matches only the intracellular domain or extracellular domain of one protein in the proteome of one species; S3, a module for identifying unique polypeptides of the extracellular domain and the intracellular domain, wherein when the count of the unique polypeptides in step S2 is ≥ 2, the unique polypeptide is identified as the extracellular domain or the intracellular domain; and S4. Analyze the free extracellular domain module of plasma membrane proteins, wherein the ratio of the number of unique extracellular domain polypeptides / the number of unique intracellular domain polypeptides of the same plasma membrane protein in the plasma membrane protein group and the secretory protein group is calculated respectively, and normalized, and the normalization formula is as follows: Among them, when Index Training When ≥2, it was defined as a free extracellular domain of plasma membrane protein.

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