A method of analyzing free plasma membrane protein ectodomains in a sample

Abstract

The present application belongs to the field of molecular diagnosis, and particularly relates to a method for analyzing free plasma membrane protein ectodomains in a sample. The present application provides a method for analyzing free plasma membrane protein ectodomains in a sample. Using the method of the present application, the free plasma membrane protein ectodomains in the sample can be accurately identified, and thus a reference can be provided for the activation of a signal pathway, and the search for a potential disease treatment and / or prevention target and marker.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular diagnosis, and in particular, relates to a method for analyzing free plasma membrane protein ectodomains in a sample. BACKGROUND

[0002] Pancreatic ductal adenocarcinoma (PDAC), also known as pancreatic cancer, is one of the most lethal 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 patients are in the middle and advanced stages when diagnosed, missing the best opportunity for radical surgical resection. At the same time, the sensitivity to comprehensive treatment such as chemotherapy and radiotherapy is poor, and its strong metastasis is also an important factor leading to high mortality in patients with pancreatic cancer.

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

[0004] However, the existing method cannot accurately analyze the ectodomain shedding of plasma membrane proteins in the sample, and further cannot find potential markers that can predict the metastasis of pancreatic cancer.

[0005] Therefore, there is a need in the art for a method for analyzing ectodomain shedding of plasma membrane proteins in a sample, and further, through this method, potential marker combinations can be found that can assist in predicting the metastasis of pancreatic cancer, providing further treatment and detection strategies for clinicians. SUMMARY

[0006] The applicant has creatively invented a method for identifying ectodomain shedding, i.e., a method for analyzing free ectodomains in a sample, and further, through this method, the level of ectodomain shedding of receptor tyrosine kinase (AXL) and the level of its ligand GAS6 have been found to have a significant correlation with pancreatic cancer metastasis.

[0007] Therefore, in a first aspect, the present application provides a method for analyzing free plasma membrane protein ectodomains in a sample, comprising the following steps:

[0008] S1, constructing a plasma membrane protein database, wherein the database comprises sequence information of ectodomains and intracellular domains of plasma membrane proteins;

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

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

[0011] S4, analyzing free plasma membrane protein extracellular domain, wherein, the ratio of the number of extracellular domain unique polypeptides / the number of intracellular domain unique 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] wherein, when Index Training ≥2, it is defined as free plasma membrane protein extracellular domain.

[0013] Further, the database constructed in the S1 step can be distinguished by the amino acid sequence information of the extracellular domain and intracellular domain of the protein in the public database.

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

[0015] In some specific embodiments, the structure of the hydrazine probe is as follows:

[0016]

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

[0018] Using the method of the present application, the free plasma membrane protein extracellular domain in the sample can be accurately identified, thereby providing a reference for the activation of the signal pathway, the search for potential disease treatment and / or prevention targets and markers.

[0019] In a second aspect, by the above method, the present application finds a marker combination for assisting in predicting pancreatic cancer metastasis, comprising:

[0020] Free extracellular domain of receptor tyrosine kinase (sAXL), and

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

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

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

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

[0025] Further, the composition further comprises:

[0026] Third reagent: a hydrazide probe.

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

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

[0029]

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

[0031] Still further, reagents required by any method capable of detecting the absolute content of the protein.

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

[0033] Further, the reagent can be an antibody used for immunoblotting or sandwich method, such as an anti-AXL extracellular domain antibody, an anti-GAS6 antibody.

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

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

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

[0037]

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

[0039] p = P GAS6 -P sAXL , wherein P GAS6GAS6 content, free receptor tyrosine kinase extracellular domain (sAXL) content;

[0040] wherein, when p>0, it indicates that the pancreatic cancer metastasis, when p≤0, it indicates that the pancreatic cancer does not metastasis.

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

[0042] Further, when both p values>0, it indicates that the pancreatic cancer metastasis, when both p≤0, it indicates that the pancreatic cancer does not metastasis.

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

[0044] at least one processor; and

[0045] a memory connected to the at least one processor in communication; wherein,

[0046] the memory stores instructions executable by the processor, the instructions being executed by the processor to implement the method of analyzing the free plasma membrane protein extracellular domain in the sample or the model of assisting in predicting the pancreatic cancer metastasis of any one of the above.

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

[0048] In a sixth aspect, the present application provides a storage medium, the storage medium stores computer instructions, the computer instructions are used to be executed by the computer to implement the method of analyzing the free plasma membrane protein extracellular domain in the sample or the model of assisting in predicting the pancreatic cancer metastasis of any one of the above.

[0049] In a seventh aspect, the present application provides a device for analyzing the free plasma membrane protein extracellular domain in the sample, comprising the following modules:

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

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

[0052] S3, Identifying unique polypeptides of extracellular and intracellular domains, wherein when the count of unique polypeptides in step S2 is ≥2, the unique polypeptide is identified as either an extracellular or intracellular domain; and

[0053] S4. Analyze the extracellular domain module of free plasma membrane proteins, wherein the ratio of the number of unique extracellular domain peptides to the number of unique intracellular domain peptides of the same plasma membrane protein in the plasma membrane proteome and secretory proteome are calculated and normalized. The normalization formula is as follows:

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

[0055] Eighthly, the present invention provides the use of the above-described composition and model in the preparation of a kit for assisting in the prediction of pancreatic cancer metastasis.

[0056] Furthermore, the present invention provides the use of the above-described composition and model in the preparation of a kit for predicting pancreatic cancer metastasis using blood-assisted methods.

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

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

[0059] Furthermore, the kit also includes the storage medium described above. Attached Figure Description

[0060] Figure 1 A schematic diagram of the method for identifying the detachment of extracellular domains of plasma membrane (PM) proteins;

[0061] Figure 2 A schematic diagram of the method for analyzing the extracellular domains of free plasma membrane proteins in a sample;

[0062] Figure 3 This is a quantitative linear graph for peptides used to quantify the intracellular domain (ICD), extracellular domain (ECD), and GAS6 of AXL. The vertical axis represents the mass spectrometry peak area of ​​the peptide, and the horizontal axis represents the absolute amount of the peptide.

[0063] Figure 4 Correlation analysis was performed on the relative levels of sAXL and GAS6 with lymph node metastasis in patients. P-values ​​were obtained from the chi-square test. Patients with consistent trends in cancer cell regions and stromal regions within the same tumor (both regions showing sAXL > GAS6 or sAXL < GAS6) were included in the statistical analysis. Detailed Implementation

[0064] The present application will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present application will be more clearly presented thereby. Those skilled in the art will understand that these specific embodiments and examples are used to illustrate the present application, but not to limit the present application.

[0065] Example 1, Identification of ectodomain shedding of plasma membrane (PM) proteins

[0066] As Figure 1 shown, in principle, because N-glycosylation modification occurs in the ECD of membrane proteins, after glycoprotein enrichment of cell samples, full-length membrane proteins (membrane proteome) are obtained, while the secretome of cells contains the ECD shed from membrane proteins. After glycoprotein enrichment of tissue samples, the S-PM proteome of tissues contains full-length membrane proteins and shed ECD (if ES occurs). The inventors collected mass spectrometry data of the secretome and membrane proteome of cell lines, and the glycosylated proteome of tumor tissues (secretome and membrane proteome), divided the amino acid sequence of plasma membrane proteins into ECD and ICD, and performed database searching on the plasma membrane data of cells and tumor tissues, thereby identifying the ECD and ICD of plasma membrane proteins in the cell and tissue data sets. By normalizing the unique peptide ratio of ECD / ICD in the secretome of cells and the S-PM proteome data set of tissues with the ratio in the membrane proteome data set of cells, and by setting a cutoff standard, the plasma membrane proteins that have shed in tumor tissues were identified.

[0067] As Figure 2The specific data analysis pipeline is shown: extracellular domain shedding PM proteins in PDAC tumor tissues were identified using secretome and PM proteome of 8 pancreatic cell lines as training set according to the following steps, (1) Quantification of full-length PM proteins in PM proteome of 8 cell lines: 1012 extracellular domain (ECD) or intracellular domain (ICD) were identified from 773 PM proteins. For both technical replicates of any PM protein, ECD and ICD strictly required at least 2 unique peptides. Strict cutoff ensured the accuracy of ratio calculation based on unique peptide number of ECD vs ICD of full-length PM protein. (2) Quantification of shed PM proteins in secretome (S) of 8 cell lines: 806 ECD or ICD were identified from 667 PM proteins. For both technical replicates of any PM protein, ECD required at least 2 unique peptides to be identified. For those shed PM proteins whose ECD were quantified but no paired ICD were identified in secretome, the missing value of unique peptide number was replaced by 1 for ratio calculation. (3) Quantification of intact and shed PM proteins in tumor: 1387 ECD or ICD were quantified from 1041 proteins. ECD or ICD required at least 1 unique peptide. (4) Identification of true shed proteins in secretome and PM proteome. ECD / ICD ratio of 107 PM proteins were quantified in both secretome and PM proteome. To determine true shed proteins in secretome, rather than contamination of full-length PM proteins in cell debris, the ratio of ECD / ICD in secretome of each cell line was normalized by the ratio of ECD / ICD in PM proteome, generating a value named Index Training . The larger the value, the higher the level of ECD shedding of PM protein. By cutoff Index Training ≥2 in at least 2 cell lines, 45 extracellular domain shedding PM proteins were identified. The same normalization standard was used to determine the shedding level of PM proteins in tumor, generating a value named Index test . Overall, 22 of 45 shed PM proteins in secretome were identified by Index test ≥2 in at least 6 tumors, and defined as shed PM proteins in tumor.

[0068] By the method of the present application, the applicant has creatively found that the shedding of AXL, i.e. the level of its free extracellular domain, in conjunction with the level of GAS6, can assist in predicting the metastasis of pancreatic cancer.

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

[0070] Therefore, further, the levels of sAXL and GAS6 in the samples were measured, using stable isotope labeled heavy peptides as internal standards (see table below), AXL intracellular and extracellular domains and the ligand of AXL, GAS6, were absolutely quantified in cancer cell and stroma regions of 50 tumor tissues by targeted proteomic method “parallel reaction monitoring (PRM)”. Cancer cell and stroma regions were harvested by LCM from frozen tumor tissue sections (15 pm thick), collecting an area of 20 mm 2 Glycoproteins were enriched by published FISGlyco method (Huang, P., Li, H., Gao, W., Cai, Z., Tian, R., (2019). A Fully Integrated Spin tip-Based Approach for Sensitive and Quantitative Profiling of Region-Resolved in Vivo Brain Glycoproteome. Analytical Chemistry 91, 9181-9189.). Some improvements were made, hydrazide microspheres were replaced by 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 attomole per peptide) were spiked into each sample, one third of each sample was loaded for PRM absolute quantification analysis. PRM-MS analysis was performed on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) and UltiMate 3000 RSLCnano chromatography system (Thermo Fisher Scientific).

[0071] Peak areas of polypeptide fragment ions were extracted from PRM raw files using Skyline (version 20.2.0.286). Data meeting the following criteria were accepted for further analysis: the retention time of endogenous light peptides and stable isotope labeled heavy peptides was the same; the retention time variation of all samples was within ±2 min; and the mass difference was within ±5 ppm. After manual inspection of each ion peak to determine that a clear peak shape could be extracted, the peak areas of all fragment ions were summed as the peak area of the polypeptide. The absolute amount of light peptides was calculated by dividing the peak area of light peptides by the peak area of their corresponding heavy peptides, then multiplying by the absolute amount of heavy peptides. Because 20 mm 2 tissue sections were co-processed and one third of the sample was loaded for nano-LC-MS / MS analysis, the absolute amount of light peptides obtained was calculated by dividing 20 by 3 to calculate the absolute amount of light peptides per square millimeter of tissue section. For proteins quantified by two polypeptides, the average value 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 Isotopically labeled amino acid AXL Extracellular domain (ECD) TATITVLPQQPR [R( 13 C6 15 N4)]]> AXL Extracellular domain (ECD) APLQGTLLGYR [R( 13 C6 15 N4) <!-- 5 -->]]> AXL Intracellular domain (ICD) HGDLHSFLLYSR [R( 13 C6 15 N4)]]> GAS6 - IAVAGDLFQPER [R( 13 C6 15 N4)]]> GAS6 - AVPLSVALVDYHSTK K, 13 C6 15 N2)]]>

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

[0075] The levels of sAXL and GAS6 were determined in 50 clinical pancreatic cancer tumor tissues according to the method of Example 2. The determined levels were then analyzed with respect to whether they had metastasized. Surprisingly, when 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 ), as can be seen from Figure 4 when the level of sAXL was less than the level of GAS6, the patient was more likely to have metastasized.

Claims

1. A method for analyzing free plasma membrane protein ectodomains in a sample, comprising the following steps: S1, constructing a plasma membrane protein database, wherein the database comprises sequence information of ectodomains and endodomains of plasma membrane proteins; S2, enriching plasma membrane protein groups on the surface of cell membranes and collecting secreted protein groups of cells, and analyzing unique polypeptides of ectodomains and endodomains of plasma membrane proteins in both, wherein the secreted protein groups contain free plasma membrane proteins, and the unique polypeptides refer to polypeptides that only match the endodomain or ectodomain of one protein in the proteome of one species; S3, identifying ectodomain and endodomain unique polypeptides, wherein when the number of unique polypeptides of step S2 is greater than or equal to 2, the unique polypeptides are identified as ectodomains or endodomains; and S4, analyzing free plasma membrane protein ectodomains, wherein the ratio of the number of ectodomain unique polypeptides to the number of endodomain unique polypeptides of the same plasma membrane protein in the plasma membrane protein group and the secreted protein group is calculated respectively, and normalized, and the normalization formula is as follows: Index Training = , wherein, when Index Training ≥ 2, is defined as the extracellular domain of a plasma membrane protein free.

2. The method of claim 1, wherein, The enrichment in the S2 step is performed by a hydrazide probe.

3. The method of claim 2, wherein, The structure of the hydrazide probe is: 。 4.The method according to any one of claims 1-3, comprising a marker combination for assisting in predicting pancreatic cancer metastasis: a free ectodomain of a receptor tyrosine kinase (sAXL), and growth arrest-specific protein 6 (GAS6). 5.The method according to any one of claims 1-3, comprising using a composition comprising: a first reagent: a reagent for detecting the content of a free ectodomain of a receptor tyrosine kinase (sAXL); and a second reagent: a reagent for detecting the content of growth arrest-specific protein 6 (GAS6).

6. The method of claim 5, wherein, The composition further comprises: a third reagent: a hydrazide probe. 7.The method according to any one of claims 1-3, comprising the following model: p = P GAS6 - P sAXL wherein, P GAS6 For the content of GAS6, P sAXL The content of the extracellular domain (sAXL) of free receptor tyrosine kinase; wherein 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.An apparatus, comprising: at least one processor; and a memory communicatively connected to the at least one processor; wherein the memory stores instructions executable by the processor, and the instructions are executed by the processor to implement the method for analyzing free plasma membrane protein ectodomains in a sample according to any one of claims 1-7. 9.A storage medium storing computer instructions for being executed by a computer to implement the method for analyzing free plasma membrane protein ectodomains in a sample according to any one of claims 1-7. 10.An apparatus for analyzing free plasma membrane protein ectodomains in a sample, comprising the following modules: S1, constructing a database module of plasma membrane proteins, wherein, the database comprises sequence information of ectodomains and endodomains of plasma membrane proteins; S2, enriching plasma membrane protein groups on the surface of cell membranes and collecting secreted protein groups of cells, and analyzing unique polypeptides of ectodomains and endodomains of plasma membrane proteins in both, wherein the unique polypeptides refer to polypeptides that only match the endodomain or ectodomain of one protein in the proteome of one species; S3, identifying extracellular domain and intracellular domain unique polypeptide modules, wherein when the number of unique polypeptides of step S2 is greater than or equal to 2, the unique polypeptides are identified as extracellular domain or intracellular domain; and S4, analyzing free plasma membrane protein extracellular domain modules, wherein the ratio of the number of extracellular domain unique polypeptides / the number of intracellular domain unique polypeptides of the same plasma membrane protein in the plasma membrane protein group and the secreted protein group is calculated respectively, and normalized, and the normalization formula is as follows: Index Training = , wherein, when Index Training ≥ 2 is defined as the extracellular domain of a plasma membrane protein.

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