A method for detecting the content of sos1 protein and use thereof

Abstract

The application discloses a kind of detection methods and purposes of SOS1 protein content, belong to biological medicine technical field.The method includes the following steps: sample to be tested enzymolysis, obtain the enzymolysis product comprising the characteristic peptide segment of SOS1 protein, the amino acid sequence of characteristic peptide segment is: FEIPEPEPTEADR and QLTLLESDLYR, stable isotope-labeled SOS1 protein characteristic peptide segment is used as internal standard, the signal value of characteristic peptide segment and internal standard peptide segment is detected using liquid chromatography-tandem mass spectrometry, the relative content of SOS1 protein is calculated by peak area ratio, and the absolute content is obtained by substituting calibration curve linear equation.The method can be used for the quantitative detection of SOS1 protein in tumor sample and other tissues, cells, blood sample, with the advantages of high sensitivity, good accuracy and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a method for detecting SOS1 protein content and application thereof. BACKGROUND

[0002] SOS1 protein (Son of Sevenless Homolog 1) is a protein encoded by the SOS1 gene, with a full length of 1333 amino acids and a molecular weight of 152464 Da. It is an important guanine nucleotide exchange factor and a key signal transduction regulatory protein. It plays a key regulatory molecular role in cell signal transduction, especially in the RAS / MAPK signaling pathway, which controls cell growth, proliferation, differentiation and survival. Therefore, the dysfunction of SOS1 protein is closely related to various diseases, especially cancer and developmental syndromes.

[0003] Studies have shown that abnormal expression or mutation of SOS1 is closely related to the occurrence and development of various cancers. In KRAS mutant cancer, even if KRAS itself has been mutated, SOS1 is still essential for KRAS activation, which makes SOS1 an attractive target for treating KRAS-driven tumors. Especially in the case of resistance to direct KRAS inhibitors, targeting SOS1 can effectively overcome or delay the occurrence of drug resistance. Mechanistically, SOS1 inhibitors or degraders inhibit the GDP / GTP exchange of KRAS by blocking the SOS1-KRAS interaction, thereby attenuating downstream signaling. In addition to its role in cancer, SOS1 is also involved in the pathological process of other diseases. For example, in Noonan syndrome, mutations in the SOS1 gene can cause changes in the function of the encoded protein, leading to symptoms such as heart abnormalities, unique facial features, and skeletal defects, which further highlights the importance of SOS1 in normal development and physiological processes.

[0004] Therefore, developing a reliable and stable method for detecting SOS1 protein concentration is of great significance in the fields of basic research, drug development, and diagnosis and mechanism research of specific diseases. For example, detecting the expression level changes of SOS1 protein under different stimuli (such as growth factors, cytokines) or in different cell types, different cell cycle stages, can help understand the fine regulation mechanism of the core signaling pathway; detecting the SOS1 protein concentration in tumor tissues and the changes in SOS1 protein concentration after drug treatment can evaluate whether the drug effectively binds to the target, induces degradation or interferes with its function; detecting the SOS1 protein concentration level of patient tumor samples can be used for patient screening in clinical trials evaluating the "KRAS target + SOS1 target" combination therapy. SUMMARY

[0005] In order to solve the problems in the prior art, the present application provides a method for detecting the content of SOS1 protein, which is suitable for determining the relative or absolute content of SOS1 protein in biological samples before and after drug treatment, and is especially suitable for determining the absolute content of SOS1 protein in tumor tissue or cell samples in the field of targeted treatment of cancer.

[0006] The method of the present application realizes quantitative analysis based on mass spectrometry multiple reaction monitoring (MRM) targeted detection of two characteristic peptide segments of SOS1 protein, and the sequences of the characteristic peptide segments are FEIPEPEPTEADR and QLTLLESDLYR, respectively. By detecting the content of the above characteristic peptide segments, the expression level of SOS1 protein can be accurately characterized, and the absolute content of SOS1 protein can be obtained according to the molecular weight conversion of the peptide segments. The method is not only suitable for tumor samples, but also can be widely applied to other clinical or scientific research model samples, including tissue, cell and blood samples, to realize high-sensitivity and high-precision quantitative detection of SOS1 protein.

[0007] The present application provides a method for detecting the content of SOS1 protein, which comprises the following steps:

[0008] (1) Sample pretreatment: trypsin enzymolysis is performed on the biological sample to be measured to obtain an enzymolysis product containing characteristic peptide segments of SOS1 protein, and the amino acid sequences of the characteristic peptide segments are shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0009] SEQ ID NO: 3: FEIPEPEPTEADR;

[0010] SEQ ID NO: 4: QLTLLESDLYR;

[0011] (2) Internal standard addition: a stable isotope-labeled SOS1 protein characteristic peptide segment with a known concentration is added to the enzymolysis product as an internal standard;

[0012] (3) Liquid chromatography-tandem mass spectrometry detection: liquid chromatography-tandem mass spectrometry is used to detect the signal values of the characteristic peptide segments and the internal standard peptide segments;

[0013] (4) Quantitative analysis: the peak area ratio is obtained by integral processing, and the relative content of SOS1 protein in the sample can be calculated; by substituting the peak area ratio into the linear equation of the calibration curve sample regression fitting, the absolute content of SOS1 protein in the sample can be calculated;

[0014] The conditions of the liquid chromatography are as follows:

[0015] Chromatographic column: octadecylsilane-bonded high-strength silica gel particle chromatographic column;

[0016] Mobile phase: Phase A is water containing 0.095%-0.105% formic acid, Phase B is methanol:acetonitrile(19-21):(76-84) mixed solution containing 0.095%-0.105% formic acid;

[0017] The liquid chromatography separation is performed according to the following gradient elution conditions:

[0018] 0.00 to 0.80 min, the proportion of B phase is 14.25%-15.75%;

[0019] 0.80 to 3.00 min, the proportion of B phase is increased from 14.25%-15.75% to 19.95%-22.05%;

[0020] 3.00 to 6.00 min, the proportion of B phase is increased from 19.95%-22.05% to 21.85%-24.15%;

[0021] 6.00 to 12.00 min, the proportion of B phase is kept at 21.85%-24.15%;

[0022] 12.00 to 13.00 min, the proportion of B phase is increased from 21.85%-24.15% to 93.1%-100%;

[0023] 13.00 to 13.80 min, the proportion of B phase is kept at 93.1%-100%.

[0024] In the liquid chromatography, the chromatographic column is preferably: ACQUITY UPLC Premier HSS T3 chromatographic column, the particle size of the filler is 1.8 μm, and the size is 2.1 mm*100 mm.

[0025] Preferably, the gradient elution conditions are as follows:

[0026] 0.00 to 0.80 min, the proportion of B phase is 15.0%;

[0027] 0.80 to 3.00 min, the proportion of B phase is increased from 15.0% to 21.0%;

[0028] 3.00 to 6.00 min, the proportion of B phase is increased from 21.0% to 23.0%;

[0029] 6.00 to 12.00 min, the proportion of B phase is 23.0%;

[0030] 12.00 to 13.00 min, the proportion of B phase is increased from 23.0% to 98.0%;

[0031] 13.00 to 13.80 min, the proportion of B phase is 98%.

[0032] 13.80 to 15.00 min, the proportion of phase B decreases from 98.0% to 15.0%.

[0033] In the liquid chromatography, the flow rate of the mobile phase is preferably 0.38-0.42 mL / min; the column temperature is preferably 66.5-73.5 °C.

[0034] In the liquid chromatography, the flow rate of the mobile phase is further preferably 0.4 mL / min; the column temperature is further preferably 70 °C.

[0035] Preferably, the sample pre-treatment comprises the following steps:

[0036] (1) mixing the biological sample to be tested with a lysis solution, carrying out tissue homogenization to obtain a tissue lysis solution sample;

[0037] (2) taking the tissue lysis solution sample containing protein, after heating and denaturation, adding a protein precipitant, freezing and centrifuging, discarding the supernatant, and collecting the precipitate;

[0038] (3) resolubilizing the precipitate in an ammonium bicarbonate solution;

[0039] (4) adding a reducing agent and incubating;

[0040] (5) adding an alkylating agent and incubating;

[0041] (6) adding trypsin and carrying out enzymatic hydrolysis;

[0042] (7) adding a termination reagent to terminate the enzymatic hydrolysis reaction, to obtain an enzymatic hydrolysate containing a characteristic peptide segment of the SOS1 protein.

[0043] In step (1), the lysis solution comprises a RIPA lysis solution, a phosphatase inhibitor, a protease inhibitor, a metal chelator, and phenylmethylsulfonyl fluoride (PMSF).

[0044] The metal chelator can be ethylenediaminetetraacetic acid (EDTA).

[0045] In the lysis solution, the concentration of the phosphatase inhibitor is preferably 1x, the concentration of the protease inhibitor is preferably 1x, the concentration of the metal chelator is preferably 1x, and the concentration of the PMSF is preferably 1 mM.

[0046] In a preferred embodiment, the composition of the lysis solution is as follows: 600 μL of the phosphatase inhibitor (50x), 1200 μL of the protease inhibitor (50x), 600 μL of the EDTA (50x), 300 μL of the PMSF (100 mM), and the rest is supplemented with RIPA lysis solution 27300 μL, so that the total volume is 30000 μL.

[0047] In step (2), the protein precipitant is preferably methanol, and the amount added is equal to the volume of the tissue lysis solution sample.

[0048] In step (3), the concentration of the ammonium bicarbonate solution is preferably 9.5-10.5 mM, for example 10 mM.

[0049] In step (4), the reducing agent is preferably dithiothreitol; the concentration of the dithiothreitol is preferably 95-105 mM, for example 100 mM; the incubation temperature is preferably 57-63°C, for example 60°C; the incubation time is preferably 28.5-31.5 minutes; for example 30 minutes.

[0050] In step (5), the alkylating agent is preferably iodoacetamide; the concentration of the iodoacetamide is preferably 190-210 mM, for example 200 mM; the incubation conditions are preferably 35-39°C, avoiding light, for 28.5-31.5 minutes, for example 37°C, avoiding light, for 30 minutes.

[0051] In step (6), the mass ratio of trypsin to protein is preferably 1:19-1:21, for example 1:20; the conditions for enzymatic digestion are preferably 23.75-26.25°C for 3.8-4.2 hours, for example 25°C for 4 hours.

[0052] In step (7), the termination reagent is preferably formic acid, and the amount of formic acid added is preferably 11-13 μL of 9-11% formic acid by volume, for example 12.4 μL of 10% formic acid by volume.

[0053] In a preferred embodiment, the sample pretreatment comprises the following steps:

[0054] Mixing the biological sample to be tested with the lysis solution, homogenizing the tissue to obtain a tissue lysis solution sample;

[0055] Taking 100 μg of protein-containing tissue lysis solution sample, and diluting to 100 μL with blank lysis solution; after heating and denaturing at 95°C for 5 minutes, adding an equal volume of pre-cooled methanol, and freezing at -80°C for 0.5 hours;

[0056] After freezing, centrifuge at 12000 rpm for 5 minutes at 4°C, discard the supernatant, and rinse the precipitate with cold methanol twice;

[0057] Resuspend the precipitate in 100 μL of 10 mM ammonium bicarbonate solution, add 5 μL of 100 mM dithiothreitol, incubate at 60°C for 30 minutes, then add 5 μL of 200 mM iodoacetamide, incubate at 37°C for 30 minutes in the dark;

[0058] Add 5 μL of 1 mg / mL trypsin at an enzyme to protein mass ratio of 1:20, and enzymatically digest at 25°C for 4 hours;

[0059] Add 12.4 μL of 10% (by volume) formic acid to terminate the enzymatic reaction, and obtain the enzymatic product containing the characteristic peptide segments of the SOS1 protein.

[0060] The internal standard peptide comprises one or more amino acid residues replaced by a polypeptide having the same sequence as FEIPEPEPTEADR and containing one or more isotopic labels; the internal standard peptide differs from the characteristic peptide segment in molecular weight by at least 4 Da.

[0061] The molecular weight of the peptide segment FEIPEPEPTEADR is 1529.6, and the molecular weight of the peptide segment QLTLLESDLYR is 1350.51. The characteristic peptide segments FEIPEPEPTEADR and QLTLLESDLYR mainly exhibit a bivalent state in the ion source.

[0062] The molecular weight of the internal standard peptide segment FEIPEPEPTEADR ( 13 C3, 15 N) is 1533.6, and it mainly exhibits a bivalent state in the ion source.

[0063] The detection method in the present application is to detect the signal value of the characteristic peptide segment of the SOS1 protein in the sample and the signal value of the stable isotope-labeled internal standard peptide segment with a known concentration added to the sample by mass spectrometry MRM detection mode, and the relative content of the SOS1 protein in the sample can be calculated by integrating the peak area ratio; the absolute content of the SOS1 protein in the sample can be obtained by substituting the peak area ratio into the linear equation of the calibration standard regression fitting.

[0064] The signal value of the characteristic peptide segment is achieved by detecting a parent ion (Q1) and a daughter ion (Q3) generated thereby.

[0065] The parent-daughter ion pair of the characteristic peptide segment FEIPEPEPTEADR is 765.6 / 570.9; the parent-daughter ion pair of the characteristic peptide segment QLTLLESDLYR is 676.1 / 782.3; and the parent-daughter ion pair of the internal standard peptide is 767.6 / 572.7. 13 C3- 15 The isotope 13 C3- 15 The internal standard peptide is labeled on the alanine residue, and the parent-daughter ion pair of the internal standard peptide is 767.6 / 572.7.

[0066] The mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry are preferably as follows: a cone voltage of 28.5-31.5 V, a capillary voltage of 2850-3150 V, an ion source temperature of 570-630 DEG C, a desolvation gas flow of 1140-1260 L / Hr, a gas flow rate of 142.5-157.5 L / Hr, and an atomization gas pressure of 6.65-7.35 Bar.

[0067] The mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry are further preferably as follows: a cone voltage of 30 V, a capillary voltage of 3000 V, an ion source temperature of 600 DEG C, a desolvation gas flow of 1200 L / Hr, a gas flow rate of 150 L / Hr, and an atomization gas pressure of 7 Bar.

[0068] The biological sample to be detected is preferably a tumor tissue sample.

[0069] The detection method used in the present application has a detection lower limit of 0.01 ng / mL.

[0070] The liquid chromatography-tandem mass spectrometry system used in the present application is preferably a liquid chromatography-tandem mass spectrometry system composed of a Waters ultra-high performance liquid chromatograph (ACQUITY Premier) and a triple quadrupole mass spectrometer (Xevo TQ-XS).

[0071] The mass spectrometry adopts a targeted mass spectrometry signal acquisition technology mode of multiple reaction monitoring (MRM) to detect the signal.

[0072] The detection method of the present application first needs to obtain theoretical enzyme digestion peptides by theoretically simulating enzyme digestion of the protein sequence of SOS1 using trypsin, and searching a species library using Skyline software to obtain unique characteristic peptides. According to certain theoretical screening criteria, the peptides with high ionization efficiency, moderate hydrophilicity (avoiding hydrophobic peptides to precipitate), appropriate length (6-25 amino acids), and avoiding containing modifiable amino acids, no enzyme cutting site, etc. are preferred, and the appropriate peptides (LFSSNDVENIFSR, LPGASSAEYR, FEIPEPEPTEADR, QLTLLESDLYR, SASVSSISLSK) are selected as candidate characteristic peptides for SOS1 protein quantification.

[0073] The candidate characteristic peptides are synthesized as standard samples, and the actual ionization efficiency, fragmentation pattern and retention time are detected by LC-MS / MS to screen peptides with high and stable signal intensity. And ensure that the mass-to-charge ratio (m / z) value of the selected ion has no obvious co-elution interference in the total ion chromatogram of the actual biological sample. Based on the above factors, the present application uses tumor samples for proteolysis in the method development, and the enzyme digestion products are analyzed by LC-MS / MS injection. By optimizing the liquid phase conditions and mass spectrometry parameters and suitable parent-daughter ion pairs (Q1 / Q3), the chromatographic behavior of the synthesized candidate characteristic peptide standard sample is compared, and the characteristic peptides with good ionization efficiency, high and stable signal intensity, and good chromatographic peak separation degree in the actual sample are selected. Finally, FEIPEPEPTEADR and QLTLLESDLYR are selected as the characteristic peptides of SOS1 protein.

[0074] The detection method measures the content of the characteristic peptides of the SOS1 protein from the proteolysis products of the tumor sample, and the content refers to the relative content or absolute content between samples. The biological samples used for detecting the content of SOS1 protein include tumor samples, cell samples, tissue samples and blood samples (such as PBMC).

[0075] The quantitative detection of the present application is based on the targeted detection of the characteristic peptides of SOS1 protein, and can perform absolute quantification of the characteristic peptides and SOS1 protein and relative quantification of SOS1 protein in different treatment samples.

[0076] The present application also provides the use of the method in the preparation of a product for detecting SOS1 protein, diagnosing related diseases, monitoring disease progression or evaluating prognosis.

[0077] Using the detection method of the present application, the content of SOS1 protein in biological samples such as tumors, cells, tissues and blood (PBMC) can be quantitatively detected, which can be used for the efficacy evaluation of targeted drugs with SOS1 protein as the target, and the diagnosis, detection and course monitoring of diseases and the prognosis evaluation.

[0078] On the basis of not violating the common sense of the art, the above-mentioned preferred conditions can be combined arbitrarily, that is, each preferred example of the present application is obtained.

[0079] The reagents and raw materials used in the present application are commercially available, except for special instructions.

[0080] The positive progress effect of the present application is:

[0081] The detection method of the present application obtains the practically applicable and uniquely representative characteristic peptide segment through the theoretical enzyme digestion of the protein sequence based on the general theoretical basis of the selection of the characteristic peptide segment, and the verification of the SOS1 protein digestion peptide segment detected in the actual sample proteolysis product. The characteristic peptide segment and the synthesized stable isotope-labeled re-labeled internal standard peptide segment are used for quantitative detection of SOS1 protein on a liquid chromatograph-mass spectrometer (LC-MS / MS) in a multiple reaction monitoring (MRM) mode. The liquid chromatograph (LC) is used for separation according to the retention time, and the mass spectrometer (MS / MS) is used for confirmation according to the mass-to-charge ratio and the fragment ion, which can exclude the interference of other co-eluted peptide segments in complex biological samples, avoid false positives, and perform high-accuracy and high-precision absolute quantification on the target SOS1 protein. The method has high specificity and selectivity. At the same time, the stable isotope-labeled re-labeled internal standard peptide segment has almost the same physical and chemical properties as the light-labeled polypeptide of SOS1 protein, and the chromatographic and mass spectrometric behaviors are also consistent. Through the normalization of the ratio of the two, the fluctuation of the detection response value caused by the matrix effect can be corrected, and absolute quantification can be realized.

[0082] The linear quantitative range of the calibration curve of the detection method of the present application is 0.01 ng / mL-5 ng / mL, which has extremely high sensitivity and wide dynamic range. The detection method of the present application does not need high-quality and high-specificity antibodies required by the immunoassay, but synthesizes a characteristic peptide segment and a stable isotope-labeled internal standard peptide segment, which has the advantages of short development cycle, high throughput, low cost, stable and reliable results, etc. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1 The parent-daughter ion mass spectrum of the characteristic peptide segment FEIPEPEPTEADR.

[0084] Figure 2 The parent-daughter ion mass spectrum of the characteristic peptide segment QLTLLESDLYR.

[0085] Figure 3The mass spectrum of the parent ion and the daughter ion of the internal standard peptide segment FEIPEPEPTEADR 13 C3, 15 N.

[0086] Figure 4 The chromatogram of the characteristic peptide segment FEIPEPEPTEADR selected by the chromatographic column ACQUITY UPLC Protein BEH C4 300A.

[0087] Figure 5 The chromatogram of the characteristic peptide segment QLTLLESDLYR selected by the chromatographic column ACQUITY UPLC Protein BEH C4 300A.

[0088] Figure 6 The chromatogram of the characteristic peptide segment FEIPEPEPTEADR selected by the chromatographic column ACQUITY Premier HSS T3 VanGuard FIT.

[0089] Figure 7 The chromatogram of the characteristic peptide segment QLTLLESDLYR selected by the chromatographic column ACQUITY Premier HSS T3 VanGuard FIT.

[0090] Figure 8 The chromatogram of the characteristic peptide segment FEIPEPEPTEADR using the gradient elution condition as shown in Table 5.

[0091] Figure 9 The chromatogram of the characteristic peptide segment QLTLLESDLYR using the gradient elution condition as shown in Table 5.

[0092] Figure 10 The chromatogram of the characteristic peptide segment FEIPEPEPTEADR using the gradient elution condition as shown in Table 6.

[0093] Figure 11 The chromatogram of the characteristic peptide segment QLTLLESDLYR using the gradient elution condition as shown in Table 6.

[0094] Figure 12 The chromatogram of the characteristic peptide segment FEIPEPEPTEADR under the condition of column temperature of 60 °C.

[0095] Figure 13 The chromatogram of the characteristic peptide segment QLTLLESDLYR under the condition of column temperature of 60 °C.

[0096] Figure 14 The chromatogram of the characteristic peptide segment FEIPEPEPTEADR under the condition of column temperature of 70 °C.

[0097] Figure 15Chromatogram of the characteristic peptide segment QLTLLESDLYR at a column temperature of 70 °C.

[0098] Figure 16 Standard curve graph of the characteristic peptide segment FEIPEPEPTEADR.

[0099] Figure 17 Standard curve graph of the characteristic peptide segment QLTLLESDLYR.

[0100] Figure 18 LLOQ chromatogram example of the characteristic peptide segment FEIPEPEPTEADR.

[0101] Figure 19 ULOQ chromatogram example of the characteristic peptide segment FEIPEPEPTEADR.

[0102] Figure 20 Chromatogram example of the characteristic peptide segment FEIPEPEPTEADR in a sample to be tested.

[0103] Figure 21 LLOQ chromatogram example of the characteristic peptide segment QLTLLESDLYR.

[0104] Figure 22 ULOQ chromatogram example of the characteristic peptide segment QLTLLESDLYR.

[0105] Figure 23 Chromatogram example of the characteristic peptide segment QLTLLESDLYR in a sample to be tested. DETAILED DESCRIPTION

[0106] The present application is further illustrated by the following examples without thereby limiting the present application to the scope of the examples. The experimental methods in the following examples, if not specified, are selected according to the conventional methods and conditions, or according to the product instructions.

[0107] The process, conditions, experimental methods, etc. for implementing the present application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the present application has no special limitations.

[0108] Example 1

[0109] In this example, the detection method of SOS1 protein content is researched and developed.

[0110] I. Screening of characteristic peptide segments

[0111] Theoretical enzyme digestion of the protein sequence of SOS1 was performed using trypsin to obtain theoretical enzyme digestion peptides. Skyline software was used to search the species library to obtain unique characteristic peptides. According to the theoretical screening criteria combined with the inventors' experience, peptides with high ionization efficiency, moderate hydrophilicity (avoiding hydrophobic peptides), appropriate length (6-25 amino acids), and avoiding the presence of modifiable amino acids, enzyme cleavage sites, etc. were selected as candidate characteristic peptides for SOS1 protein quantification.

[0112] Table 1: Candidate characteristic peptides

[0113]

[0114] Synthetic candidate characteristic peptides were used as standard samples to detect their ionization efficiency, fragmentation pattern and retention time by LC-MS / MS. Peptides with high and stable signal intensity were selected. The mass-to-charge ratio (m / z) values of the selected ions were ensured to have no significant co-elution interference in the total ion chromatogram of the actual biological sample. The preliminary mass spectrometry information of the five candidate characteristic peptides is shown in the following table.

[0115] Table 2: Mass spectrometry information of candidate characteristic peptides

[0116]

[0117] In the method development, tumor samples were pre-selected for proteolysis. The enzyme digestion products were analyzed by LC-MS / MS. By optimizing the liquid chromatography conditions and mass spectrometry parameters, as well as suitable parent-daughter ion pairs (Q1 / Q3), the chromatographic behavior of synthetic candidate characteristic peptide standards was compared. The characteristic peptides with good ionization efficiency, high and stable signal intensity, and good chromatographic peak separation in actual samples were selected.

[0118] After considering various factors, FEIPEPEPTEADR (peptide 3) and QLTLLESDLYR (peptide 4) were selected as the characteristic peptides of SOS1 protein.

[0119] Further, the inventors optimized and screened the mass spectrometry conditions of the above characteristic peptides and corresponding heavy internal standard peptides FEIPEPEPTEADR (peptide 3) 13 C3, 15 N). The mass spectrometry conditions are shown in the following table, which have the advantages of high signal intensity and good stability. The parent-daughter ion mass spectra of peptide 3, peptide 4 and heavy internal standard peptide are shown in Figures 1-3 .

[0120] Table 3: Mass spectrometry parameter conditions after optimization

[0121]

[0122] II. Optimization of LC-MS conditions

[0123] To ensure the accuracy and repeatability of the quantitative results, the LC-MS conditions were optimized and confirmed in this example. The mass spectrometry parameters such as selection of Q1 / Q3, cone voltage, capillary voltage, ion source temperature, the liquid chromatography conditions such as column type, gradient elution, and the pretreatment conditions of the sample before proteolysis were all optimized.

[0124] 1. Optimization of LC conditions

[0125] During the establishment of the LC-MS / MS quantitative detection method for the characteristic peptides FEIPEPEPTEADR and QLTLLESDLYR of SOS1 protein, the applicant conducted multiple rounds of screening and optimization of the liquid separation conditions.

[0126] Firstly, the applicant attempted to use a conventional C18 column and a short gradient program (e.g. 5-10 minutes) for detection. It was found that under such conditions, isobaric interference peaks with the same or similar parent ions and daughter ions as the target peptide FEIPEPEPTEADR appeared. The interference peaks could not be effectively separated on the chromatogram, resulting in significant amplification of the target peak area, high quantitative results, and extremely large CV%, and stable quantitative results could not be obtained.

[0127] To overcome the interference, the applicant optimized the liquid chromatography conditions such as column type, column temperature, and elution gradient, as follows:

[0128] (1) Column selection: Considering the strong hydrophilicity of the characteristic peptides, the retention ability on conventional columns is weak and the peak shape is poor. For example, the results of the previous screening test are shown in FIGS. 3 and 4, which are the chromatograms of peptide 3 and peptide 4, respectively, using the column ACQUITY UPLC Protein BEH C4 300A (1.7 μm, 2.1 x 100 mm). Figures 4-5 Figures 4-5

[0129] Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in methanol: acetonitrile (20:80) solution; the gradient elution conditions are shown in Table 4, and the column temperature is 40°C.

[0130] Table 4 Gradient elution conditions

[0131]

[0132] ​​After multiple comparisons, Waters ACQUITY UPLC HSS T3 chromatographic column was selected, which has high-strength silica gel matrix and three-end sealing tail characteristics, which endows it with better hydrophilic compound retention performance, and can effectively improve the separation degree and peak shape stability of the target peptide segment. For example Figures 6-7 as shown in Figures 6-7 The chromatograms of peptide segment 3 and peptide segment 4, respectively, were selected using the chromatographic column ACQUITY Premier HSS T3 VanGuard FIT (1.8 μm, 2.1 x 100 mm), and the gradient elution conditions are shown in Table 4, and the column temperature is 40°C.

[0133] (2) Gradient program optimization: Since there are many proteins and peptide segments in the sample, which interfere with the peptide segments, under the conventional isocratic elution or gradient elution conditions, the target peptide segments and interfering peptide segments cannot be well separated. For example Figures 8-11 as shown in Figures 8-9 The chromatograms of peptide segment 3 and peptide segment 4, respectively, were selected using the chromatographic column ACQUITY Premier HSS T3 VanGuard FIT (1.8 μm, 2.1 x 100 mm), and the gradient elution conditions are shown in Table 5, and the column temperature is 40°C. Figures 10-11 The chromatograms of peptide segment 3 and peptide segment 4, respectively, were selected using the chromatographic column ACQUITY Premier HSS T3 VanGuard FIT (1.8 μm, 2.1 x 100 mm), and the gradient elution conditions are shown in Table 6, and the column temperature is 40°C.

[0134] Table 5 Gradient elution conditions

[0135]

[0136] Table 6 Gradient elution conditions

[0137]

[0138] After repeated experimental adjustment of the elution conditions by the inventors, an elution gradient program with a total duration of about 15 minutes was established, in which the interval of 3-12 minutes (the proportion of phase B slowly increased from about 21% to 23%) used a very gentle gradient slope.

[0139] This setting is specially designed to achieve complete separation of the target peptide segment and the interfering peak. In contrast, the conventional steep gradient elution conditions cannot achieve similar separation effect.

[0140] (2) Column temperature optimization: The inventors found that under the standard column temperature (30-50°C), the target peptide segment and the interfering peak overlap, and cannot be separated. The chromatograms of peptide segment 3 and peptide segment 4 injected under the condition of column temperature of 40°C are shown in Figures 6-7shown (chromatographic column was ACQUITY Premier HSS T3 VanGuard FIT (1.8 pm, 2.1 x 100 mm), gradient elution conditions were shown in Table 4). The chromatograms of Peptide 3 and Peptide 4 injected at 60 °C were shown in Figures 12-13 shown (chromatographic column was ACQUITY Premier HSS T3 VanGuard FIT (1.8 pm, 2.1 x 100 mm), gradient elution conditions were shown in Table 4), which showed that the target peptide peaks had been well separated.

[0141] The inventors unexpectedly found that when the column temperature was increased to 70 °C and the elution gradient was fine-tuned, the retention behaviors of the two were significantly different, thereby achieving baseline separation. As shown in Figures 14-15 Figures 14-15 The chromatograms of Peptide 3 and Peptide 4 injected at 70 °C, respectively, were shown in

[0142] The optimized LC-MS / MS liquid phase conditions were shown in Table 7. The LC-MS / MS liquid chromatography gradient used after optimization was shown in Table 8.

[0143] Table 7 Information table of LC-MS / MS liquid phase conditions used after optimization

[0144]

[0145] Table 8 Table of LC-MS / MS liquid chromatography gradient used after optimization

[0146]

[0147] III. Sample pretreatment condition optimization before enzymolysis

[0148] The sample to be tested in this example was a solid tumor sample. The sample to be tested was weighed, and a suitable proportion of lysis solution was added for tissue homogenization to obtain a uniform tissue lysate. The tissue lysate sample was subjected to the following operations in turn (protein precipitation, resuspension, disulfide bond reduction, mercaptoalkylation, and trypsin enzymolysis) to obtain an enzymolysis product sample containing characteristic peptides of SOS1 protein, which was then subjected to quantitative analysis by LC-MS / MS. The following methods were used:

[0149] 1. Lysis

[0150] ​Respectively, a certain amount of tumor sample was taken, and equal proportion of lysis solution was added according to the mass volume ratio of 1 g / 5 mL to extract protein by tissue homogenate. The total protein concentration was determined using BCA kit. The composition and preparation ratio of lysis solution are as follows.

[0151] Table 9 Composition and preparation ratio of lysis solution

[0152]

[0153] Take the solution containing 100 μg of total protein (diluted to 100 μL with blank lysis solution), heat denaturation at 95°C metal bath for 5 minutes.

[0154] 2, Precipitation

[0155] Add equal volume of pre-cooled methanol or acetonitrile or methanol: acetonitrile (1:1), vortex for 2 minutes, centrifuge at 12000 rpm for 5 minutes at 4°C to precipitate protein, and discard the supernatant. Corresponding 200 μL of pre-cooled methanol or acetonitrile or methanol: acetonitrile (1:1) is used to further wash the precipitate twice. The peak area is used as the screening index to compare the effects of the three different precipitants and the corresponding washing conditions, and the results are shown in the following table.

[0156] Table 10 Optimization of precipitant selection

[0157]

[0158] The above results show that methanol as a precipitant has better effect.

[0159] 3, Resuspension

[0160] Respectively, 100 μL of different concentrations of NH4HCO3 resuspension solution was added to the above precipitate to investigate the effects of 10 mM NH4HCO3 solution, 25 mM NH4HCO3 and 50 mM NH4HCO3 resuspension solution, and the test results are shown in the following table.

[0161] Table 11 Optimization of NH4HCO3 salt concentration of resuspension solution

[0162]

[0163] The above results show that 10 mM NH4HCO3 as a resuspension solution has better effect.

[0164] 4, Reduction

[0165] Respectively, 5 μL of reducing agent DTT (dithiothreitol) was added to the sample to investigate the concentration of 50 mM, 100 mM and 200 mM. Incubate at 60°C for 30 minutes. The test results of the three concentrations of reducing agent are shown in the following table.

[0166] Table 12 Optimization data table of reducing agent DTT concentration

[0167]

[0168] The above results show that 100 mM DTT as a reducing agent has better effect.

[0169] 5. Alkylation

[0170] 5 μL of alkylation reagent IAA (iodoacetamide) was added to the sample, respectively, and the concentrations of 50 mM, 100 mM and 200 mM were investigated. Incubate at 37°C for 30 minutes in the dark. The test results of the three concentrations of alkylation reagent are shown in the table below.

[0171] Table 13 Concentration investigation of alkylation reagent IAA

[0172]

[0173] The above results show that the IAA concentration has a significant effect on the test results, and 200 mM alkylation reagent IAA has better effect.

[0174] 6. Enzymolysis

[0175] (1) Enzyme addition amount investigation

[0176] 5, 2, and 1 μL of 1 mg / mL trypsin solution (trypsin purchased from Thermo Fisher Scientific, product type 90059) were added, respectively, and the trypsin was added at a ratio of 1:20, 1:50 and 1:100 according to the enzyme / total protein ratio (w / w) to investigate the enzymeolysis. The test results of the three enzyme concentrations of enzymeolysis are shown in the table below.

[0177] Table 14 Trypsin enzymeolysis concentration screening results

[0178]

[0179] The above results show that the trypsin is added at a ratio of 1:20 for enzymeolysis, which has better effect.

[0180] (2) Temperature investigation

[0181] During the trypsin enzymeolysis process, the incubation reaction at 1000 rpm was investigated at 25°C, 37°C and 60°C, respectively. The test results of the three enzymeolysis process incubation temperatures are shown in the table below.

[0182] Table 15 Optimization data table of trypsin enzymeolysis temperature

[0183]

[0184] The above results show that the enzymolysis at 25°C has good effect.

[0185] (3) Incubation time investigation

[0186] During the use of trypsin enzymolysis, three incubation times of 2 hours, 4 hours and 16 hours (overnight) were investigated respectively. The test results of three enzymolysis reaction times are shown in the following table.

[0187] Table 16 Optimization data table of trypsin enzymolysis temperature

[0188]

[0189] The above results show that the increase of incubation time helps to improve the content of target peptide segment, but too long time may also cause degradation of the peptide segment, and incubation for 4 hours has good effect.

[0190] 7, Sample post-processing

[0191] After trypsin enzymolysis to the predetermined time, the enzymolysis was terminated by adding 12.4 μL of 10% formic acid.

[0192] Then the sample was centrifuged at 12,000 rpm at 4°C for 5 minutes, 90 μL of the enzymolysis solution and 10 μL of 10 ng / mL internal standard (isotope-labeled peptide segment FEIPEPEPTEADR (N)) were taken to a 96 deep well plate, vortexed, and then analyzed by LC-MS / MS injection. 13 C3, 15 N)

[0193] Based on the optimization conditions of each sample pretreatment, the following sample pretreatment process was determined:

[0194] Accurately pipette 100 μg of tissue lysate sample into a 1.5 mL microcentrifuge tube, and dilute to 100 μL with blank lysate, vortex, and heat denature at 95 °C in a metal bath for 5 minutes. Then add an equal volume of pre-chilled methanol, vortex, and freeze at -80 °C for 0.5 hour. After freezing, centrifuge at 12,000 rpm for 5 minutes at 4 °C, discard the supernatant, and further rinse the pellet with cold methanol twice, and re-dissolve in 100 μL of 10 mM ammonium bicarbonate. Then add 5 μL of 100 mM dithiothreitol, incubate at 60 °C for 30 minutes, then add 5 μL of 200 mM iodoacetamide, and incubate the sample at 37 °C for 30 minutes in the dark. After alkylation, add 5 μL of 1 mg / mL trypsin solution at an enzyme / protein mass ratio of 1:20 (w / w), and enzymatically digest the sample at 25 °C for 4 hours. Terminate the enzymatic digestion by adding 12.4 μL of 10% (v / v) formic acid. Finally, pipette 90 μL of the digested sample and 10 μL of 10 ng / mL internal standard (isotope-labeled peptide FEIPEPEPTEADR (Peptide 3) and QLTLLESDLYR (Peptide 4)) into a 96-deep well plate, vortex, and then use LC-MS / MS for sample analysis. 13 C3, 15 N) as the internal standard, and quantitatively analyze the concentration of endogenous protein SOS1 in the sample. The standard curve is linearly fitted by the weighted least squares method (W = 1 / X 2 ), and the standard curve regression equation is obtained, which is substituted into the ratio of the peak area of the test substance to the peak area of the internal standard for quantification. The recalculated concentration of the calibration standard is within ±20.0% of the theoretical concentration (±25.0% at the LLOQ level); the calibration standards excluded from the standard curve are not more than 25%, and at least six acceptable concentration values are included. There are at least 5 concentration data at each concentration level for the quality control sample, and the average accuracy of at least 3 data at each concentration is within ±20.0% (±25.0% at the LLOQ).

[0195] Example 2

[0196] This example verifies the method for detecting the content of SOS1 protein.

[0197] 1. Method overview

[0198] The calibration standard and quality control sample evaluation used the characteristic peptides FEIPEPEPTEADR (Peptide 3) and QLTLLESDLYR (Peptide 4) of the SOS1 endogenous protein as standard analyte, used 10 mM ammonium bicarbonate solution containing 1% FA as blank matrix, and used isotope-labeled peptide FEIPEPEPTEADR (Peptide 3) as internal standard to quantitatively analyze the concentration of endogenous protein SOS1 in the sample. The standard curve was linearly fitted by the weighted least squares method (W = 1 / X 13 C3, 15 N), and the standard curve regression equation was obtained, which was substituted into the ratio of the peak area of the test substance to the peak area of the internal standard for quantification. The recalculated concentration of the calibration standard was within ±20.0% of the theoretical concentration (±25.0% at the LLOQ level); the calibration standards excluded from the standard curve were not more than 25%, and at least six acceptable concentration values were included. There were at least 5 concentration data at each concentration level for the quality control sample, and the average accuracy of at least 3 data at each concentration was within ±20.0% (±25.0% at the LLOQ).

[0199] 2. Standard curve

[0200] The results of the back-calculated concentrations of each concentration point of the standard curve of the characteristic peptide segment FEIPEPEPTEADR are shown in Table 17. The results show that the deviation of the calibration standard is in the range of -9.8% ~ 8.8%, which meets the acceptance criteria. The regression equation of the standard curve is shown in Table 17, and the fitted standard curve graph is shown in Figure 16 .

[0201] Table 17 Standard curve data table of characteristic peptide segment FEIPEPEPTEADR

[0202]

[0203] *Out of the accepted range, not included in the mean calculation

[0204] The results of the back-calculated concentrations of each concentration point of the standard curve of the characteristic peptide segment QLTLLESDLYR are shown in Table 18. The results show that the deviation of the calibration standard is in the range of -17.5% ~ 13.8%, which meets the acceptance criteria. The regression equation of the standard curve is shown in Table 18, and the fitted standard curve graph is shown in Figure 17 .

[0205] Table 18 Standard curve data table of characteristic peptide segment QLTLLESDLYR

[0206]

[0207] *Out of the accepted range, not included in the mean calculation

[0208] 3. Sensitivity, accuracy and precision

[0209] The results of the characteristic peptide segment FEIPEPEPTEADR quality control sample are shown in Table 19. The results show that the average accuracy deviation of the peptide segment FEIPEPEPTEADR quality control sample from the low concentration level (LQC) to the high concentration level (HQC) and the sensitivity (LLOQ) is -0.7%, -3.1%, 7.5%, 8.5% and 3.4% respectively, and the precision is 4.3%, 4.1%, 2.1%, 1.9% and 9.9% respectively, all of which meet the acceptance criteria.

[0210] Table 19 Sensitivity, accuracy and precision of characteristic peptide segment quality control

[0211]

[0212] The results of the characteristic peptide segment QLTLLESDLYR quality control sample are shown in Table 20. The results show that the average accuracy bias of the peptide segment FEIPEPEPTEADR quality control sample from the low concentration level (LQC) to the high concentration level (HQC) and the sensitivity (LLOQ) is -0.6%, -7.2%, 13.2%, 14.6% and -1.9%, respectively, and the precision is 5.4%, 5.0%, 2.7%, 2.8% and 9.4%, respectively, all of which meet the acceptance criteria.

[0213] Table 20. Quality control sensitivity, accuracy and precision of the characteristic peptide segment QLTLLESDLYR

[0214]

[0215] *Out of the acceptance range, not included in the mean calculation

[0216] The above results show that the method has been verified, and the indicators such as linearity, accuracy, precision and stability meet the acceptance criteria, and the method verification is qualified, which can be used for sample detection.

[0217] Example 3

[0218] In this embodiment, the detection method of the content of SOS1 protein screened in Example 1 is used to detect mouse tumor samples.

[0219] According to the optimized sample pretreatment conditions in Example 1, the CDX model mouse tumor samples are treated, and the confirmed liquid chromatography parameters are used for injection detection on the liquid chromatography-mass spectrometry instrument. The data are collected by MassLynx V4.2, and the chromatographic peaks of the analytes (characteristic peptide segments of SOS1 protein) and internal standards (stable isotope markers of characteristic peptide segments) in each MRM channel are integrated to obtain the peak area and peak area ratio. The peak area ratio of the analyte to the internal standard is taken as the vertical coordinate (Y axis), and the known concentration of the analyte is taken as the horizontal coordinate (X axis), and a standard curve regression equation is obtained by linear fitting (least squares method). The peak area ratio of the characteristic peptide segment of SOS1 protein in the detection sample to the peak area of the stable isotope marker internal standard peptide segment is substituted into the regression equation of the standard curve, and the concentration of the analyte in the detection sample can be calculated, realizing the absolute quantification of the characteristic peptide segment. The concentration of the characteristic peptide segment is converted into the concentration of SOS1 protein through the molecular weight information of the characteristic peptide segment, so as to realize the absolute quantification of the content of SOS1 protein in the detection sample. The sample detection can also not use standard curve samples, and different types of samples can be directly analyzed and detected, and the peak area ratio is compared to realize relative quantification.

[0220] After obtaining the relative concentration information of them, corresponding mathematical processing, plotting or statistical analysis can be performed to obtain the correlation of SOS1 among different samples. The absolute quantitative value of SOS1 protein in the test sample is shown in the following table. Figures 17 to 23 The following is a representative chromatogram.

[0221] Table 21: Quantitative concentration of characteristic peptide segment 3 and its converted SOS1 protein concentration data table

[0222]

[0223] Table 22: Quantitative concentration of characteristic peptide segment 4 and its converted SOS1 protein concentration data table

[0224]

[0225] Table 23: Comparison table of SOS1 protein concentration data quantified using characteristic peptide segment 3 and peptide segment 4

[0226]

[0227] The average concentration of SOS1 protein obtained by using the characteristic peptide segment FEIPEPEPTEADR for quantitative detection of the test sample is 0.167 fmol / μg, and the CV is 5.8%; the average concentration of SOS1 protein obtained by using the characteristic peptide segment QLTLLESDLYR for quantitative detection of the test sample is 0.190 fmol / μg, and the CV is 12.5%; the difference value of SOS1 protein concentration quantified by selecting the two characteristic peptide segments FEIPEPEPTEADR and QLTLLESDLYR is 12.8%.

[0228] The above data results show that the detection method of the present SOS1 protein has good accuracy and precision. The method simultaneously uses two characteristic peptide segments for detection, which mutually confirm each other and are indispensable. Only when the detection results of the two characteristic peptide segments have a difference within an acceptable range (difference%<30%), the analysis result is valid, and the obtained quantitative result has reliability.

[0229] The protection scope of the present application is not limited to the above embodiments. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.

Claims

1. A method for detecting the content of SOS1 protein, characterized by, The detection method comprises the following steps: (1) sample pretreatment: trypsin enzyme hydrolysis is performed on the biological sample to be measured to obtain an enzyme hydrolysis product containing a characteristic peptide segment of SOS1 protein, and the amino acid sequence of the characteristic peptide segment is shown in SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 3: FEIPEPEPTEADR; SEQ ID NO: 4: QLTLLESDLYR; (2) internal standard addition: a characteristic peptide segment of SOS1 protein labeled with stable isotopes with a known concentration is added to the enzyme hydrolysis product as an internal standard; (3) liquid chromatography-tandem mass spectrometry detection: the signal values of the characteristic peptide segment and the internal standard peptide segment are detected by liquid chromatography-tandem mass spectrometry; (4) quantitative analysis: the peak area ratio is obtained by integral processing, and then the relative content of SOS1 protein in the sample can be calculated; the peak area ratio is substituted into the linear equation of the sample regression fitting of the calibration curve, and then the absolute content of SOS1 protein in the sample can be calculated; The conditions of the liquid chromatography are as follows: Chromatographic column: octadecylsilane bonded high-strength silica gel particle chromatographic column; Mobile phase: A phase is a water solution containing 0.095%-0.105% formic acid, and B phase is a mixed solution of methanol: acetonitrile (19-21):(76-84) containing 0.095%-0.105% formic acid; The liquid chromatography separation is performed according to the following gradient elution conditions: 0.00-0.80 min, the proportion of B phase is 14.25%-15.75%; 0.80-3.00 min, the proportion of B phase is increased from 14.25% to 15.75% to 19.95%-22.05%; 3.00-6.00 min, the proportion of B phase is increased from 19.95%-22.05% to 21.85%-24.15%; 6.00-12.00 min, the proportion of B phase is kept at 21.85%-24.15%; 12.00-13.00 min, the proportion of B phase is increased from 21.85%-24.15% to 93.1%-100%; 13.00-13.80 min, the proportion of B phase is kept at 93.1%-100%.

2. The method of claim 1, wherein the SOS1 protein content is detected by an antibody. In the liquid chromatography, the chromatographic column is an ACQUITY UPLC Premier HSS T3 chromatographic column, the packing particle size is 1.8 μm, and the specification is 2.1 mm x 100 mm; The gradient elution conditions are as follows: 0.00 to 0.80 min, B phase ratio is 15.0%; 0.80 to 3.00 min, B phase ratio is raised from 15.0% to 21.0%; 3.00 to 6.00 min, B phase ratio is raised from 21.0% to 23.0%; 6.00 to 12.00 min, B phase ratio is 23.0%; 12.00 to 13.00 min, B phase ratio is raised from 23.0% to 98.0%; 13.00 to 13.80 min, B phase ratio is 98%; 13.80 to 15.00 min, B phase ratio is reduced from 98.0% to 15.0%.

3. The method of claim 1, wherein the SOS1 protein content is detected by an antibody against the SOS1 protein. In the liquid chromatography, the flow rate of the mobile phase is 0.38-0.42 mL / min; the column temperature is 66.5-73.5°C.

4. The method of claim 1, wherein the SOS1 protein content is detected by an antibody against the SOS1 protein. The sample pretreatment comprises the following steps: (1) mixing the biological sample to be tested with a lysis solution, homogenizing the tissue to obtain a tissue lysis solution sample; (2) taking the tissue lysis solution sample containing protein, heating and denaturing, adding a protein precipitant, freezing and centrifuging, discarding the supernatant, and collecting the precipitate; (3) resuspending the precipitate in an ammonium bicarbonate solution; (4) adding a reducing agent and incubating; (5) adding an alkylating agent and incubating; (6) adding trypsin and enzymatically digesting; (7) adding a termination reagent to terminate the enzymatic digestion reaction to obtain an enzymatic digestion product containing a characteristic peptide segment of the SOS1 protein.

5. The method for detecting the content of the SOS1 protein according to claim 4, wherein in step (1), the lysis solution comprises RIPA lysis solution, phosphatase inhibitor, protease inhibitor, metal chelator and phenylmethylsulfonyl fluoride; in step (2), the protein precipitant is methanol, and the amount added is equal to the volume of the tissue lysis solution sample; in step (3), the concentration of the ammonium bicarbonate solution is 9.5-10.5 mM; in step (4), the reducing agent is dithiothreitol, the concentration of the dithiothreitol is 95-105 mM, the incubation temperature is 57-63°C, and the incubation time is 28.5-31.5 minutes; in step (5), the alkylating agent is iodoacetamide, the concentration of the iodoacetamide is 190-210 mM, and the incubation conditions are 35-39°C, avoiding light for 28.5-31.5 minutes; in step (6), the mass ratio of trypsin to protein is 1:19-1:21, and the enzymatic digestion conditions are 23.75-26.25°C for 3.8-4.2 hours; in step (7), the termination reagent is formic acid, and the amount of formic acid added is 11-13 μL of 9-11% formic acid by volume.

6. The method for detecting the content of the SOS1 protein according to any one of claims 1-5, wherein the internal standard peptide comprises one or more amino acid residues replaced by a polypeptide with one or more isotopic labels and the same sequence as FEIPEPEPTEADR; and the molecular weight of the internal standard peptide and the characteristic peptide segment differs by at least 4 Da. ​ 7. The method of claim 6, wherein the SOS1 protein content is detected by Western blotting. The parent-daughter ion pair of the characteristic peptide segment FEIPEPEPTEADR is 765.6 / 570.9; the parent-daughter ion pair of the characteristic peptide segment QLTLLESDLYR is 676.1 / 782.3; and the internal standard peptide is 13 C3- 15 N-labeled FEIPEPEPTEADR, the isotopic 13 C3- 15 N-labeled on the alanine residue, and the parent-daughter ion pair of the internal standard peptide is 767.6 / 572.

7.

8. The method of claim 1, wherein the SOS1 protein content is detected by an antibody that specifically binds to the SOS1 protein. The mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry were as follows: cone voltage of 28.5–31.5 V, capillary voltage of 2850–3150 V, ion source temperature of 570–630 °C, desolvation gas flow rate of 1140–1260 L / Hr, gas flow rate of 142.5–157.5 L / Hr, and nebulizer pressure of 6.65–7.35 Bar.

9. The method of claim 1, wherein the SOS1 protein content is detected by an antibody that specifically binds to the SOS1 protein. The biological sample to be tested is a tumor tissue sample.

10. The method for detecting SOS1 protein content as described in any one of claims 1-9, used in the preparation of products for the detection of SOS1 protein, diagnosis of related diseases, disease monitoring, or prognosis assessment.

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