Application of 73 histidine methylation of serum actin as marker in preparation of reagent for assisting early prognosis evaluation of sepsis

Abstract

The invention provides application of 73rd histidine methylation of serum actin as a marker in preparation of a reagent for assisting early prognosis evaluation of sepsis, and belongs to the technical field of biomarkers. Tests show that the 73rd site histidine of serum actin is subjected to methylation modification, and the differences among a Control group, a Survivor group and a Non Survivor group are remarkable; the secondary spectrum of the target peptide fragment shows that the peptide spectrum matching effect of the modified peptide fragment is good, and the modification identification is credible; and the area under the ROC curve and the 95% confidence interval are 73.56% (60.17%-82.68%). Under the optimal cutoff value of 30.29, the sensitivity is 0.792, the specificity is 0.639, the positive predicted value is 0.656, the negative predicted value is 0.780, the positive likelihood ratio is 2.198, and the negative likelihood ratio is 0.325. Therefore, the 73 histidine methylation of serum actin is an independent predictive factor of sepsis prognosis, can be used as a reliable and potential biomarker for early prognosis evaluation of sepsis, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomarker technology, and in particular to the application of serum actin methylation at histidine position 73 as a biomarker in the preparation of reagents to aid in the early prognostic assessment of sepsis. Background Technology

[0002] Sepsis is a common complication following severe infection, severe trauma, burns, shock, and surgery, and can lead to septic shock and multiple organ dysfunction syndrome (MODS). It has become one of the leading causes of death among critically ill patients, characterized by high morbidity, high mortality, and high treatment costs.

[0003] Actin is a highly conserved, multifunctional protein that forms microfilaments and is present in almost all eukaryotic cells. In recent years, actin has attracted widespread attention due to its important roles in cell apoptosis, proliferation, migration, phagocytosis, and immune regulation. Methylation of histidine at position 73 of mammalian actin has been known for fifty years, and this methylation plays a crucial role in controlling the cellular function of actin filaments and the release pathway of phosphate groups. Currently, the relationship between histidine methylation at position 73 and sepsis has not been reported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of serum actin methylation at histidine position 73 as a marker in the preparation of reagents for early prognostic assessment of sepsis.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of serum actin methylation at histidine position 73 as a biomarker in the preparation of reagents to aid in the early prognostic assessment of sepsis. If the signal intensity of serum actin methylation at histidine position 73 is >1.538 × 10⁻⁶, the biomarker is considered effective. 9 This suggests a poor prognosis for patients with sepsis; if the signal intensity of histidine methylation at position 73 of serum actin is <1.538 × 10⁻⁶, it indicates a poor prognosis. 9 This suggests that patients with sepsis have a good prognosis.

[0006] Preferably, the detection method for serum actin histidine methylation at position 73 is a rapid mass spectrometry detection, including non-targeted detection and parallel reaction monitoring (PRM) targeted detection. It can also be applied to routine biochemical detections such as enzyme-linked immunosorbent assays using specific antibodies.

[0007] Preferably, the reagents include protein precipitation reagents, protein oxidation inhibitors, enzymatic hydrolysis reagents, peptide desalting reagents, and liquid chromatography mobile phases.

[0008] Compared with existing technologies, this invention has the following beneficial effects: This invention provides the application of serum actin methylation at position 73 (histidine) as a biomarker in the preparation of reagents to aid in the early prognostic assessment of sepsis. Statistical analysis of serum samples after pretreatment showed that the methylation modification at position 73 significantly differed among the Control, Survivor, and Nonsurvivor groups. Secondary peptide spectroscopy of the target peptides indicated good peptide match, making the modification identification reliable. ROC curves showed a sensitivity of 75.4% and a specificity of 67.2%. Therefore, serum actin methylation at position 73 (histidine) can serve as a reliable and promising biomarker for the prognosis of sepsis, demonstrating excellent value in the early prognostic assessment of the disease. Attached Figure Description

[0009] Figure 1 The association between the abundance of histidine methylated peptide at position 73 of actin and the prognosis of sepsis was investigated, including (A) the intergroup distribution of normalized histidine methylated peptide abundance at position 73 of actin; (B) the intergroup distribution of histidine methylated peptide abundance at position 73 of actin normalized to β-actin abundance; and (C) the ROC curve for assessing the 14-day prognosis of sepsis based on the normalized histidine methylated peptide abundance at position 73 of actin.

[0010] Figure 2 The association between the abundance of actin histidine methylated peptide at position 73 and sepsis survival time was investigated, including (A) a correlation analysis of the abundance of prognostic-related modification features and the survival time of sepsis patients; and (B) a correlation analysis of the abundance of actin histidine methylated peptide at position 73 and the survival time of sepsis patients.

[0011] Figure 3 Survival curves for prognostic stratification of sepsis using the actin methylated peptide at histidine position 73.

[0012] Figure 4 To compare the PRM spectra of the synthesized standard peptide with those of the standard actin histidine methylated peptide at position 73, the cosine similarity was 0.967, demonstrating a very high degree of matching and supporting the application of the PRM method for biomarker quantification.

[0013] Figure 5 This study aimed to validate the prevalence-reduction (PRR) of the methylated peptide at histidine position 73 of actin. (A) Abundance distribution of the methylated peptide at histidine position 73 of actin across groups; (B) ROC curves for early assessment of sepsis prognosis based on the abundance of the methylated peptide at histidine position 73 of actin.

[0014] Figure 6Structural equation modeling of the abundance of histidine methylated peptide at actin position 73 and the concentrations of β-actin and F-actin, as well as the prognosis of sepsis. Detailed Implementation

[0015] This invention provides an application of a reagent combination for detecting histidine methylation at position 73 of actin in the preparation of a reagent for early prognostic assessment of sepsis; the reagent combination includes a protein precipitation reagent, a protein oxidation inhibitor, an enzymatic digestion reagent, a peptide desalting reagent, and a liquid chromatography mobile phase; If the signal intensity of serum actin methylation is >1.538×10 9 This suggests a poor prognosis for patients with sepsis; if the signal intensity of serum actin methylation is <1.538×10, it indicates a poor prognosis. 9 This indicates a good prognosis for patients with sepsis. In this invention, the area under the curve corresponding to the parent ion peak response value in the signal intensity mass spectrometry detection is defined as...

[0016] In this invention, the method for detecting the degree of methylation of histidine at position 73 of actin in the serum sample is rapid mass spectrometry detection.

[0017] In this invention, the protein precipitation reagent is preferably ammonium bicarbonate and acetone.

[0018] In this invention, the protein oxidation inhibitor is preferably dithiothreitol.

[0019] The preferred protein digestive enzymes used in the mass spectrometry pretreatment of this invention are Trypsin(semi)[KR|P]; Trypsin / p[KR|-]; TrypsinK[K|P]; TrypsinR[R|P]; ChymoTrypsin[FWYL|P]; ArgC[R|P]; AspN[-|D]n-term; Clostripain[R|-]; CNBr[M|P]; Elastase[GVLIA|P]; Formic Acid[D|P]; GluC[DE|P] ;GluCbicarb[E|P];Iodosobenzoate[W|-];LysC[K|P];LysC / P[K|-];LysN[-|K]n-term;LysNpromisc[-|KASR]n-term ; PepsinA[FL|P]; Proteinendopeptidase[P|-]; Staphprotease[E|-]; Trypsin-CNBr[KRM|P]; Trypsin-GluC[DEKR|P].

[0020] In this invention, the polypeptide desalting reagent is preferably formic acid (FA) and acetonitrile.

[0021] In this invention, the liquid chromatography mobile phase includes mobile phase A and mobile phase B; mobile phase A is preferably an aqueous solution of formic acid, and mobile phase B is preferably an aqueous solution of acetonitrile containing formic acid.

[0022] In this invention, specific antibodies can also be used for the quantitative detection of biomarkers in routine biochemical assays such as enzyme-linked immunosorbent assays (ELISA). Specifically, based on currently used methods for preparing specifically modified antibodies (synthesizing the peptide KYPIEHGIVTN corresponding to amino acids 68-78 of β-actin, methylating it, purifying it by chromatography, and binding it to KLH as an antigen to immunize rabbits, using the corresponding unmodified peptide as a negative control, and purifying the immobilized antigen peptide to prepare a specific antibody), the biomarker can be quantitatively detected using an ELISA.

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1

[0025] I. Serum Collection

[0026] A total of 164 samples were used, including 47 serum samples from healthy individuals (Control group), 64 samples from sepsis survivors (Survivor group), and 53 sepsis patients who died within 14 days (Nonsurvivor group).

[0027] Normal human serum samples: collected from age- and sex-matched patients undergoing physical examinations at the Physical Examination Center of the First Affiliated Hospital of Zhengzhou University.

[0028] Sample of sepsis survivors: Patients hospitalized in the General ICU of the First Affiliated Hospital of Zhengzhou University between October 2019 and August 2020.

[0029] Diagnostic criteria: (1) Meets the Sepsis-3.0 diagnostic criteria set by the American Society of Critical Care Medicine / European Society of Critical Care Medicine: a. Suspected or confirmed infection: diagnosis by a clinician; b. Evidence of acute organ dysfunction: for patients without a history of chronic organ dysfunction, assume a baseline SOFA score of 0 and a SOFA score ≥ 2; for patients with a history of chronic organ dysfunction, the SOFA score should be determined based on the baseline condition and the SOFA score should be ≥ 2 points higher than the baseline; (2) Sepsis is diagnosed within 48 hours of admission.

[0030] Exclusion criteria: (1) Age less than 18 years or older than 80 years; (2) Pregnancy or breastfeeding; (3) New York Heart Association grade 4 congestive heart failure, non-infectious cardiogenic shock, or uncontrolled acute blood loss; (4) Advanced pulmonary fibrosis or having received non-invasive ventilation prior to study enrollment; (5) Recipient of solid organ or bone marrow transplant; (6) Received cardiopulmonary resuscitation within 72 hours prior to study enrollment; (7) Neutrophil count less than 0.5 × 10⁻⁶. 9 / L (excluding those secondary to sepsis); (8) known positive for human immunodeficiency virus serology; (9) malignant tumor with a projected survival of less than 3 months; (10) chronic renal failure stage V; (11) the patient's serum sample shows obvious hemolysis or chylous blood.

[0031] II. Serum Sample Pretreatment

[0032] Add 190 μL of 25 mM ammonium bicarbonate to 10 μL of serum sample, then add 1000 μL of pre-cooled acetone. Vortex for 20 seconds, then incubate at -40°C for 1 hour to precipitate proteins. Centrifuge at 10,000g at 4°C for 10 minutes, and carefully remove the supernatant.

[0033] Add 600 μL of 25 mM ammonium bicarbonate to the protein precipitate to dissolve it, and determine the protein concentration using BCA reagent. Take out the protein solution containing 100 μg of protein, and then dilute it to 200 μL with 25 mM ammonium bicarbonate. At this point, the protein concentration is 0.5 μg / μL.

[0034] Add 5 mM DTT (dithiothreitol) to 200 μl of protein solution, mix well, and incubate at 55 °C and 700 rpm for 30 min. Add trypsin (trypsin:protein = 1:50 w / w) and incubate at 37 °C and 700 rpm with shaking for 12 h. Add trypsin again (trypsin:protein = 1:100 w / w) and incubate at 37 °C and 700 rpm with shaking for 2 h.

[0035] Peptide desalting and elution drying: Buffer A: 0.1% FA (H2O); Buffer B: 70% ACN (H2O); FA is formic acid, ACN is acetonitrile. Add 500 μL of ACN to equilibrate the C18 desalting column and let it stand for 10 minutes. Add 1 mL of Buffer A to wash away residual ACN. Pass the sample through the desalting column, then equilibrate the desalting column twice with 500 μL of Buffer A. Elute the peptide with 50 μL of 70% ACN. After vacuum centrifugation to dry the eluent, add 100 μL of Buffer A to dissolve the peptide. Centrifuge at 20,000 g, 4 °C for 10 min, then transfer 15 μL to a sample vial for LC-MS analysis.

[0036] Separation was performed using an EASY-nLC 1200 liquid chromatography system, and detection was performed using an online Thermo QE HF-X mass spectrometer in DDA mode.

[0037] Parameters of liquid phase separation system: Pre-column: 150μm 3cm C18 column, 1.9μm Reprosil-Pur120; Column: 150 μm 25cm C18 column 1.9μm Reprosil-Pur120; Flow rate: 600 nl / min; Mobile phase A: 0.1% formic acid (water); Mobile phase B: 0.1% formic acid + 80% acetonitrile (water); Elution gradient: 4%-7% B for 1 min, 7%-25% B for 94 min, 25%-40% B for 16 min, 40%-100% B for 5 min, 100% B for 4 min; Loading volume: 5 μl.

[0038] Mass spectrometry detection parameters: Scan range: 150-2000 m / z; Data acquisition mode: DDA acquisition method, top 20 fragment maps; MS1 resolution (200m / z): 60,000; MS1 AGCtarget: 3e6, maxIT: 50ms; MS2 resolution (200m / z): 15,000; MS2 AGCtarget: 5e4, maxIT: 45ms; Normalized collision energy (NCE): 28%; isolation window: 1.6 m / z.

[0039] After the detection is completed, mass spectrometry data are obtained.

[0040] II. Data Analysis of Histidine Methylation at Actin Position 73

[0041] 1. Data statistical analysis

[0042] The data obtained were analyzed using R 4.3.1 statistical software. Wilcoxon tests were used for intergroup comparisons. Feature selection was employed to identify the most important characteristic modifications for the survival group and the group that died within 14 days. The data analysis results are as follows: Figure 1 As shown. By Figure 1The methylation modification of histidine at position 73 was significantly different among the Control, Survivor, and Nonsurvivor groups (P<0.001), and this change remained significant after adjustment based on β-actin protein abundance. This indicates that this change is statistically highly significant between the serum of sepsis patients and the control group, and is associated with the occurrence of sepsis and poor prognosis. ROC analysis showed that the area under the ROC curve and the 95% confidence interval for the actin methylated peptide to identify the 14-day prognosis of sepsis patients were 73.56% (60.17%-82.68%). At the optimal cutoff value of 30.29, the sensitivity was 0.792, the specificity was 0.639, the positive predictive value was 0.656, the negative predictive value was 0.780, the positive likelihood ratio was 2.198, and the negative likelihood ratio was 0.325. Conclusion Analysis: The area under the ROC curve (AUC) ranged between 1.0 and 0.5. When AUC > 0.5, the closer the AUC was to 1, the better the diagnostic accuracy. AUC values ​​between 0.5 and 0.7 showed lower accuracy, between 0.7 and 0.9 showed some accuracy, and above 0.9 showed high accuracy. These results indicate that using serum actin methylation at position 73 (histidine) as a biomarker can accurately distinguish between patients with good and poor prognoses in sepsis. Serum actin methylation at position 73 (histidine) can serve as a reliable and promising biomarker for sepsis prognosis and has good clinical diagnostic value for sepsis.

[0043] Spearman rank correlation analysis was performed on all modification features that showed persistently elevated levels in the Control-Sepsis-Nonsurvivor group and their correlation with survival time to identify the features most strongly associated with survival time. Actin methylation was found to be the most strongly associated feature. The correlation analysis results are as follows: Figure 2 As shown, the association between the abundance of actin histidine methylated peptide at position 73 and sepsis survival time is analyzed, including (A) the correlation analysis between the abundance of prognostic modification features and the survival time of sepsis patients; and (B) the correlation analysis between the abundance of actin histidine methylated peptide at position 73 and the survival time of sepsis patients. It is evident that among blood protein modification features, the abundance of actin histidine methylated peptide at position 73 shows the most significant negative correlation with sepsis survival time (Spearman's...). r =-0.254, P=0.014), suggesting that its abundance dynamics can be used for prognostic assessment of sepsis.

[0044] Survival curves of actin histidine methylation peptide at position 73 for prognostic stratification in sepsis ( Figure 3The KM curve showed that, based on the abundance of histidine methylated peptide at actin position 73, the survival rate of patients with an abundance higher than the median was significantly reduced (P<0.001).

[0045] 2. Secondary spectra of the target peptide

[0046] The secondary spectrum of the peptide YPIEH (+14.01565 Da)GIVTNWDDMEK containing histidine methylation modification at actin position 73 is shown below. Figure 4 As shown.

[0047] The original mass spectrometry data revealed the following information regarding the modifications: Protein to which the modification pertains: P60709 (Actin β); Position of the modification in actin: 73; Peptide to which the modification pertains: YPIEHGIVTNWDDMEK; Molecular weight of the modification: +14.01565 Da; Highest original peptide score: 23.778105 (a higher score generally indicates more reliable peptide identification, >1); Lowest false positive rate: 0 (a lower false positive rate generally indicates more reliable peptide identification, <0.01); Number of supporting spectra: 53 (in a single study, the more supporting spectra of modified peptides with a false positive rate <0.01, the higher the likelihood of the modification; generally, more than 10 spectra indicate high reliability). In conclusion, the peptide mapping of the modified peptides showed good results, and the modification identification is reliable.

[0048] The PRM spectrum of the standard actin histidine methylated peptide at position 73 was compared with that of the standard actin, and the cosine similarity was 0.967, which proved that the two were highly matched and supported the application of the PRM method for biomarker quantitative detection.

[0049] Example 2

[0050] A prognostic method for sepsis based on serum actin 73-methylhistidine as a biomarker

[0051] In this embodiment, the feasibility of a rapid mass spectrometry detection method for histidine methylation at position 73 of serum actin as a clinical diagnostic method was verified by PRM targeted detection.

[0052] The mass spectrometry data obtained in Example 1 were analyzed using Skyline software to obtain the chromatographic retention time of the target peptide YPIEH (+14.01565 Da) GIVTNWDDMEK. This retention time was combined with the monovalent and divalent precursor ions of the target peptide and their corresponding fragment ions to construct an ion list for PRM (see Table 1. Ion List. Note: Each precursor ion has multiple fragment ions, and each fragment ion can exist in multiple valence states. This patent only lists conventionally selected and relatively abundant monovalent and divalent fragment ions.)

[0053] Table 1. Monovalent and divalent precursor ions of the target peptide and their corresponding fragment ions

[0054] The validation cohort (which included blood samples from 80 ICU patients diagnosed with sepsis, of whom 39 died within 28 days (Nonsurvivor group) and 41 survived (Survivor group), with inclusion and exclusion criteria consistent with Example 1).

[0055] The collected serum samples were processed according to the "Serve Sample Pretreatment" procedure in Example 1. Separation was performed using an EASY-nLC1200 liquid chromatography system, and detection was performed using an online Thermo QE HF-X mass spectrometer.

[0056] The parameters for the liquid phase separation system are as follows: pre-column: 75 μm 2cm PepMap C18 3μm; Column: 75μm 15cm PepMap C18 2μm; Flow rate: 500 nl / min; Mobile phase A: 0.1% formic acid (water); Mobile phase B: 0.1% formic acid 80% acetonitrile (water); Elution gradient: 6%-25% B for 21 min, 25%-40% B for 4 min, 40%-100% B for 3 min, 100% B for 2 min; Sample loading volume: 3 μl.

[0057] Mass spectrometry detection parameters, data acquisition mode: PRM acquisition mode, top 20 fragment spectra; MS2 resolution (200m / z): 30,000; MS2 AGCtarget: 2e5, max IT: 70ms; Normalized collision energy (NCE): 28%; isolation window: 1.6 m / z.

[0058] Mass spectrometry parameters were adjusted to enable rapid sample detection according to Table 1. The mass spectrometry results were analyzed using software such as Skyline. The absolute amount of histidine methylation at position 73 of actin in the patient's serum, as determined by the software analysis, was used to predict the patient's prognosis.

[0059] The results are as follows Figure 5As shown, (A) the abundance distribution of actin methylated peptide at position 73 in each group; (B) the ROC curve of actin methylated peptide abundance for early prognosis of sepsis. PRM quantitative results showed that the early plasma abundance of actin methylated peptide at position 73 (after correction based on internal standard peptide) was significantly higher in the Nonsurvivor group than in the Survivor group. At the optimal cutoff value of 27.167, the sensitivity was 0.821, the specificity was 0.631, the positive predictive value was 0.681, the negative predictive value was 0.788, the positive likelihood ratio was 2.245, and the negative likelihood ratio was 0.283.

[0060] Example 3

[0061] Serum β-actin and F-actin concentrations were simultaneously quantified using enzyme-linked immunosorbent assay (ELISA). These concentrations, along with the SOFA score and serum lactate concentration—the most commonly used prognostic assessment methods for sepsis—were incorporated into a structural equation model (the model was constructed using the plspm package in R (v4.3.1)). The associations between the biomarkers and the independence of the abundance of actin histidine methylated peptide at position 73 as a prognostic biomarker for sepsis were evaluated. The results are as follows: Figure 6 The results show a significant causal relationship between the abundance of histidine methylated peptide at position 73 of actin and the prognosis of sepsis patients (β=0.387, P<0.001), independent of SOFA score and blood lactate concentration, and significantly better than serum β-actin concentration (β=0.268, P=0.016) and F-actin concentration (β=-0.054, P=0.623). This demonstrates that the abundance of histidine methylated peptide at position 73 of actin can serve as an independent predictor of sepsis prognosis.

[0062] As can be seen from the above embodiments, the present invention provides that the methylation of histidine at position 73 of actin can serve as a reliable and promising biomarker for the prognosis of sepsis, and has good clinical diagnostic value for sepsis.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of serum actin histidine methylation at position 73 as a biomarker in the preparation of reagents for early prognostic assessment of sepsis, characterized in that, If the signal intensity of histidine methylation at position 73 of serum actin is >1.538 × 10⁻⁶ 9 This suggests a poor prognosis for patients with sepsis; if the signal intensity of histidine methylation at position 73 of serum actin is <1.538 × 10⁻⁶, it indicates a poor prognosis. 9 This suggests that patients with sepsis have a good prognosis.

2. The application according to claim 1, characterized in that, The detection method for serum actin histidine methylation at position 73 is mass spectrometry or biochemical detection based on specific antibodies.

3. The application according to claim 1, characterized in that, The reagents include protein precipitation reagents, protein oxidation inhibitors, enzymatic hydrolysis reagents, peptide desalting reagents, and liquid chromatography mobile phases.

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