Apparatus and methods to correlate spike protein urinary metabolism with body health

Abstract

The present invention relates to a novel apparatus and method for evaluating urinary spike proteins and isoforms, specifically focusing on the detection of unique, unsaturated peptide species generated by trypsin digestion of spike proteins. In particular, the invention relates to a method for evaluating urinary spike proteins, isoforms and post-translational modifications thereof and their association with protein spike sequences and their variants, the method comprising the following steps in sequence: a) trypsin digestion of a urinary sample of a subject; b) analyzing the digested sample of step a) using Liquid Chromatography-Mass Spectrometry (LC-MS) in an alternating tandem mass spectrometry (MS / MS) analysis and acquiring data in autofragmentation mode (data-dependent scan) so that the most abundant ions in the spectra of the chromatographic analysis are progressively fragmented; c) processing the data acquired at step b) to correlate peptide modifications in terms of post-translational modifications such as phosphorylation or glycosylation and chemical modifications such as peptide unsaturation or reduction.

Description

[0001]APPARATUS AND METHODS TO CORRELATE SPIKE PROTEIN URINARY METABOLISM WITH BODY HEALTH Description The present invention relates to a novel apparatus and method for evaluating urinary spike proteins and isoforms, specifically focusing on the detection of unique, unsaturated peptide species generated by trypsin digestion of spike proteins. Background art The detection of spike proteins in biological fluids has been a subject of intense research in recent years, with a particular focus on identifying a reliable method for detecting these proteins in urine. Various techniques, including enzyme-linked immunosorbent assay (ELISA) and mass spectrometry, have been developed to detect and quantify spike proteins in biological fluids. However, despite these efforts, no existing method has successfully detected spike proteins in urine and reliably associated them with the subject's health status. ELISA is a widely used method for the detection and quantification of proteins in biological fluids, but it has limitations in terms of sensitivity and specificity, particularly when applied to urine samples. Additionally, it can be affected by interference from other substances present in the sample. Mass spectrometry is a powerful analytical tool used for identifying and quantifying proteins in complex biological samples. Its sensitivity and specificity make it a valuable tool for diagnosing and monitoring various diseases, including metabolic disorders. However, its widespread use is limited by the need for sophisticated instrumentation and expertise in data analysis, as well as the complexity of sample preparation and the expense of mass spectrometers. Furthermore, mass spectrometry data analysis can be challenging and requires specialized software and expertise in data interpretation. The data generated from mass spectrometry experiments can be complex and often necessitate extensive bioinformatic analysis to identify and quantify proteins of interest. For these reasons, at our knowledge, this technique has not been used yet for detecting spike proteins in urine. To address the current lack of a reliable method for detecting spike proteins in urine, the present inventor investigated a novel approach based on trypsin digestion of urine samples, followed by analysis using liquid chromatography - mass spectrometry and a dedicated bioinformatic approach for detecting protein isoforms. This innovative method offers numerous advantages over ELISA, including increased sensitivity and specificity for detecting spike proteins in urine at low concentrations, and the ability to identify protein isoforms and post-translational modifications, which may be indicative of the subject's health status. Summary of the invention It is therefore an object of the present invention an apparatus and a method as outlined in the annexed claims, the definitions of which form an integral part of the present description. The present invention is an apparatus and method for evaluating urinary spike proteins and isoforms and their association with host metabolites. The method focuses on detecting unsaturated peptides that possess a different number of proton species, ranging from 1 to 9, which is the basis for the efficiency of the approach. This non-invasive and efficient method enables the detection and monitoring of metabolic disorders, particularly liver disease, a significant cause of morbidity and mortality worldwide. The present invention overcomes previous limitations by utilizing a novel approach involving trypsin digestion of urinary samples, followed by liquid chromatography mass spectrometric analysis and a dedicated bioinformatic approach for detecting spike protein isoforms, correlating their expression to the subject's metabolic health status. The non-invasive nature of the present invention makes it highly attractive for routine monitoring of metabolic disorders, especially liver diseases induced by spike proteins and their metabolically induced variants. Additionally, the ability to detect spike protein isoforms and post-translational modifications allows for a more precise and accurate evaluation of a subject's metabolic health, ultimately aiding in the development of personalized treatment plans associated with circulating spike proteins. The apparatus required for this method includes instrumentation for sample preparation, analysis, and data processing. The equipment comprises a liquid chromatography system coupled with a mass spectrometer for separating and detecting proteins in the sample (Cristoni S, Bernardi LR. Expert Rev Proteomics. 2004 Dec;1(4):469-83. doi: 10.1586 / 14789450.1.4.469). The apparatus is designed to automate sample preparation and data analysis, providing a fast, efficient, and reproducible method for the non-invasive detection and monitoring of metabolic disorders. A first object of the invention is a method for evaluating urinary spike proteins, isoforms and post- translational modifications thereof and their association with protein spike sequences and their variants, the method comprising the following steps in sequence: a) trypsin digestion of a urinary sample of a subject; b) analyzing the digested sample of step a) using Liquid Chromatography-Mass Spectrometry (LC- MS) in an alternating tandem mass spectrometry (MS / MS) analysis and acquiring data in autofragmentation mode (data-dependent scan) so that the most abundant ions in the spectra of the chromatographic analysis are progressively fragmented; c) processing the data acquired at step b) to correlate peptide modifications in terms of post- translational modifications such as phosphorylation or glycosylation and chemical modifications such as peptide unsaturation or reduction Another object of the invention is a kit for the method of evaluating urinary spike proteins and isoforms and their association with host metabolites, comprising a trypsin solution at a concentration of 5 µg / mL or lower and an activation buffer solution containing NH4HCO3at a concentration of 50-200 mmol / L, wherein the trypsin solution and the buffer solution are preferably in separate containers. Still another object of the invention is a Liquid Chromatography-Mass Spectrometry (LC-MS) apparatus designed for the above defined method, comprising a data processing system wherein a software for performing steps c1) to c5) is running. Further characteristics and advantages of the present invention will become clearer from the description of some embodiments, made hereinafter for indicative and non-limiting purposes, with reference to the following figures: Figure 1 is a schematic view of an LC-MS apparatus structure and components; Figure 2 shows an example of a modified peptide MS / MS spectrum and characterization; Figure 3 shows an example of metabolism activity evaluation according to the invention; Figure 4 shows an example of database search associated to modified and unmodified spike and to potential metabolomic functionality score according to the invention. Detailed description of the invention The present invention encompasses an apparatus and method for evaluating urinary spike proteins and isoforms, and their association with host metabolites. The apparatus employed is a Liquid Chromatography Mass Spectrometer (LC-MS) capable of analyzing the sample in tandem mass spectrometry mode. The LC-MS is specifically designed to detect all protein spike sequences reported in Table 1 and its variants obtained through post-translational modifications and chemical modifications, such as unsaturated peptides with proton differences ranging from 1 to 9. Table 1 SEQ. Sequenza ID no. 1 DPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWR 2 FNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYR 3 SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTK 4 DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYR NFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFK WPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLK AGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPR SSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTK TSVDCTMYICGDSTECSNLLLQYGSFCTQLNR GYHLMSFPQSAPHGVVFLHVTYVPAQEK SYLTPGDSSSGWTAGAAAYYVGYLQPR VYSSANNCTFEYVSQPFLMDLEGK NHTSPDVDLGDISGINASVVNIQK DLPQGFSALEPLVDLPIGINITR YNENGTITDAVDCALDPLSETK ISNCVADYSVLYNSASFSTFK LPDDFTGCVIAWNSNNLDSK VCEFQFCNDPFLGVYYHK FDNPVLPFNDGVYFASTEK CVNFNFNGLTGTGVLTESNK DFGGFNFSQILPDPSKPSK FPNITNLCPFGEVFNATR QLSSNFGAISSVLNDILSR LNDLCFTNVYADSFVIR VVVLSFELLHAPATVCGPK LQDVVNQNAQALNTLVK EGVFVSNGTHWFVTQR FNGIGVTQNVLYENQK TQSLLIVNNATNVVIK LQSLQTYVTQQLIR NLNESLIDLQELGK TQLPPAYTNSFTR IQDSLSSTASALGK NFTTAPAICHDGK VYSTGSNVFQTR FDEDDSEPVLK QYGDCLGDIAAR LIANQFNSAIGK SFIEDLLFNK GWIFGTTLDSK VGGNYNYLYR NTQEVFAQVK ALTGIAVEQDK FLPFQQFGR FASVYAWNR FQTLLALHR GIYQTSNFR MSECVLGQSK DIADTTDAVR SWMESEFR VEAEVQIDR VTLADAGFIK VQPTESIVR SNLKPFER GCCSCGSCCK HTPINLVR QCVNLTTR QIAPGQTGK IADYNYK NIDGYFK CYGVSPTK ASANLAATK YEQYIK GVYYPDK DLICAQK SFTVEK LNEVAK EFVFK VDFCGK STNLVK EELDK AHFPR TFLLK SNIIR QGNFK GDEVR 77 LITGR 78 AAEIR 79 TPPIK 80 QIYK 81 LHYT 82 EIDR 83 IYSK The correlation between the found m / z values generated in the LC-MS analysis and the above sequences is made according to the conventional interpretation rules of a skilled expert. The LC-MS analysis is capable of detecting and monitoring urinary biomarkers correlated to the metabolism that leads to the production of spike protein metabolic variants form. The LC-MS analysis provides high sensitivity and specificity in detecting target peptides and enables the development of personalized treatment plans by identifying the specific metabolic disorders present in the patient and monitoring their progression over time. The method according to the invention comprises the following steps in sequence: a) trypsin digestion of a urinary sample of a subject; b) analyzing the digested sample of step a) using Liquid Chromatography-Mass Spectrometry (LC- MS) in an alternating tandem mass spectrometry (MS / MS) analysis and acquiring data in autofragmentation mode (data-dependent scan) so that the most abundant ions in the spectra of the chromatographic analysis are progressively fragmented; c) processing the data acquired at step b) to correlate peptide modifications in terms of post- translational modifications such as phosphorylation or glycosylation and chemical modifications such as peptide unsaturation or reduction. Step a) is based on a kit composed of a trypsin solution at a concentration of 5 µg / mL or lower and an activation buffer comprising a NH4HCO3 50-200 mmol / L, preferably 50-100 mmol / L solution. The kit may comprise the trypsin solution and the activation buffer solution in separate containers, and optionally suitable means for transferring metered amounts of these solutions into the sample. To prepare the urinary sample, an amount of the urine sample is mixed with an equal amount of the above trypsin solution and with an amount of NH4HCO3 buffer in a ratio of 4:1 to 6:1, preferably about 5:1, by volume with respect to the sample. The pH of the resulting solution should be in the range between 7 and 8.2. The sample undergoes digestion for 4-8 hours at about 37°C. Step a) of trypsin digestion ensures specific cleavage of proteins, facilitating the detection of unique peptide fragments. Step b) is performed with a Liquid Chromatography - Mass Spectrometer (LC-MS) system, which consists of an apparatus (A) composed of a liquid chromatographer (B) and a mass spectrometer (C) (see figure 2). The flow rate for the LC analysis is comprised between 0.1 ml / min and 0,3 ml / min, preferably about 0.2 ml / min, and the solvent gradient consists of two solvents: Solvent A (water + 0.2% formic acid) and Solvent B (CH3OH, methanol). According to a preferred embodiment, the gradient profile for the LC analysis is performed over a 15-minute period, with the following specific gradient steps: 0-2 minutes: B% (percentage of Solvent B)= 2 2-5 minutes: B%= 30 5-8 minutes: B%= 70 8-10 minutes: B%= 70 (maintaining the same level of Solvent B) 10-10.5 minutes: B%= 2 (rapid decrease in the level of Solvent B) 10.5-15 minutes: B%= 2 (maintaining the initial level of Solvent B for column re- equilibration). According to preferred embodiments, the mass spectrometric conditions of the LC-MS analysis are as follows. The data are acquired using an LTQ ion trap (e.g., Thermo Fisher, San Jose, USA) in data- dependent scan mode. An ESI (electrospray ionization) source is utilized for ionization, preferably with a spray voltage of 2750 V and a nebulizer gas of 50 amu (atomic mass unit). The full scan analysis is conducted in the m / z range of 400-1800, while the MS / MS scans are performed under dynamic range conditions. According to preferred embodiments, dynamic exclusion is implemented, which allows each peak to be fragmented twice and inserted into an exclusion list containing a maximum of 50 ions for 2 minutes. The collision gas is preferably helium (He), with a pressure of 1E-5 Torr. The collision energy can be set at 35% of its maximum value (5V peak-to- peak) and normalized with respect to the m / z ratio. In step c), a software specifically calibrated to detect all protein spike sequences reported in Figure 1, and its variants obtained through post- translational modifications and chemical modifications, such as the critical unsaturated peptides with proton differences between 1 and 9, can be used. Data Processing of step c) utilizes sophisticated software to correlate m / z values with post-translational modifications and disease states. The procedure for generating the comparison score and evaluating the degree of correlation between the modified spike peptides and hepatic and total body metabolite functionality is shown in Figures 4 and 5. The figure displays a statistical model that was utilized to develop a comparison score between the patient's LC-MS data and the control group's LC-MS data. The statistical model takes into account the level of modified peptides in the patient's urine sample and compares it to the levels found in the control group. This generates a comparison score that is used to evaluate the degree of correlation between modified spike peptides and hepatic and total body metabolite functionality. Thus, step c) comprises: c1) detecting m / z modified peptides in a subject’s sample; c2) determining punctual modifications and proton rearrangements of the modified peptides of the subject’s sample; c3) searching a database containing spectra of control and patient subjects that present circulating spike proteins and comparing the subject’s sample data with the database spectra to generate a comparison score between punctual modifications and diseases; c4) searching a database containing spectra of control and patient subjects that present circulating spike proteins and comparing the subject’s sample data with the database spectra to generate a comparison score between proton rearrangements and diseases; c5) evaluating the degree of correlation between modified spike peptides of the subject’s sample with hepatic and total body metabolite functionality on the base of the comparison score of step c3) and c4). Examples of publicly accessible databases used in step c3) are: - Human Metabolome Database (HMDB): used to correlate modified peptides with various metabolic disorders, providing a comprehensive and detailed database of observed metabolic changes; - PRIDE Archive: a public resource for proteomic data, used to compare spectrometric data obtained with control and patient samples, generating accurate and reliable comparison scores. The comparison score (CS) of steps c3) can be calculated by the formula CS = 1 / Sum|(Xi-Yi)| where Xi represents the intensity of the m / z ratios obtained in the MS / MS spectrum obtained by analyzing the peptides (MS / MS with and without proton rearrangement) and Yi those present in the MS / MS spectra database. The evaluation of step c) is based on a statistical model using a t-test between the level of expression of the peptides in healthy and unhealthy subjects. The invention also relates to a method of monitoring the progression of metabolic disorders in a subject, comprising performing steps a) to c) on subject samples at regular intervals and comparing the obtained LC-MS data to previously obtained data. The invention also allows to set a personalized treatment plan for a subject with a metabolic disorder, comprising performing steps a) to c) on a subject’s sample and utilizing the obtained LC-MS data to identify the specific metabolic disorder present in the subject. EXAMPLE 1 A urine sample from a patient with liver disease was analysed using the apparatus and method described in the present invention. The LC-MS system (Figure 2) utilized in this example consisted of an autosampler injector (A), a liquid chromatographer (B) and a mass spectrometer (C). 10 µL of the urine sample was mixed with 10 µL of a trypsin solution (I) and 50 µL of an activation buffer solution (ii) containing NH4HCO3. The sample was left to digest for 4 hours at 37°C and the digested solution was analysed through LC-MS alternated tandem mass spectrometric (MS / MS) analysis acquiring the data in autofragmentation mode (data dependent scan). The LC-MS data obtained from the analysis were then analysed using a dedicated software that is set to search for peptide modifications in terms of post- translational modification and chemical modification. SANIST Shift-Correlation software was used (Cristoni et al. SANIST: optimization of a technology for compound identification based on the European Union directive with applications in forensic, pharmaceutical and food analyses. J Mass Spectrom. 2017 Jan;52(1):16-21. doi: 10.1002 / jms.3895). This software has been obtained by modifying the Xtandem open source software (Muth T. et al. XTandem Parser: an open-source library to parse and analyse X!Tandem MS / MS search results. Proteomics 2010 Apr;10(7):1522- 4. doi: 10.1002 / pmic.200900759). Figure 3 shows the results obtained from the LC-MS analysis. The modified peptides correspond to the peaks at values 1035.2, 1233.1 and 1288.8, indicating the post-translational modifications that have been detected. The following interpretation is made: - Sequence: SEQ. ID No. 5 (NFYEPQIITTDNTFVSGNCD) - m / z Values: 1035.2, 1233.1, 1288.8 - Pathology Correlation: These m / z values correspond to post-translational modifications like phosphorylation and glycosylation, which are indicative of liver dysfunction. The peptide with m / z 1035.2 was identified as a key indicator for the metabolic disorder associated with reduced glutathione production, correlated specifically via the HMDB database. Similarly, the peptide with m / z 1233.1 was correlated with liver disorders using PRIDE Archive data. EXAMPLE 2 Similarly to EXAMPLE 1, a urine sample of another patient was analysed according to the inventive method to give the following interpretation: - Sequence: SEQ. ID No. 22 (FPNITNLCPFGEVFNATR) - m / z Values: 1500.4, 1600.6 - Pathology Correlation: The detection of these m / z values in urine samples can indicate altered protein metabolism associated with metabolic syndrome. EXAMPLE 3 - Correlation Between m / z and Sequences from Claim 2, Particularly Regarding Proton Rearrangements That Confer Selectivity to the Method The selectivity of the method is enhanced by the specific detection of proton rearrangements in the peptide sequences. This involves identifying unique m / z (mass-to-charge) ratios that correspond to specific spike protein sequences and their proton rearranged forms. For example, sequences listed in SEQ. ID No. 1 to SEQ. ID No. 83 can undergo proton rearrangements, leading to different m / z values. This allows for the discrimination between modified and unmodified peptides, thus providing a more accurate analysis. The proton rearrangement usually involves multiple of 2 Da with respect to the parent molecule considering even the multicharged state. For example, in SEQ. ID No. 1, proton rearrangements can create a series of detectable m / z values such as 646, 686, and others. These m / z values are then used to identify specific modifications related to metabolic conditions. EXAMPLE 4 - Broad Correlation of Sequence- Pathology Using Databases The correlation between sequences and pathologies is achieved through the use of comprehensive databases containing spectra from both control and patient samples, such as the databases described above. These databases allow for the comparison of detected m / z values with known profiles associated with various diseases. The comparison generates scores that indicate the likelihood of specific pathologies. The m / z values such as 646 and 686 are critical markers for specific metabolic activities and proton rearrangements. These values are indicative of particular peptide modifications that are often associated with metabolic disorders. A database search reveals that elevated levels of m / z 646 and 686 in the urine sample of a patient correlate with the presence of certain liver disease biomarkers. This correlation is supported by statistical analysis comparing the patient's data with those of healthy controls. Description of Significance: - m / z 646: May indicate a single proton rearrangement in a peptide sequence, often correlating with early-stage metabolic changes. - m / z 686: Represents multiple proton rearrangements, suggesting advanced modifications typically seen in severe metabolic dysfunctions. In a patient with liver disease, the detection of m / z 646 and 686 can signify different stages or types of metabolic impairment. These markers help in monitoring disease progression and tailoring personalized treatment plans. LC ANALYSIS The LC (liquid chromatography) part of the LC-MS (liquid chromatography-mass spectrometry) system is described in more detail. The flow rate for the LC analysis was set at 0.2 ml / min, and the solvent gradient consisted of two solvents: Solvent A (water + 0.2% formic acid) and Solvent B (CH3OH, methanol). The gradient profile for the LC analysis was performed over a 15-minute period, with the following specific gradient steps: 0-2 minutes: B% (percentage of Solvent B) = 2 2-5 minutes: B% = 30 5-8 minutes: B% = 70 8-10 minutes: B% = 70 (maintaining the same level of Solvent B) 10-10.5 minutes: B% = 2 (rapid decrease in the level of Solvent B) 10.5-15 minutes: B% = 2 (maintaining the initial level of Solvent B for column re- equilibration). The injection volume for the LC analysis was set at 10 µL. This detailed description of the LC part provides a clearer understanding of the gradient profile and solvent composition, which allows for more accurate and reproducible analysis of the urine samples. The mass spectrometric conditions of the LC-MS analysis are described in greater detail. The data were acquired using an LTQ ion trap (Thermo Fisher, San Jose, USA) in data-dependent scan mode. An ESI (electrospray ionization) source was utilized for ionization, with a spray voltage of 2750 V and a nebulizer gas of 50 amu. The full scan analysis was conducted in the m / z range of 400-1800, while the MS / MS scans were performed under dynamic range conditions. Dynamic exclusion was implemented, which allowed each peak to be fragmented twice and inserted into an exclusion list containing a maximum of 50 ions for 2 minutes. The collision gas used was helium (He), with a pressure of 1E-5 Torr. The collision energy was set at 35% of its maximum value (5V peak-to-peak) and normalized with respect to the m / z ratio. Based on the LC-MS data and the dedicated software analysis described above, the full spike characterization was obtained, including the identification of protein isoforms and post- translational modifications. The results showed that the patient had elevated levels of a specific spike isoform that is known to be associated with liver disease. This example demonstrates the efficacy of the present invention in the non-invasive detection and monitoring of spike protein associated metabolic disorders, particularly liver disease. The method is capable of detecting and monitoring urinary biomarkers of various metabolic disorders, providing a more comprehensive evaluation of the subject's metabolic health, and enabling the development of personalized treatment plans. Figure 3 reports the comparison score expressed in terms of log(e). Only the identification score with Log(e)<-3 are considered valid. In summary, the present invention offers a simple, efficient, and non-invasive method for detecting and monitoring urinary biomarkers of various metabolic disorders, including liver disease. The method relies on a kit that provides all the necessary components for preparing urinary samples for analysis using LC-MS and dedicated software capable of detecting protein isoforms and post-translational modifications. In particular, proton rearrangements can emphasize the uniqueness of the method. This specificity can underline the selectivity and accuracy in detecting disease- associated modifications in spike proteins. This innovative approach has the potential to revolutionize the field of diagnostic medicine by providing a fast, efficient, and non-invasive method for detecting and monitoring metabolic disorders connected to circulating spike protein, particularly liver disease, and for developing personalized treatment plans. It is evident that only some particular embodiments of the present invention have been described, to which the expert in the art will be able to make all those modifications necessary for its adaptation to particular applications, without however departing from the scope of protection of the present invention as defined in the attached claims.

Claims

Claims l. A method for evaluating urinary spike proteins, isoforms and post-translational modifications thereof and their association with protein spike sequences and their variants, the method comprising the following steps in sequence: a) trypsin digestion of a urinary sample of a subject; b) analyzing the digested sample of step a) using Liquid Chromatography-Mass Spectrometry (LC- MS) in an alternating tandem mass spectrometry (MS / MS) analysis and acquiring data in autofragmentation mode (data-dependent scan) so that the most abundant ions in the spectra of the chromatographic analysis are progressively fragmented; c) processing the data acquired at step b) to correlate peptide modifications in terms of post- translational modifications such as phosphorylation or glycosylation and chemical modifications such as peptide unsaturation or reduction.

2. The method of claim 1, wherein the urinary spike proteins, isoforms and post-translationalmodifications thereof and their association with protein spike sequences and their variants are selected from SEQ ID No. 1 to SEQ ID No. 83 as shown in the following table: SEQ. Sequenza ID no. 1 DPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWR 2 FNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYR 3 SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTK 4 DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYR 5 NFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFK 6 WPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLK 7 AGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPR 8 SSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTK 9 TSVDCTMYICGDSTECSNLLLQYGSFCTQLNR 10 GYHLMSFPQSAPHGVVFLHVTYVPAQEK 11 SYLTPGDSSSGWTAGAAAYYVGYLQPR 12 VYSSANNCTFEYVSQPFLMDLEGK 13 NHTSPDVDLGDISGINASVVNIQK 14 DLPQGFSALEPLVDLPIGINITR 15 YNENGTITDAVDCALDPLSETK 16 ISNCVADYSVLYNSASFSTFK 17 LPDDFTGCVIAWNSNNLDSKVCEFQFCNDPFLGVYYHK FDNPVLPFNDGVYFASTEK CVNFNFNGLTGTGVLTESNK DFGGFNFSQILPDPSKPSK FPNITNLCPFGEVFNATR QLSSNFGAISSVLNDILSR LNDLCFTNVYADSFVIR VVVLSFELLHAPATVCGPK LQDVVNQNAQALNTLVK EGVFVSNGTHWFVTQR FNGIGVTQNVLYENQK TQSLLIVNNATNVVIK LQSLQTYVTQQLIR NLNESLIDLQELGK TQLPPAYTNSFTR IQDSLSSTASALGK NFTTAPAICHDGK VYSTGSNVFQTR FDEDDSEPVLK QYGDCLGDIAAR LIANQFNSAIGK SFIEDLLFNK GWIFGTTLDSK VGGNYNYLYRNTQEVFAQVK ALTGIAVEQDK FLPFQQFGR FASVYAWNR FQTLLALHR GIYQTSNFR MSECVLGQSK DIADTTDAVR SWMESEFR VEAEVQIDR VTLADAGFIK VQPTESIVR SNLKPFER GCCSCGSCCK HTPINLVR QCVNLTTR QIAPGQTGK IADYNYK NIDGYFK CYGVSPTK ASANLAATK YEQYIK GVYYPDK DLICAQK66 SFTVEK 67 LNEVAK 68 EFVFK 69 VDFCGK 70 STNLVK 71 EELDK 72 AHFPR 73 TFLLK 74 SNIIR 75 QGNFK 76 GDEVR 77 LITGR 78 AAEIR 79 TPPIK 80 QIYK 81 LHYT 82 EIDR 83 IYSK 3. The method according to claim 1 or 2, wherein step a) is performed with a trypsin solution at a concentration of 5 µg / mL or lower and an activation buffer, the activation buffer being preferably a NH4HCO350-200 mmol buffer.

4. The method according to claim 3, wherein an amount of the urine sample is mixed with an equal amount of the trypsin solution and with an amount of NH4HCO3 buffer in a ratio of 4:1 to 6:1, preferably about 5:1, by volume with respect to the urine sample.

5. The method according to any one of claims 1 to 4, wherein step a) is conducted for 4-8 hours at about 37°C and at a pH in the range from 7 to 8.

2.

6. The method according to any one of claims 1 to 5, wherein step b) is performed with a flow rate for the LC analysis comprised between 0.1 ml / min and 0,3 ml / min, preferably about 0.2 ml / min, and a solvent gradient consisting of two solvents: Solvent A = water + 0.2% formic acid; and Solvent B = methanol.

7. The method according to claim 6, wherein the gradient profile for the LC analysis is performed over a 15-minute period, with the following specific gradient steps: 0-2 minutes: B% (percentage of Solvent B)= 2 2-5 minutes: B%= 30 5-8 minutes: B%= 70 8-10 minutes: B%= 70 (maintaining the same level of Solvent B)10-10.5 minutes: 2 (rapid decrease in the level of Solvent B) 10.5-15 minutes: B%= 2 (maintaining the initial level of Solvent B for column re-equilibration).

8. The method according to any one of claims 1 to 7, wherein the mass spectrometry analysis in step b) is performed in tandem mass spectrometry mode using a mass isolation of m / z 50 or lower and wherein, preferably, preferably the isolation windows is lower than m / z 20.

9. The method according to claim 8, wherein the mass spectrometric conditions of the LC-MS analysis are the following: - acquisition with an LTQ ion trap in data-dependent scan mode - ionization with an ESI (electrospray ionization) source, preferably with a spray voltage of 2750 V and a nebulizer gas of 50 amu (atomic mass unit) - full scan analysis in the m / z range of 400-1800, the MS / MS scans being performed under dynamic range conditions, preferably implementing dynamic exclusion wherein each peak is fragmented twice and inserted into an exclusion list containing a maximum of 50 ions for 2 minutes- preferably, collision gas = helium, with a pressure of 1E-5 Torr, the collision energy being set at 35% of its maximum value (5V peak-to-peak) and normalized with respect to the m / z ratio.

10. The method according to any one of claims 1 to 9, wherein step c) comprises: c1) detecting m / z modified peptides in a subject’s sample; c2) determining punctual modifications and proton rearrangements of the modified peptides of the subject’s sample; c3) searching a database containing spectra of control and patient subjects that present circulating spike proteins and comparing the subject’s sample data with the database spectra to generate a comparison score between punctual modifications and diseases; c4) searching a database containing spectra of control and patient subjects that present circulating spike proteins and comparing the subject’s sample data with the database spectra to generate a comparison score between proton rearrangements and diseases; c5) evaluating the degree of correlation between modified spike peptides of the subject’s samplewith hepatic and total body metabolite functionality on the base of the comparison score of steps c3) and c4).

11. The method of claim 10, wherein the comparison score (CS) of step c3) can be calculated by the formula CS =where Xi represents the intensity of the m / z ratios obtained in the MS / MS spectrum obtained by analysing the peptides (MS / MS with and without proton rearrangement) and Yi those present in the MS / MS spectra database.

12. The method according to any one of claims 1 to 11, wherein an evaluation of the association of the modified spike peptides with hepatic and total body metabolite functionality is performed with a statistical model using a t-test between the level of expression of the peptides in healthy and unhealthy subjects.

13. The method according to any one of claims 1 to 12, wherein the method comprises monitoring the progression of metabolic disorders in a subject, comprising performing steps a) to c) on subject samples at regular intervals and comparing the obtained LC-MS data to previously obtained data.

14. A kit for the method of evaluating urinary spike proteins and isoforms and their association withhost metabolites as defined in any one of claims 1 to 13, comprising a trypsin solution at a concentration of 5 µg / mL or lower and an activation buffer solution containing NH4HCO3 at a concentration of 50-200 mmol / L, wherein the trypsin solution and the buffer solution are preferably in separate containers.

15. A method of developing a personalized treatment plan for a subject with a metabolic disorder, comprising performing the method of any one of claims 1 to 12 and utilizing the obtained LC-MS data to identify the specific metabolic disorder present in the subject.

16. The method of claim 15, further comprising utilizing the comparison score generated in step c) to evaluate the degree of association of modified spike peptides with hepatic and total body metabolite functionality and developing a personalized treatment plan based on the evaluation.

17. A Liquid Chromatography-Mass Spectrometry (LC-MS) apparatus designed for the method of any one of claims 1 to 13, comprising a data processing system wherein a software for performing steps c1) to c5) is running.

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