| Spike glycoprotein of SARS-Co V-2 as a key step for the virus to invade the hosts is an important target for drug design and virus detection in the study of coronavirus.Up to now,China has got its nucleic acid and pseudo virus ribonucleic acid reference materials.However,there is still no reference material for the spike glycoprotein which has important function in SARS-Co V-2.The critical aspect of the development process of reference materials is the accurate quantification.At present,the main techniques for protein quantification are immunoassay,Surface Plasmon Resonance and Mass Spectrometry methods.Isotope dilution mass spectrometry（IDMS）is widely used for the quantification of Reference Materials since it is a fundamental method with the highest level of metrological properties and directly traceability to SI units.The aim of the assay was to establish a method for the accurate quantification of SARS-Co V-2 spike protein S1 subunit based on specific peptides,and to use this to develop the reference materials.The experiment steps are divided into 4 parts following:1)Firstly,we conduct qualitative analysis of the synthetic candidate（molecular weight,amino acid sequence,biological activity and purity）.2)Amino acid-isotope dilution mass spectrometry was chosen as the first quantification method,and the accuracy of the method was validated with reference material of BSA.3)The second approach is to quantify the candidates using selected specific peptides in combination with isotope dilution mass spectrometry after optimization of experimentally relevant parameters（buffer,addition times of enzyme and reaction time）.In addition,the feasibility of the method（specificity,matrix effect,precision and accuracy）was also investigated.Comparing whether there is a difference in quantitative results between the two methods.4)Finally,we investigate the homogeneity and stability of the Reference Materials candidates and fix the value of the reference materials candidates with the calculation of uncertainty.The experimental results indicate that the synthesized SARS-Co V-2 spike protein S1 subunit raw material has the correct protein structure and physiological activity that can be used as the object of study for quantitative analysis.The amino acid combined isotope dilution mass spectrometry method was verified to have the ability to quantify accurately.T6(GIYQTSNFR,GIYQTSNF(13C9,15N)R)was chosen as the specific peptide,and the protein was hydrolyzed in Tris buffer composed of 30%acetonitrile,10 m M calcium chloride,p H=8 for 48 h.The enzyme was added every 4 h for the first12 h and every 12 h for the later period.The candidates were quantified by isotope dilution mass spectrometry.Comparing the results of the two quantification methods in terms of equivalence indicates no difference,and the quantification method based on the specific peptides combined with isotope dilution mass spectrometry is feasible.The reference materials candidates are highly homogeneous,and the short-term stability data shows that they should not be left for more than 36 h at 4°C and degrade rapidly at 25°C.The candidates were not significantly degraded after freezing and thawing for three times.The long-term stability data shows that the shelf life of the reference materials candidates could be set to 36 months.Finally,we combine the two quantitative methods of amino acids and specific peptides to synthesize the uncertainty of the candidates.The final value of this reference materials candidates was measured as 1.008 mg/g with an uncertainty of 0.039 mg/g and an expansion factor k=2 with an expansion uncertainty of 0.078 mg/g.In this project,we developed the spike glycoprotein S1 subunit with good traceability and accurate quantitative value.The value of each test kit can be calibrated,and the results are compatible.It also provides quality control for the development of kits and recombinant protein vaccines to ensure life safety. |
