| Cardiovascular disease is a type of high-mortality diseases, besides relying on clinical symptoms and signs and other indicators, detecting various types of protein biomarkers of myocardial injury is also a very reliable method. In serum indicators of infection, the direct examination of some interleukins and tumor necrosis factor-loaded is a common way, but checking the acute phase response proteins can help early determination of coronary artery disease (CAB) of the lesion, help clinicians to take appropriate therapeutic measures. Acute phase protein (APR) is usually divided into two categories:one type of concentration with the degree of injury increases; another type of concentration with the degree of injury decreases. In recent years, the first category APR has been widespread concern, while the study of the second category APR has rarely been reported. In this paper, accurate determination of human serum albumin (hALB) and human transferrin (hTRF) help to determine the extent of myocardial injury, which can guide treatment and follow-up treatment in the diagnosis, and will play an important role in the field of heart disease, risk assessment and efficacy prognosis estimation.First, a mass balance method was established to measure moisture, ash and organic purity of two proteins and the fraction of hALB and hTRF were 0.861 g/g and 0.825 g/g, respectively. At the same time, an amino acid-based isotope dilution mass spectrometry accurate measurement of both proteins were established to determine the repeatability, reproducibility and accuracy of pure hALB and hTRF by optimizing protein hydrolysis time. The quantification result of hALB and hTRF were 0.853 g/g and 0.830 g/g, respectively. Besides that, the uncertainty of isotope dilution mass spectrometry was evaluated, uncertainty of the determination of the two proteins were both 1.7%(k= 2). Compared with mass balance method, the isotope dilution mass spectrometry is simple, high sensitive, high accuracy, reproducible well, and the quantitative results can be traceable to SI units, which can be used as the valuation method of pure protein standard material and the foundation work for the accurately determination of follow-up matrix protein.Secondly, an isotope dilution mass spectrometry based on TMT labeling techniques by labeling target protein was established to accurate quantitaive human serum hTRF. Firstly, hTRF and human serum were reduced and alkylated, and each was labeled with one TMT reagent, mixed the TMT labeled samples and digested to give a mixture of peptide segments by Glu-C. Then the digestion was separated at nanoliter liquid phase spectrometry and the different components will be collected on the target plate, then the target plate was analyzed by MALDI-TOF-MS/MS, the mass fraction will obtained by calculation of the ratios of the intensity of reporter ions of MS/MS spectrum. Secondly, during the analysis of the peptide mixture, three specific peptides were selected; the optimization result of the amount of labeled reagent was 64 g labeling reagent per gram of protein, the labeling efficiency of label reagents was improved to nearly 100%, the average apparent purity of reporter ions was corrected to 94.91%. Finally, the fraction of human serum hTRF was accurate quantitated, and the results was 2.08 mg/g. The research on spiked recoveries of human serum indicated that the recoveries scope of the method is 97.15% to 105.10%. Since the pure hTRF was used as standard, which was quantified by amino acid-based dilution mass spectrometry method, the quantitative results of serum hTRF were accurate, reliable and SI units traceable. While commercial ELISA kit was used to determine human serum hTRF content and the measurement results was 2.03 mg/g. The t-test compared with isotope dilution mass spectrometry was analyzed with p value of 0.1543, it was confirmed that the established method has the advantage of high accuracy, excellent repeatability for the accurate determination of different proteins in other matrix.Finally, a fully automated chemiluminescent Western blot method for the determination of human serum hTRF was established. The pure hTRF whose value was quantified by isotope dilution mass spectrometry was used as an external standard. A specific strong antibody HTF-14 was selected in a variety of antibodies, the optimization of antibody dilution was 1:25, the optimization of sample dilution was 1:20, and the sample should be denatured and reduced before analysis on Wes. The human serum hTRF was quantified by the peak area of target peak in chemiluminescence spectra with a result of 2.03 mg/g. Compared with commercial ELISA kits and isotope dilution mass spectrometry method by t-test (p>0.5), it was confirmed that the newly established method has the advantage of high accuracy and high sensitivity. Meanwhile, the effect of protein polymer and metabolic fragment of hTRF to detect the mass fraction of hTRF in human serum was researched. It is found that the effect was so small and the result has the advantage of excellent reproducibility, high accuracy, high sensitivity and excellent spiked recovery. In addition, as the external pure hTRF standard was traceable to SI units, the quantitative results of serum hTRF were also SI traceability, which laid the foundation for the immunoassay method of other protein biomarkers. |
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