Identification and quantitative analysis of human blood albumin based on characteristic peptide and liquid technology
Objective To screen species-specific peptide segments of human serum albumin and establish a HPLC-MS method for qualitative and quantitative analysis of human serum albumin based on this characteristic peptide.Methods After trypsin hydrolysis of the sample,it was analyzed using a nano HPLC orbiter fusion.The Proteome Discoverer mass spectrometry database was searched to screen for specific characteristic peptides of human serum albumin;And a liquid chromatography triple quadrupole mass spectrometry multiple reaction monitor(MRM)mode was used to establish and validate the specificity identification method and quantitative analysis method.The method used Waters ACQUITY UPLC BEH C18(50 mm×2.1 mm,1.7 μm)as the chromatographic column,0.1%formic acid aqueous solution as mobile phase A,and 0.1%formic acid acetonitrile solution as mobile phase B for gradient elution;The flow rate is 0.2 mL·min-1,and the qualitative and quantitative ion pairs are m/z 507.5→189.2 and m/z 507.5→132.1.Results The characteristic peptide with the amino acid sequence LVAASQALGL was selected as the detection peptide for human serum albumin.The detection limit of the method was 3.75 ng·mL-1,and the quantification limit was 2.5 ng·mL-1.The peptide had a good linear relationship within the concentration range of 12.5～100 ng·mL-1(r=0.9964);The RSD of precision,repeatability,and stability tests were all<5%,with a recovery rate of 92.3%to 99.7%and RSD of 2.9%(n=6).Conclusion The selected characteristic peptide segments of human serum albumin have strong specificity and can accurately identify the species source of human serum albumin;The established analytical method is accurate and reliable,and can be used for qualitative identification and quantitative analysis of human serum albumin.
Human albuminCharacteristic peptidesTrypsinEnzymatic hydrolysisHPLC-MS
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维普
