Utilization Of Liquid Chromatography-Tandem Mass Spectrometry For The Quantification Of Biological Macromolecules Taking Human Serum Transferrin As A Representative And Its Clinical Application

Liquid chromatography-tandem mass spectrometry (LC/MS/MS) is a fast andeffective technology based on the combination of liquid chromatography and tandemmass spectrometry, which could offer high throughput, good sensitivity, remarkablespecificity, low limit of quantification and less cost. It is now widely used in the areaof pharmaceutical, environmental and food detections. Recently, as massspectrometry is making a lot of progresses, this technology is now being utilized tothe detection of peptides and proteins in complex biological matrix.Nodularin-R is a hepatotoxic cyclic peptide that consists of5amino acids, whichbelongs to a kind of cyanotoxins and could lead to functional disturbance of the liverin both humans and animals. Cyanotoxins are produced by cyanobacteria, which havespread across China’s east coastline and into major Chinese lakes, and caused waterpollution, thus did harm to human body. However, most current methods are ill-suitedto precisely quantify nodularin-R in biological matrix. Therefore, it is vital to developa method to accurately quantify nodularin-R.Human transferrin (hTRF) is the principal iron-transporting protein in the body.Its main function is to bind circulating iron and transport it to a range of cell types.Therefore, the absolute quantification of hTRF is usually performed in clinical situations where ion metabolism is evaluated (e.g., anemia). In addition, a number ofstudies have indicated that hTRF in serum and other body fluids is a potentialbiomarker for the early detection of certain cancers. These findings indicated theimportance for developing an effective and accurate quantitative assay.As immunological and electrochemical technologies have limitations both inspecificity and reproducibility in the detection of these two kinds ofbiomacromolecules, LC/MS/MS assays were developed and validated for thedetermination of hTRF as well as nodularin-R in biological matrix (mainly humanserum).In the nodularin-R quantitative analysis, a stable isotope-labeled nodularin-Rwas selected as an internal standard. Samples were processed with solid phaseextraction (SPE) cleanup and enrichment. A method validation was performed. Inaddition, this assay was applied to the quantification of nodualrin-R in clinicalsamples and compared with a commercially available ELISA.In the assay of hTRF quantification, we selected the tryptic peptide108EDPQTFYYAVAVVK121as the surrogate analyte for quantification and used astable isotope-labeled synthetic peptide with this sequence as an internal standard.Sample cleanup and enrichment were achieved using SPE. Afterwards, this assay wasapplied to the quantitative analysis of hTRF in clinical samples and compared with acommercially available immunoturbidimetric assay.The nodularin-R quantification results showed that the validated calibrationrange was from0.50to100.0ng/mL. The intra-and inter-day precisions were less than6.0%and9.8%, respectively. The accuracy at the lower limit of quantification(LLOQ) was2.0%, while for other quality control (QC) levels, it was3.4%. Thecomparison between LC/MS/MS and ELISA indicated that the absoluteconcentrations of nodularin-R determined by the LC/MS/MS assay were significantlylower than those measured using a commercial immunoassay. Unfortunately, there isa poor correlation between LC/MS/MS and ELISA (y=1.09x+0.847，r=0.5276(p>0.05)), it may be explained by the limitations of specificity of ELISA, whichcould only detect a total amount of nodularins, but not nodularin-R alone.The hTRF quantification results showed that the validated calibration range wasfrom500to5000ng/mL. The intra-and inter-day precisions were less than4.9%and9.0%, respectively. The accuracy for the QC samples was less than5.4%. Therecoveries of low, middle and high QC samples were103.0%,82.2%and98.1%,respectively. Finally, we successfully used this assay to quantify hTRF in clinicalsamples. The obtained values were assessed by independently measuring hTRF in thesame samples using a commercially available immunoturbidimetric assay. As a result,the absolute concentrations determined by the LC/MS/MS assay compared well withthose obtained with the immunoturbidimetric method (y=1.194x-0.457, r=0.9096,p <0.05).In conclusion, LC/MS/MS assay showed its advantages as followings: moreeffective, less cost, high sensitivity, better selectivity, lower limit of quantification(LOQ) and especially an offer of more reliable data when analyzing samples withlower concentrations and in more complicated matrices. Moreover, it was also proved to be an effective quantification technology for clinical samples.