期刊
ANALYTICA CHIMICA ACTA
卷 1184, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.aca.2021.339022
关键词
Bidimensional liquid chromatography; Multiple heart-cutting; Isotope dilution; Targeted proteomics; Protein biomarkers; Glaucoma
资金
- Spanish Ministry of Science and Innovation [PGC2018-097961-B-I00]
- Plan de Ciencia, Tecnologia e Innovacion (PCTI) of Gobierno del Principado de Asturias, European FEDER
- FICYT [FC-GRUPIN-IDI/2018/00 0239]
- Principado de Asturias through Plan de Ciencia, Tecnologia e Innovacion 2013-2017
- Fondo de Investigacion Sanitaria (FIS)-Instituto de Salud Carlos III [PI13/01961]
The study evaluated the use of 2D-LC combined with isotope dilution tandem mass spectrometry for the direct analysis of tryptic digests of serum samples, demonstrating a robust quantification method for potential candidate biomarkers. The results showed high accuracy, reproducibility, and satisfactory outcomes using this approach for the quantification of target peptides.
We evaluate here the combination of two-dimensional liquid chromatography (2D-LC) in the multiple heart cutting mode and isotope dilution tandem mass spectrometry for the direct analysis of tryptic digests of serum samples. As a proof of concept, we attempt the quantification of proteotypic peptides of Apolipoprotein AIV (APOA4), Complement C3 (C3) and Vitronectin (VTN) which have been previously identified as potential candidate biomarkers of glaucoma. Using this 2D-LC strategy, analyte enrichment steps are avoided and the sample preparation involved after enzymatic digestion amounted to a simple centrifugation, evaporation of the supernatant and reconstitution in the 1D mobile phase. A mobile phase not compatible with the ESI source (10 mM KH2PO4 at pH 2.7) was used in the first dimension as it provided a satisfactory chromatographic resolution of the peptides and a high buffering capacity avoiding changes in retention times when analyzing complex matrices like human serum. We also demonstrate that using coeluting labelled analogues of the target peptides, protein concentrations were not affected by slight retention time shifts affecting the amount of target peptides transferred to the second dimension. Satisfactory results were obtained when analyzing fortified serum samples (re-coveries from 98 to 113%). Precisions in the range of 1-9% RSD were obtained when replicating the analysis of a pooled serum sample. The comparative analysis of serum samples from n = 94 control subjects and n = 91 patients diagnosed with primary open-angle glaucoma did not show significant differences in the APOA4, VTN and C3 concentrations in contrast with previous studies using immunoassays. (C) 2021 The Author(s). Published by Elsevier B.V.
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