Absolute quantification of DcR3 and GDF15 from human serum by LC‐ESI MS

Journal of Cellular and Molecular Medicine

Ioana Lancrajan¹  Regine Schneider-Stock³  Elisabeth Naschberger⁴  Vera S. Schellerer²  Michael Stürzl⁴ 

[1] Institute of Biochemistry (Emil-Fischer-Centre), Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany;Department of Surgery, University Medical Centre Erlangen, Erlangen;Experimental Tumorpathology, Institute of Pathology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany;Division of Molecular and Experimental Surgery, University Medical Centre Erlangen, Erlangen, Germany

关键词: absolute quantification;    serum;    biomarker;    low‐abundance proteins;    partial denaturation;    mass spectrometry;    LC‐ESI MS;    proteomics;   

DOI  :  10.1111/jcmm.12540

来源: Wiley

【 摘 要 】

Abstract

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high-abundant serum proteins by partial denaturation and enrichment of low-abundant biomarkers by size exclusion chromatography. The recovery of low-abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using ¹⁵N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody-based strategies, and offers the possibility of multiplexing. Our proof-of-principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.

【 授权许可】

CC BY   
© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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