title: Absolute quantification of DcR3 and GDF15 from human serum by LC-ESI MS.
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 (15) N 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.
authors.affiliation: Institute of Biochemistry (Emil-Fischer-Centre), Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Ioana
authors.initials: I
authors.lastname: Lancrajan
authors.affiliation: Experimental Tumorpathology, Institute of Pathology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Regine
authors.initials: R
authors.lastname: Schneider-Stock
authors.affiliation: Division of Molecular and Experimental Surgery, University Medical Centre Erlangen, Erlangen, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Naschberger
authors.affiliation: Department of Surgery, University Medical Centre Erlangen, 91054, Erlangen.
authors.forename: Vera S
authors.initials: VS
authors.lastname: Schellerer
authors.affiliation: Division of Molecular and Experimental Surgery, University Medical Centre Erlangen, Erlangen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Stürzl
authors.affiliation: Institute of Biochemistry (Emil-Fischer-Centre), Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Enz
mesh_terms: D000595:Amino Acid Sequence; D015415:Biomarkers / Q000097:blood; D001798:Blood Proteins / Q000378:metabolism; D002850:Chromatography, Gel; D055436:Growth Differentiation Factor 15 / Q000097:blood* / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D057230:Limit of Detection; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011489:Protein Denaturation; D059748:Proteolysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053323:Receptors, Tumor Necrosis Factor, Member 6b / Q000097:blood* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D015415:Biomarkers; D001798:Blood Proteins; C526140:GDF15 protein, human; D055436:Growth Differentiation Factor 15; D012333:RNA, Messenger; D053323:Receptors, Tumor Necrosis Factor, Member 6b; C505911:TNFRSF6B protein, human
keywords: LC-ESI MS; absolute quantification; biomarker; low-abundance proteins; mass spectrometry; partial denaturation; proteomics; serum
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