title: Quantitative antibody-free LC-MS/MS analysis of sTRAIL in sputum and saliva at the sub-ng/mL level.
abstract: Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) induces apoptosis via the extrinsic death receptor pathway and may be a biomarker in the pathogenesis of a broad range of diseases. To investigate the role of sTRAIL in asthma, we developed a quantitative LC-MS/MS method with a lower limit of quantitation (LLOQ) of ≈3pM in induced sputum (174pg/mL) and saliva (198pg/mL) without the use of antibodies. sTRAIL was enriched by immobilized metal affinity chromatography (IMAC) solid-phase extraction (SPE) followed by tryptic digestion and subsequent enrichment of a signature peptide by strong cation exchange (SCX) SPE. The method was validated with respect to stability, accuracy and precision using the standard addition approach and fully metabolically (15)N-labelled hrTRAIL as internal standard. Our results indicate that it is possible to quantify cytokines like sTRAIL at the pM level by LC-MS/MS without the use of antibodies, which has, to our knowledge, never been shown before.
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wilffert
authors.affiliation: Department of Pathology, University of Pisa, Via Roma 55, 56126 Pisa, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Donzelli
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Angela
authors.initials: A
authors.lastname: Asselman
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Jos
authors.initials: J
authors.lastname: Hermans
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Natalia
authors.initials: N
authors.lastname: Govorukhina
authors.affiliation: Department of Pulmonology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
authors.forename: Nick H
authors.initials: NH
authors.lastname: Ten Hacken
authors.affiliation: Pharmaceutical Biology, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Quax
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands; PRA Health Sciences, Bioanalytical Laboratory, Amerikaweg 18, 9407 TK Assen, The Netherlands.
authors.forename: Nico C
authors.initials: NC
authors.lastname: van de Merbel
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. Electronic address: r.p.h.bischoff@rug.nl.
authors.forename: Rainer
authors.initials: R
authors.lastname: Bischoff
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D057230:Limit of Detection; D012463:Saliva / Q000737:chemistry*; D052616:Solid Phase Extraction / Q000379:methods; D013183:Sputum / Q000737:chemistry*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000032:analysis*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D053221:TNF-Related Apoptosis-Inducing Ligand
keywords: (15)N-Metabolically labeled internal standard; Antibody-free LC–MS/MS; Immobilized metal affinity (IMAC) enrichment; Induced sputum; Saliva; Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL)