Quantitative antibody-free LC-MS/MS analysis of sTRAIL in sputum and saliva at the sub-ng/mL level.

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.

Highly sensitive antibody-free µLC-MS/MS quantification of rhTRAIL in serum.

BACKGROUND: We describe an antibody-free approach to quantify rhTRAIL(WT) (wild-type) and its closely related death receptor 4 selective variant rhTRAIL(4C7) in human and murine serum by multiplex LC-MS/MS on a microfluidics interface. METHODOLOGY: Enrichment of rhTRAIL was performed by strong cation-exchange (SCX) followed by immobilized metal affinity (IMAC) solid-phase extraction. This was followed by trypsin digestion and using methionine-containing signature peptides after fully oxidizing the methionine residue with 0.25% (w/w) hydrogen peroxide. CONCLUSION: Absolute quantification was reaching down to 0.5 ng/ml for rhTRAIL(WT) (8.5 pM) and 2 ng/ml for rhTRAIL(4C7) (34 pM) in 100 µl human serum. To support preclinical studies in mice, the analysis was optimized further, for a sample volume of 20 µl murine serum.

Antibody-free workflows for protein quantification by LC-MS/MS.

Antibody-free approaches for quantitative LC-MS/MS-based protein bioanalysis are reviewed and critically evaluated, and compared with the more widely used immunoaffinity-based approaches. Antibody-free workflows will be divided into four groups and discussed in the following order: direct analysis of signature peptides after proteolytic digestion; enrichment of target proteins and signature peptides by fractionated protein precipitation; enrichment of target proteins and signature peptides by reversed-phase and ion-exchange solid-phase extraction; and enrichment of target proteins and signature peptides by (antibody-free) affinity-solid-phase extraction.

Antibody-free LC-MS/MS quantification of rhTRAIL in human and mouse serum.

The major challenge in targeted protein quantification by LC-MS/MS in serum lies in the complexity of the biological matrix with regard to the wide diversity of proteins and their extremely large dynamic concentration range. In this study, an LC-MS/MS method was developed for the simultaneous quantification of the 60-kDa biopharmaceutical proteins recombinant human tumor necrosis factor-related apoptosis-inducing ligand wild type (rhTRAIL(WT)) and its death receptor 4 (DR4)-specific variant rhTRAIL(4C7) in human and mouse serum. Selective enrichment of TRAIL was accomplished by immobilized metal affinity chromatography (IMAC), which was followed by tryptic digestion of the enriched sample and quantification of a suitable signature peptide. For absolute quantification, (15)N-metabolically labeled internal standards of rhTRAIL(WT) and rhTRAIL(4C7) were used. Since the signature peptides that provided the highest sensitivity and allowed discrimination between rhTRAIL(WT) and rhTRAIL(4C7) contained methionine residues, we oxidized these quantitatively to their sulfoxides by the addition of 0.25% (w/w) hydrogen peroxide. The final method has a lower limit of quantification of 20 ng/mL (ca. 350 pM) and was fully validated according to current international guidelines for bioanalysis. To show the applicability of the LC-MS/MS method for pharmacokinetic studies, we quantified rhTRAIL(WT) and rhTRAIL(4C7) simultaneously in serum from mice injected intraperitoneally at a dose of 5 mg/kg for each protein. This is the first time that two variants of rhTRAIL differing by only a few amino acids have been analyzed simultaneously in serum, an approach that is not possible by conventional enzyme-linked immuno-sorbent assay (ELISA) analysis.