Quantification of surfactant protein D (SPD) in human serum by liquid chromatography-mass spectrometry (LC-MS)Klont, F., Pouwels, S. D., Bults, P., van de Merbel, N. C., Ten Hacken, N. H. T., Horvatovich, P. & Bischoff, R., 1-Sep-2019, In : Talanta. 202, p. 507-513 7 p.
Research output: Contribution to journal › Article › Academic › peer-review
Quantification of intact proteins in complex biological matrices by liquid chromatography-mass spectrometry (LC-MS) is a promising analytical strategy but is technically challenging, notably for concentrations at or below the ng/mL level. Therefore, MS-based protein quantification is mostly based on measuring protein-specific peptides, so-called 'surrogate peptides', that are released through proteolysis. While quantitative protein bioanalysis based on peptide LC-MS is much more sensitive, not every peptide is suitable in this respect. For example, some peptides are too small to be unique for a protein while others are too large to be measured with sufficient sensitivity, so careful selection of appropriate peptides is essential. Here we present a validated LC-MS method for quantification of surfactant protein D (SPD) at clinically relevant levels between 5 and 500 ng/mL using 50 mu L. of serum. This method targets two SPD-specific peptides in the C-type lectin, ligand binding domain of the SPD protein. One of these peptides contains a methionine residue which would typically be avoided because of its unstable nature. Some quantitative methods do target methionine-containing peptides, and corresponding workflows feature an oxidation step at the peptide level using hydrogen peroxide (H2O2) to convert all methionine residues to more stable methionine sulfoxides. For our method, such a procedure was associated with peptide loss, hence we developed an oxidation procedure at the protein level using H2O2 to oxidize methionine residues and the enzyme catalase to quench excess H2O2. This procedure may be applicable to other quantitative methods based on a surrogate peptide-based approach and may potentially also be useful for MS-based workflows targeting intact proteins.
|Number of pages||7|
|Publication status||Published - 1-Sep-2019|
- Biomarker, COPD, Immunoaffinity enrichment, LC-MS, Methionine oxidation, Quantification, TARGETED PROTEOMICS, PEPTIDES, OPPORTUNITIES