Quantitative analysis of complex proteins is a challenging task in modern bioanalytical chemistry. Commonly available isotope labels are still suffering from limitations and drawbacks, whereas new metal labels open numerous possibilities in mass spectrometric analyses. In this work, we have developed a new metal labeling strategy to tag glycan structures of proteins, more particularly antibodies. The oligosaccharide glycans were selectively trimmed to the last N-acetylglucosamine to which an artificial azide containing galactose residue was bound. This azide can be used for subsequent cycloaddition of an alkyne. Therefore, we developed a lanthanide-containing macrocyclic reagent to selectively connect to this azido galactose. In summary, the glycan structures of an antibody can be labeled with a metal functionality using this approach. Furthermore, the functionality of the antibodies can be fully maintained by labeling the Fc glycans instead of using labeling reagents that target amino or thiol groups. This approach enables the possibility of using elemental, besides molecular mass spectrometry, for quantitative analyses or imaging experiments of antibodies in complex biological samples. Graphical abstract Antibody labeling at sugar moieties with rare earth elements to enable application in elemental mass spectrometry.
Objectives To develop an isotope dilution-liquid chromatography-tandem mass spectrometry-(ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for quantification of methotrexate in human serum and plasma. Methods Quantitative nuclear magnetic resonance (qNMR) was used to determine absolute methotrexate content in the standard. Separation was achieved on a biphenyl reversed-phase analytical column with mobile phases based on water and acetonitrile, both containing 0.1% formic acid. Sample preparation included protein precipitation in combination with high sample dilution, and method validation according to current guidelines. The following were assessed: selectivity (using analyte-spiked samples, and relevant structural-related compounds and interferences); specificity and matrix effects (via post-column infusion and comparison of human matrix vs. neat samples); precision and accuracy (in a five-day validation analysis). RMP results were compared between two independent laboratories. Measurement uncertainty was evaluated according to current guidelines. Results The RMP separated methotrexate from potentially interfering compounds and enabled measurement over a calibration range of 7.200–5,700 ng/mL (0.01584–12.54 μmol/L), with no evidence of matrix effects. All pre-defined acceptance criteria were met; intermediate precision was ≤4.3% and repeatability 1.5–2.1% for all analyte concentrations. Bias was −3.0 to 2.1% for samples within the measuring range and 0.8–4.5% for diluted samples, independent of the sample matrix. RMP results equivalence was demonstrated between two independent laboratories (Pearson correlation coefficient 0.997). Expanded measurement uncertainty of target value-assigned samples was ≤3.4%. Conclusions This ID-LC-MS/MS-based approach provides a candidate RMP for methotrexate quantification. Traceability of methotrexate standard and the LC-MS/MS platform were assured by qNMR assessment and extensive method validation.
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