Eight key rules for successful data-dependent acquisition in mass spectrometry-based metabolomics.

In recent years, metabolomics has emerged as a pivotal approach for the holistic analysis of metabolites in biological systems. The rapid progress in analytical equipment, coupled to the rise of powerful data processing tools, now provides unprecedented opportunities to deepen our understanding of the relationships between biochemical processes and physiological or phenotypic conditions in living organisms. However, to obtain unbiased data coverage of hundreds or thousands of metabolites remains a challenging task. Among the panel of available analytical methods, targeted and untargeted mass spectrometry approaches are among the most commonly used. While targeted metabolomics usually relies on multiple-reaction monitoring acquisition, untargeted metabolomics use either data-independent acquisition (DIA) or data-dependent acquisition (DDA) methods. Unlike DIA, DDA offers the possibility to get real, selective MS/MS spectra and thus to improve metabolite assignment when performing untargeted metabolomics. Yet, DDA settings are more complex to establish than DIA settings, and as a result, DDA is more prone to errors in method development and application. Here, we present a tutorial which provides guidelines on how to optimize the technical parameters essential for proper DDA experiments in metabolomics applications. This tutorial is organized as a series of rules describing the impact of the different parameters on data acquisition and data quality. It is primarily intended to metabolomics users and mass spectrometrists that wish to acquire both theoretical background and practical tips for developing effective DDA methods.

Towards a simple on-line coupling of ion exchange chromatography and native mass spectrometry for the detailed characterization of monoclonal antibodies.

This work describes the direct hyphenation of cation exchange chromatography (CEX) with a compact, easy-to-use benchtop Time of Flight mass spectrometer (ToF/MS) for the analytical characterization of monoclonal antibodies (mAbs). For this purpose, a wide range of commercial mAb products (including expired samples and mAb biosimilars) were selected to draw reliable conclusions. From a chromatographic point of view, various buffers and column dimensions were tested. When considering pH response, buffer stability over time and MS compatibility, the best compromise is represented by the following recipe: 50 mM ammonium acetate, titrated to pH 5.0 (mobile phase A) and 160 mM ammonium acetate, titrated to pH 8.5 (mobile phase B). Despite the broader peaks observed with the 2.1 mm i.d. CEX column, this was preferentially selected for CEX-MS operation, since the efficiency loss (caused by extra-column dispersion) was still acceptable while MS compatibility was strongly enhanced (thanks to low flow rate). In terms of MS, it was important to avoid the use of glass-bottled mobile phases, laboratory glassware and glass vials to minimize loss of MS resolution, sensitivity, and mass accuracy due to metal contaminants. With this new CEX-MS setup, straightforward and rapid analysis (in less than 10 min) of charge variants was possible, allowing the separation and identification of several charge variants.

Sub-picomolar quantification of PTH 1-34 in plasma by UHPLC-MS/MS after subcutaneous injection of teriparatide and identification of PTH 1-33, its degradation product.

Teriparatide (PTH 1-34, Forsteo®) is a bioactive N-terminal fragment of the native endogenous parathyroid hormone (PTH 1-84) recommended for the treatment of osteoporosis in patients with high risk of fracture. Since PTH 1-34 may undergo proteolysis we have validated an ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for unambiguously measuring intact PTH 1-34 with the same sensitivity as ELISA, at subpicomolar level (LLOQ at 0.4 pM). The full chromatographic run was achieved in 16.5 min. The method validation showed satisfactory intra- and inter-assay precision (CV < 13%) and excellent trueness (<5%), and almost no matrix effect (recoveries 78-92%). We found that after subcutaneous injection in two volunteers, PTH 1-34 half-life was shorter with UHPLC-MS/MS and that ELISA was overestimating PTH 1-34 late concentrations in both volunteers. Qualitative mass spectrometry was performed and led to the discovery of PTH 1-33, a fragment of PTH 1-34 with unknown function. This study emphasized the importance of switching from immunoassays to mass spectrometry when measuring bioactive peptides prompt to proteolysis into fragments that may exhibit altered bioactivity and duration of action.