On Mass Ambiguities in High-Resolution Shotgun Lipidomics

Mass-spectrometry-based lipidomics aims to identify as many lipid species as possible from complex biological samples. Due to the large combinatorial search space, unambiguous identification of lipid species is far from trivial. Mass ambiguities are common in direct-injection shotgun experiments, where an orthogonal separation (e.g., liquid chromatography) is missing. Using the rich information within available lipid databases, we generated a comprehensive rule set describing mass ambiguities, while taking into consideration the resolving power (and its decay) of different mass analyzers. Importantly, common adduct species and isotopic peaks are accounted for and are shown to play a major role, both for perfect mass overlaps due to identical sum formulas and resolvable mass overlaps. We identified known and hitherto unknown mass ambiguities in high- and ultrahigh resolution data, while also ranking lipid classes by their propensity to cause ambiguities. On the basis of this new set of ambiguity rules, guidelines and recommendations for experimentalists and software developers of what constitutes a solid lipid identification in both MS and MS/MS were suggested. For researchers new to the field, our results are a compact source of ambiguities which should be accounted for. These new findings also have implications for the selection of internal standards, peaks used for internal mass calibration, optimal choice of instrument resolution, and sample preparation, for example, in regard to adduct ion formation

Tuning the Extent and Depth of Penetration of Flexible Liposomes in Human Skin

In this work we made an attempt to assess the effect of drug-induced changes of flexibility on the penetration of deformable vesicles into the human skin. Eight cationic liposomes with different degrees of flexibility were obtained by entrapping unfractionated heparin, enoxaparin, and nadroparin. The deformability was studied by a novel, facile, and reliable extrusion assay appositely developed and validated by means of quantitative nanoscale mechanical AFM measurements of vesicle elastic modulus (log₁₀(YM)). The proposed extrusion assay, determining the forces involved in vesicles deformation, resulted very sensitive to evidence of minimal changes in bilayer rigidity (σ) and vesicle deformation (<i>K</i>). The drug loading caused a reduction of liposome flexibility with respect to the reference plain liposomes and in accordance to the heparin type, drug to cationic lipid (DOTAP) ratio, and drug distribution within the vesicles. Interestingly, the σ and log₁₀(YM) values perfectly correlated (<i>R</i>² = 0.935), demonstrating the reliability of the deformability data obtained with both approaches. The combination of TEM and LC–MS/MS spectrometry allowed the pattern of the penetration of the entire vesicles into the skin to be followed. In all cases, intact liposomes in the epidermis layers were observed and a relationship between the depth of penetration and the liposome flexibility was found, supporting the hypothesis of the whole vesicle penetration mechanism. Moreover, the results of the extent (<i>R</i>₂₄) of vesicle penetration in the human skin samples showed a direct relation to the flexibility values (σ₁ = 0.65 ± 0.10 MPa → R₂₄ = 3.33 ± 0.02 μg/mg; σ₂ = 0.95 ± 0.04 MPa → R₂₄ = 1.18 ± 0.26 μg/mg; σ₃ = 1.89 ± 0.30 MPa → R₂₄ = 0.53 ± 0.33 μg/mg)