This rapid LC-MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions.
The simultaneous quantification of protein concentrations via proteotypic peptides in human blood by liquid chromatography coupled to quadrupole MS/MS is an important field of bioanalytical research with a high potential for routine diagnostic applications. This review summarizes currently available sample preparation procedures and trends for absolute protein quantification in blood using LC-MS/MS. It discusses approaches of transferring established qualitative protocols to a quantitative analysis regarding their reliability and reproducibility. Techniques used to enhance method sensitivity such as the depletion of high-abundant proteins or the immunoaffinity enrichment of proteins and peptides are described. Furthermore, workflows for (i) protein denaturation, (ii) disulfide bridge reduction and (iii) thiol alkylation as well as (iv) enzymatic digestion for absolute protein quantification are presented. The main focus is on the tryptic digestion as a bottleneck of protein quantification via proteotypic peptides. Conclusively, requirements for a high-throughput application are discussed.
3,4‐Methylenedioxymethamphetamine (MDMA, Ecstasy) tablets are widely used recreationally, and not only vary in appearance, but also in MDMA content. Recently, the prevalence of high‐content tablets is of concern to public health authorities. To compare UK data with other countries, we evaluated MDMA content of 412 tablets collected from the UK, 2001–2018, and investigated within‐batch content variability for a sub‐set of these samples. In addition, we investigated dissolution profiles of tablets using pharmaceutical industry‐standard dissolution experiments on 247 tablets. All analyses were carried out using liquid chromatography−tandem mass spectrometry (LC–MS/MS). Our data supported other studies, in that recent samples (2016–2018) tend to have higher MDMA content compared to earlier years. In 2018, the median MDMA content exceeded 100 mg free‐base for the first time. Dramatic within‐batch content variability (up to 136 mg difference) was also demonstrated. Statistical evaluation of dissolution profiles at 15‐minutes allowed tablets to be categorized as fast‐, intermediate‐, or slow‐releasing, but no tablet characteristics correlated with dissolution classification. Hence, there would be no way of users knowing a priori whether a tablet is more likely to be fast or slow‐releasing. Further, within‐batch variation in dissolution rate was observed. Rapid assessment of MDMA content alone provides important data for harm reduction, but does not account for variability in (a) the remainder of tablets in a batch, or (b) MDMA dissolution profiles. Clinical manifestations of MDMA toxicity, especially for high‐content, slow‐releasing tablets, may be delayed or prolonged, and there is a significant risk of users re‐dosing if absorption is delayed.
Albeit achieving the X-ray diffraction structure of dimeric photosystem II core complexes (dPSIIcc) at the atomic resolution, the nature of the detergent belt surrounding dPSIIcc remains ambiguous. Therefore, the solution structure of the whole detergent−protein complex of dPSIIcc of Thermosynechococcus elongatus (T. elongatus) solubilized in n-dodecyl-ß-D-maltoside (ßDM) was investigated by a combination of small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) with contrast variation. First, the structure of dPSIIcc was studied separately in SANS experiments using a contrast of 5% D 2 O. Guinier analysis reveals that the dPSIIcc solution is virtually free of aggregation in the studied concentration range of 2−10 mg/mL dPSIIcc, and characterized by a radius of gyration of 62 Å. A structure reconstitution shows that dPSIIcc in buffer solution widely retains the crystal structure reported by Xray free electron laser studies at room temperature with a slight expansion of the entire protein. Additional SANS experiments on dPSIIcc samples in a buffer solution containing 75% D 2 O provide information about the size and shape of the whole detergent− dPSIIcc. The maximum position of P(r) function increases to 68 Å, i.e., it is about 6 Å larger than that of dPSIIcc only, thus indicating the presence of an additional structure. Thus, it can be concluded that dPSIIcc is surrounded by a monomolecular belt of detergent molecules under appropriate solubilization conditions. The homogeneity of the ßDM−dPSIIcc solutions was also verified using dynamic light scattering. Complementary SAXS experiments indicate the presence of unbound detergent micelles by a separate peak consistent with a spherical shape possessing a radius of about 40 Å. The latter structure also contributes to the SANS data but rather broadens the SANS curve artificially. Without the simultaneous inspection of SANS and SAXS data, this effect may lead to an apparent underestimation of the size of the PS II−detergent complex. The formation of larger unbound detergent aggregates in solution prior to crystallization may have a significant effect on the crystal formation or quality of the ßDM−dPSIIcc.
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