Vitamin D plays a vital role in successful pregnancy outcomes for both the mother and fetus. Vitamin D is bound to vitamin D binding protein (VDBP) in blood and is carried to the liver, kidneys and other target tissues. Accurate measurements of the clinically measured metabolite of vitamin D, 25-hydroxyvitamin D [25(OH)D], depend on complete removal from the binding protein. It has been found that VDBP concentrations increase in maternal serum during pregnancy, obfuscating the accuracy of 25(OH)D concentration measurements in pregnant women. Additionally, measurements of VDBP concentrations during pregnancy have been performed using immunoassays, which suffer from variations due to differences in antibody epitopes, making clinical comparisons difficult. Quantification of VDBP is also of interest because changes in VDBP expression levels may indicate negative outcomes during pregnancy, such as preterm delivery and restricted fetal growth. To address the need for accurate measurement of VDBP during pregnancy, a method using liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was developed to quantify VDBP using isotopically labeled peptides as internal standards. This method was used to quantify VDBP in Standard Reference Material® (SRM) 1949 Frozen Human Prenatal Serum, which was prepared from separate serum pools of women who were not pregnant and women during each trimester of pregnancy. VDBP concentrations were found to be lowest in the serum pool from non-pregnant women and increased in each trimester. These data had good repeatability and were found to be suitable for reference value assignment of VDBP in SRM 1949.
Rapid identification of methicillin-resistant Staphylococcus could ensure appropriate medical care. A total of 409 Staphylococcus spp. strains were used to develop a reliable MALDI-TOF method for species identification. We tested twelve S. aureus strains to compare three different sample preparation methods and the reproducibility of the methicillin-resistant m/z 2414 ± 2 indicator peak with direct method in triplicate. A total of 65 Staphylococcus spp. strains (including 37 methicillin-resistant strains) from clinical and hospital environment isolates were used to confirm the presence of phenol-soluble modulin (PSM-mec) peptide. All 272 S. aureus strains from 409 samples were correctly identified at species level by MALDI-TOF. The samples prepared by three methods gave spectra with differences in the intensities and presence of certain peaks. The PSM-mec peak was not visible after the extraction method. The peak m/z 2414 ± 2 was only detected in 61% of the methicillin-resistant strains and in none of the methicillin-sensitive strains. The peak reproducibility for the five analyzed S. aureus strains showing the peak at m/z 2414 ± 2 was 87%. The delta-toxin was observed in 49 out of 65 samples regardless of methicillin susceptibility, as well as in all the samples exhibiting the PSM-mec peak. The peak m/z 2414 ± 2 is specific to methicillin-resistant strains carrying the mecA gene, but the absence of peak m/z 2414 ± 2 does not exclude the possibility of resistance to methicillin. Thus, implementing MALDI-TOF analysis in routine laboratory work, especially with clinical samples, would in many cases provide rapid warning about the presence of methicillin-resistant strains.
As in drug discovery and development, mass spectrometry has become essential at all stages for establishing the safety and efficacy of botanical dietary supplements. Applications of mass spectrometry to the development of botanical dietary supplements include preclinical studies of the mechanisms of action (e.g., proteomic target identification and validation); identification of active natural products using high resolution tandem mass spectrometry; chemical standardization using UHPLC-MS/MS; and studies of metabolism, absorption and toxicity of active compounds using high resolution and UHPLC-MS/MS. Clinical applications of mass spectrometry include evaluation of the potential for drug-botanical interactions; investigation of the pharmacokinetics of active compounds; and quantitative analysis of biomarkers of efficacy during Phase I and II and clinical trials of safety and efficacy of botanical dietary supplements.
Mass spectral data from multiple samples are suitable for a hypothesis-free development of clinically useful multivariate tests using modern machine learning techniques. However, the transition from discovery to adoption of proteomic tests has proved challenging. Slow adoption of these tests in clinical practice is, in part, related to insufficient understanding of the biological mechanisms underlying multivariate tests developed based on correlative studies. While identification of individual proteins may provide important insights, elucidation of concerted relationships of sets of proteins with biological pathways can better reflect complex phenomena, such as cancerogenesis and response to treatment. Protein set enrichment analysis (PSEA) allows identification of associations of mass spectral features or test classifications with biological function by looking for consistent correlations across a group of proteins. We evaluated the utility of PSEA for exploring the biological information content of mass spectra, using a sample set with mass spectral data and matched protein expression information. This made it possible to detect significant biological associations with mass spectral peaks without identifying their protein constituents. We demonstrated that the method produces reproducible associations and can be used for elucidation of the mechanisms of action associated with two previously developed multivariate mass spectrometry-based tests. Significant correlations with several host immune response-related processes were found on the level of individual mass spectral features and with test classifications. The results illustrate the utility of the PSEA approach applied to mass spectral data as a method for elucidating biological mechanisms underlying phenotypes related to different physiological states of the organism. (C) 2019 The Author(s). Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL).
Phthalates and bisphenol A interfere with the synthesis, secretion, transport, binding, metabolism, and excretion of endogenous hormones and, for this reason, are classified as endocrine disruptors. We are here presenting an analytical method for the simultaneous detection of six phthalates metabolites and bisphenol A in different biological fluids (urine, serum and follifular fluid) by liquid chromatography coupled to tandem mass spectrometry. The quantification was carried out in negative electrospray ionization mode using selected reaction monitoring as acquisition mode. Different extraction protocols, using either solid phase or liquid/liquid extraction, were comparatively evaluated to optimize the sample preparation procedure. Solid-phase extraction was chosen as it ensured the best recovery and overall sensitivity. The method was successfully validated: recovery varying in the range 71 +/- 2%-107 +/- 6% depending on the target analyte and the matrix considered, intra-assay and inter-assay precision <= 12% for follicular fluid, <= 11% for serum and <= 10% for urine and accuracy <= 115% for follicular fluid, <= 113% for serum <= 115% for urine , linearity with R-2 > 0.99, with the exception of MEP (recovery 64 +/- 8%, intra-assay precision <= 20%, inter-assay precision <= 16% for follicular fluid). The actual applicability of the method developed and validated in this study was assessed by the analysis of real samples, including 10 specimens of follicular fluid, serum and urine samples, that showed the presence of phthalates metabolites and Bisphenol A, and confirming that the newly developed method can be applied in the routine clinical laboratory for the identification and quantitation of these endocrine-disrupting chemicals. (C) 2020 Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL).
