Clinical Mass Spectrometry最新文献

(R)-2-hydroxyglutarate (R-2-hg) is a metabolite produced under physiologic conditions but also by tumors harboring isocitrate dehydrogenase (IDH) mutations. Detection is challenging as it must be distinguished from its enantiomer (S)-2-hydroxyglutarate (S-2-hg), which is also produced in the body but can increase under hypoxic conditions. A chiral gas chromatography-tandem mass spectrometry (GC–MS/MS) assay was developed, which separated enantiomers using a chiral column and quantified levels using a stable-isotope internal standard. The assay improves upon current methods by avoiding chiral derivatization and implementing a simplified sample extraction procedure. The assay was validated and serum 2-hg levels from healthy patients were measured, establishing a new, comprehensive reference range for normal levels of each enantiomer. Differences in basal levels were observed between races, but not sex. Age also correlated with S-2-hg levels, but not R-2-hg levels. Finally, serum levels of 2-hg enantiomers were measured in a pilot study of 11 patients with and without IDH mutant gliomas. An increase in R-2-hg levels was observed in 2/3 patients with actively growing IDH mutant gliomas. S-2-hg levels were increased in 4/11 patients, irrespective of IDH status. The results presented demonstrate the feasibility of a GC–MS/MS assay for measurement of 2-hg enantiomer levels for clinical use.

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.

Misregulated chromatin remodeling via erroneous histone acetylation has been linked with multiple diseases including many age-associated maladies. Previous studies on histone acetylation have utilized specific histone acetylation antibodies as a primary quantification tool. However, antibodies that target specific histone modifications frequently lack sufficient sensitivity and specificity, making signal quantification and data interpretation difficult. Here, we used mass spectrometry to quantify the acetylation patterns of histone H4 in peripheral blood mononuclear cells (PBMCs) from human patients of varying age. We observed that mono-acetylation of H4K16ac is lower in midlife human PBMCs, and that various changes occur in the H4 acetylation signature using PBMCs prepared from old patients. Our data corroborates previous work suggesting that ageing might be characterized by specific changes in the acetylation states of histone H4 and shows that targeted mass spectrometry together with appropriate quantification methods can be used to sensitively measure such changes.

In the field of Alzheimer’s disease, there is an urgent need for novel analytical tools to identify disease-specific biomarkers and to evaluate therapeutics. Preclinical trials commonly employ amyloid beta (Aβ) peptide signatures as a read-out. In this paper, we report a simplified and detailed protocol for robust immunoprecipitation of Aβ in brain tissue prior to mass spectrometric detection exemplified by a study using transgenic mice. The established method employed murine monoclonal and rabbit polyclonal antibodies and was capable of yielding well-reproducible peaks of high intensity with low background signal intensities corresponding to various Aβ forms.

Pharmacological treatment of the attention-deficit/hyperactivity disorder (ADHD) includes use of the psychostimulant amphetamine. Non-adherence to medication is a well-documented problem in ADHD treatment and a cause of treatment failure. The study evaluated the possibility of using oral fluid for compliance monitoring during treatment with lisdexamphetamine (Elvanse®). UPLC-MS/MS methods for general oral fluid drug testing, lisdexamphetamine and amphetamine quantification and chiral analysis of amphetamine were used. The applied measuring ranges were 1–500 ng/mL for amphetamine and 0.01–15 ng/mL for lisdexamphetamine. Amphetamine (racemic) was detected and quantified in 98 (96%) of the 102 samples. The concentrations ranged from 2 to 8410 ng/mL. In 17 of these, the chiral analysis demonstrated intake of illicit amphetamine because L-amphetamine was present. The median D- + L-amphetamine concentration in the compliant group was 280 ng/mL, while the median concentration in the non-compliant group was statistically higher, 1677 ng/mL. In the non-compliant cases where L-amphetamine was detected, the L/D-amphetamine ratios ranged from 0.75 to 13.1 with a median of 1.0. Lisdexamphetamine was detected and quantified in 76 of the 102 cases, which represent 79% of the 98 cases with detected oral fluid amphetamine. The concentrations ranged from 0.01 to 6895 ng/mL. Drug testing had a positive rate of 23% in patients not taking illicit amphetamine and 82% among non-compliant patients with detected L-amphetamine. In conclusion, the study demonstrated the value of measuring amphetamine with a chiral method to detect intake of illicit amphetamine and to perform drug testing in oral fluid as a mean for compliance monitoring.

Background

Therapeutic drug monitoring (TDM) of antidepressants is important to ensure compliance and to rule out pharmacokinetic abnormalities. Therefore, reliable methods for quantification are important for clinical laboratories. Most of the currently used mass spectrometry methods use triple quadrupoles as mass analyzers. We aimed to develop a method using high-resolution mass spectrometry (HRMS) and wanted to test the suitability of this analyzer for quantitative TDM assays. This would be beneficial since HRMS instruments can also be used for metabolomics and protein analysis and, thus, many different analyses could be run on one instrument.

Methods

After manual protein precipitation of serum samples, further sample clean-up was achieved using a Turbo Flow column preconnected to the analytical LC column. Stable isotope-labelled counterparts of the target analytes were used as internal standards. For detection, we used a Q Exactive Focus Orbitrap mass spectrometer operating in full-scan mode. Ionization was performed in positive ESI.

Results

Accuracy, recovery, and matrix effect were acceptable for all analytes. The method demonstrated outstanding precision (within-run imprecision <4.5%, between-run imprecision <7.5%). The selectivity of the method was ensured by chromatographic separation of all isobaric compounds. Close agreement between Orbitrap and triple stage based quantification was observed in a comparison measurement of leftover patient samples.

Conclusions

We have established a selective method for the quantification of antidepressants with outstanding precision using a high-resolution Orbitrap mass spectrometer. The applicability of HRMS instruments to TDM was demonstrated.

Cerebrospinal fluid (CSF) tau and phospho-tau are well established biomarkers of Alzheimer’s disease. While these measures are conventionally referred to as ‘total tau’ (T-tau) and ‘phospho-tau’ (P-tau), several truncated and modified tau forms exist that may relay additional diagnostic information. We evaluated the diagnostic performance of an endogenous tau peptide in CSF, tau 175–190, in the phosphorylated and non-phosphorylated state. A liquid chromatography-mass spectrometry (LC-MS) method was established to measure these peptides in CSF and was used to analyze two independent clinical cohorts; the first cohort included patients with Alzheimer’s disease (AD, n = 15), Parkinson’s disease (PD, n = 15), progressive supranuclear palsy (PSP, n = 15), and healthy controls (n = 15), the second cohort included AD patients (n = 16), and healthy controls (n = 24). In both cohorts T-tau and P-tau concentrations were determined by immunoassay. While tau 175–190 and P-tau 175–190 did not differentiate the study groups, the separation of AD and controls by T-tau (area under the ROC Curve (AUC) = 95%) and P-tau (AUC = 92%) was improved when normalizing the ELISA measurements to the concentrations of the endogenous peptides: T-tau/tau 175–190 (AUC = 100%), P-tau/P-tau 175–190 (AUC = 95%). The separation between patients and controls by T-tau (AUC = 88%) and P-tau (AUC = 82%) was similarly improved in the second cohort by taking the ratios of T-tau/tau 175–190 (AUC = 97%) and P-tau/P-tau 175–190 (AUC = 98%). In conclusion, our results suggest that the performance of the AD biomarkers T-tau and P-tau could be improved by normalizing their measurements to the endogenous peptides tau 175–190 and P-tau 175–190, possibly because these endogenous tau peptides serve to normalize for physiological, and disease-independent, secretion of tau from neurons to the extracellular space and the CSF. Finally, the observations made here add to the general applicability of mass spectrometry as a tool for rapid identification and accurate quantification of biomarker candidates.

Transactive response DNA-binding protein 43 kDa (TDP-43) is a highly conserved and widely expressed protein in human tissues that regulates nucleic acid processing. In frontotemporal dementia and amyotrophic lateral sclerosis, however, TDP-43 forms insoluble aggregates in central nervous tissues. These pathological deposits of TDP-43 have been primarily studied by ligand binding, namely western blot analysis, and, thus, methods with greater structural resolution are needed to aid in our understanding of the pathological processes associated with TDP-43 misfolding and aggregation. Toward this goal, we have developed a selective and multiplex method for the detection and characterization of TDP-43 using liquid chromatography tandem mass spectrometry. As proof-of-concept, the method was applied to the detection and characterization of TDP-43 in human cell lines and human brain tissue.

Background

Parathyroid hormone-related protein (PTHrP) is involved in intracellular calcium regulation, neural cell proliferation and synaptic transmission. To date, no studies have been performed to evaluate the potential of PTHrP concentrations in cerebrospinal fluid (CSF) as a biomarker of brain pathophysiology. In this study we evaluated the association between CSF concentrations of PTHrP with the core CSF biomarkers of Alzheimer’s disease (AD).

Methods

PTHrP and calcium were analysed using validated mass spectrometry based methods in a set of CSF samples that tested positive (n = 45) and negative (n = 45) for the AD biomarkers, including total tau protein (T-tau), phosphorylated tau protein (P-tau) and amyloid-β 42 (Aβ42). The measured CSF concentrations of PTHrP and calcium (Ca) were evaluated for association with AD CSF biomarkers.

Results

PTHrP and Ca concentrations in CSF samples ranged between 25 and 137 pmol/L and 0.92–1.53 mmol/L, respectively. Higher concentrations of PTHrP were observed in association with increased concentrations of T-tau and P-tau in the AD and the control group; while a stronger correlation was observed in the control group (ρ = 0.6, p < 0.0001; and ρ = 0.72, p < 0.0001, for T-tau and P-tau, respectively). Negative correlation was observed between concentrations of PTHrP and Aβ42 in the AD group (ρ = 0.27, p = 0.015). A statistically significantly lower ratio Aβ42/PTHrP was observed in the AD group (p < 0.0001).

Conclusion

In the current study, we observed an association of PTHrP concentrations with concentrations of clinically used CSF biomarkers of AD. Concentrations of PTHrP were positively correlated with concentrations of T-tau and P-tau, suggesting an association with neuronal secretion and function, which is reduced upon progression to AD pathology. Our data suggest potential utility of the Aβ42/PTHrP ratio in assessment of AD progression.