Towards reproducible MRM based biomarker discovery using dried blood spots.
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ABSTRACT:
SUBMITTER: Ozcan S
PROVIDER: S-EPMC5366927 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundMucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage. Recently approved enzyme replacement therapy renders the easy and early identification of MPS IVA of out-most importance.MethodologyWe propose a completely new assay for the stable and reproducible detection of GALNS deficiency in dry blood spots (DBS). For the validation blood samples were taken from 59 healthy individuals and 24 randomly selected genetically confirmed MPS IVA patients. The material extracted from DBS was incubated with a 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate as a specific substrate. Final enzymatic product, 4-methylumbelliferone, obtained after adding exogenous beta-galactosidase, was quantified by LC/MRM-MS (liquid-chromatography/multiple-reaction-monitoring mass-spectrometry). 4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one was used as internal standard, a compound with a similar molecular structure and fragmentation pattern in negative ion mode as 4-methylumbelliferone.FindingsThe enzymatic assay yielded a positive and negative predictive value of 1.0 for genetically confirmed MPS IVA patients (GALNS activity of 0.35 ± 0.21 μmol/L/h) and for controls with normal GALNS activity (23.1 ± 5.3 μmol/L /h). With present enzymatic conditions, the reaction yield in dried blood spots is at least 20 fold higher than any previously reported data with other assays.InterpretationThe present LC/MRM-MS based assay for MPS IVA diagnosis provides an easy, highly-standardized, accurate and innovative quantification of the enzymatic product in vitro and distinguishes perfectly between MPS IVA affected patients and normal controls. This technique will significantly simplify the early detection of MPS IVA patients.
| S-EPMC4492791 | biostudies-literature
Project description:In this study, a rapid multi-mycotoxin approach was developed for biomonitoring and quantification of 27 important mycotoxins and mycotoxin metabolites in human blood samples. HPLC-MS/MS detection was used for the analysis of dried serum spots (DSS) and dried blood spots (DBS). Detection of aflatoxins (AFB1, AFB2, AFG1, AFG2, AFM1), trichothecenes (deoxynivalenol, DON; DON-3-glucoronic acid, DON-3-GlcA; T-2; HT-2; and HT-2-4-GlcA), fumonisin B1 (FB1), ochratoxins (OTA and its thermal degradation product 2'R-OTA; OTα; 10-hydroxychratoxin A, 10-OH-OTA), citrinin (CIT and its urinary metabolite dihydrocitrinone, DH-CIT), zearalenone and zearalanone (ZEN, ZAN), altenuene (ALT), alternariols (AOH; alternariol monomethyl ether, AME), enniatins (EnA, EnA1, EnB, EnB1) and beauvericin (Bea) was validated for two matrices, serum (DSS), and whole blood (DBS). HPLC-MS/MS analysis showed signal suppression as well as signal enhancement due to matrix effects. However, for most analytes LOQs in the lower pg/mL range and excellent recovery rate were achieved using matrix-matched calibration. Besides validation of the method, the analyte stability in DBS and DSS was also investigated. Stability is a main issue for some analytes when the dried samples are stored under common conditions at room temperature. Nevertheless, the developed method was applied to DBS samples of a German cohort (n = 50). Besides positive findings of OTA and 2'R-OTA, all samples were positive for EnB. This methodical study establishes a validated multi-mycotoxin approach for the detection of 27 mycotoxins and metabolites in dried blood/serum spots based on a fast sample preparation followed by sensitive HPLC-MS/MS analysis. Graphical Abstract ᅟ.
| S-EPMC5395583 | biostudies-literature
Project description:The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs.The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.
| S-EPMC7050112 | biostudies-literature
Project description:Neonatal dried blood spots (DBS) are routinely utilized in the clinical setting as a diagnostic tool for various genetic disorders and infectious diseases. DBS allow for minimally invasive, small volume blood collection and are stored at room temperature. Neonatal whole blood and serum samples can be important in determining genetic risk factors and predicting infantile disease; however, at the present time, limited methods exist for rapidly analyzing DBS samples for their proteomic profile, years after samples have been collected. A novel method is presented for the extraction and analysis of target proteins and peptides from neonatal DBS using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Extraction parameters were optimized to achieve ideal signal intensity and resolution to obtain protein identifications. Samples were extracted from filter paper with 0.1% TFA in H2O for 72 h. The extract was subjected to enzymatic digestion, spotted on an ITO-coated glass slide, and washed in order to remove salts. Analysis of extracted blood spots from ten newborns was completed. Similarities and differences in the proteomic profile of the washed extracts are presented, herein, to verify the viability of this method for analysis of dated DBS samples. This method allows for analysis of DBS samples years after collection and can be utilized to correlate diseases or disorders manifesting later in life with potential risk factors presenting in the proteomic profile of the DBS collected at time of birth.
| S-EPMC8620524 | biostudies-literature
Project description:Purpose: Cerebrotendinous xanthomatosis (CTX) is a treatable hereditary disorder caused by the deficiency of sterol 27-hydroxylase, which is encoded by the CYP27A1 gene. Different newborn screening biomarkers for CTX have been described, including 7?,12?-dihydroxy-4-cholesten-3-one (7?12?C4), 5?-cholestane-3?,7?,12?,25-tetrol glucuronide (GlcA-tetrol), the ratio of GlcA-tetrol to tauro-chenodeoxycholic acid (t-CDCA) (GlcA-tetrol/t-CDCA), and the ratio of tauro-trihydroxycholestanoic acid (t-THCA) to GlcA-tetrol (t-THCA/GlcA-tetrol). We set out to evaluate these screening methods in a research study using over 32,000 newborn dried blood spots (DBS).Methods: Metabolites were extracted from DBS with methanol containing internal standard, which was then quantified by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).Results: The measurement of 7?12?C4 was complicated by isobaric interferences and was discontinued. A total of 32,737 newborns were screened based on the GlcA-tetrol concentration in DBS. GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios were also calculated. Newborns displaying both elevated GlcA-tetrol and GlcA-tetrol/t-CDCA ratio were considered to be screen positives. The t-THCA/GlcA-tetrol ratio was used to further distinguish CTX screen positives from Zellweger Spectrum Disorder (ZSD) screen positives. Only one newborn displayed both elevated GlcA-tetrol concentration in DBS and a typical CTX biochemical profile. This newborn was interpreted as a CTX-affected patient as CYP27A1 gene sequencing identified two known pathogenic variants.Conclusion: The results indicate that both GlcA-tetrol and the GlcA-tetrol/t-CDCA ratio are excellent CTX biomarkers suitable for newborn screening. By characterizing the relationship of GlcA-tetrol, t-CDCA, and t-THCA as secondary markers, 100% assay specificity can be achieved.
| S-EPMC7529987 | biostudies-literature
Project description:The quantitation of metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT) - referred to as metanephrines -- by LC-MS/MS is the gold-standard for screening for pheochromocytoma and paragangliomas (PPGLs), tumours of the adrenal gland and the peripheral nervous system. An assay for metanephrines from dried blood spots (DBSs) would be of high clinical utility as it simplifies sample collection, enables remote sampling, and could increase compliance with the clinical recommendation for supine sampling. Moreover, DBS sampling facilitates the measurement of blood-derived metanephrines in pediatric patients - where DBSs are well-established - in order to diagnose neuroblastomas. Here, we adapted an established derivatization-based LC-MRM-MS assay for plasma catecholamines, and optimized the sample extraction, LC, and MS parameters to produce a fast, sensitive, and robust method for the measurement of metanephrines from DBSs, including 3-methoxytyramine. The DBS samples were excised, derivatized with phenyl isothiocyanate (PITC) on-spot, extracted, and measured by LC-MRM-MS. To validate assay suitability and performance, we assessed the linearity, precision, accuracy, recovery, and matrix effects of the method, and determined the stability of metanephrines in DBSs under different storage conditions. Assay performance for NMN, MN, and 3-MT was sufficient for quantitation from a single DBS within a linear range from 40 to 2000 pg/mL. MN and NMN were stable in DBSs for 2 weeks, whereas 3-MT was stable for one week regardless of storage temperature. Altogether, this work represents the first quantitative LC-MS/MS method for metanephrines from DBSs and provides a novel opportunity for the diagnosis of PPGLs and neuroblastomas in the future.
| S-EPMC7346842 | biostudies-literature
Project description:Each year thousands of babies are born with rare genetic disorders not identified by current NBS panels, due to programs which are not yet optimal. Next-generation sequencing technologies have the potential to overcome many NBS drawbacks and provide large amounts of molecular data, broadening the number of diseases investigated. Here, we design and set up an NGS-based approach to evaluate the feasibility of NGS from dried blood spot starting from 34 DBSs. After assessing gDNA yield and integrity, libraries were performed using three target enrichment approaches, sequenced on NS500 platform, and analyzed on commercial platform. Specifically, we focus on virtual gene panels related to highly actionable neonatal/pediatric disorders. WES show that amount and quality of DBS-extracted gDNA are suitable for high-throughput sequencing. We obtain 500-1500 ng for each specimen, 1.7-1.8 260/280 wavelength, and DIN of 7 resulting DNA integrity, on par with traditional venous blood collection. A high read depth with 94.3% coverage uniformity is achieved for all samples. Data results on mean coverage are comparable among the different workflows tested and demonstrate that DBS from newborn collected at birth is a suitable material for the developing of gNBS programs.
| S-EPMC10966309 | biostudies-literature
Project description:The reproducibility of experiments has been a long standing impediment for further scientific progress. Computational methods have been instrumental in drug discovery efforts owing to its multifaceted utilization for data collection, pre-processing, analysis and inference. This article provides an in-depth coverage on the reproducibility of computational drug discovery. This review explores the following topics: (1) the current state-of-the-art on reproducible research, (2) research documentation (e.g. electronic laboratory notebook, Jupyter notebook, etc.), (3) science of reproducible research (i.e. comparison and contrast with related concepts as replicability, reusability and reliability), (4) model development in computational drug discovery, (5) computational issues on model development and deployment, (6) use case scenarios for streamlining the computational drug discovery protocol. In computational disciplines, it has become common practice to share data and programming codes used for numerical calculations as to not only facilitate reproducibility, but also to foster collaborations (i.e. to drive the project further by introducing new ideas, growing the data, augmenting the code, etc.). It is therefore inevitable that the field of computational drug design would adopt an open approach towards the collection, curation and sharing of data/code.
| S-EPMC6988305 | biostudies-literature
Project description:Raw files were created using XCalibur 3.0.63 (Thermo Scientific) software and analysed with Thermo Proteome Discoverer 2.2.0.388 software suite (Thermo Scientific). The raw data files were processed as previously described 26 with the following modifications: the peak lists were submitted to Mascot 2.4.1and Sequest servers using the UniProt human database 28 which contained 20,338 protein sequences. The enzyme was set to Trypsin (Full) with max missed cleavage set to 2. The fixed modification was set to carbamidomethyl and variable modifications were set as oxidation (M), deamidation (NQ), and N-terminal methionine loss. Precursor and fragment mass tolerances were set at 8 ppm and 0.8 Da, respectively. Peptide spectral match validation was performed using Percolator setting the maximum Delta Cn to 0.05 and decoy database searches at 0.01 for target FDR (Strict), 0.05 (relaxed) and validation based on q-Value
2019-11-27 | MSV000084629 | MassIVE
Project description:Dried blood spots (DBSs), a micro-sampling technique whereby a drop of blood is collected on filter paper has multiple advantages over conventional blood sampling regarding the sampling itself, as well as transportation and storage. This is the first paper describing the development and validation of a method for the determination of 23 mycotoxins and phase I metabolites in DBSs from pigs and broiler chickens using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The targeted mycotoxins belong to groups for which the occurrence in feed is regulated by the European Union, namely, aflatoxins, ochratoxin A and several Fusarium mycotoxins, and to two groups of unregulated mycotoxins, namely Alternaria mycotoxins and Fusarium mycotoxins (enniatins and beauvericin). The impact of blood haematocrit, DBS sampling volume and size of the analysed DBS disk on the validation results was assessed. No effects of variation in size of the analysed disk, haematocrit and spotted blood volume were observed for most mycotoxins, except for the aflatoxins and ?-zearalanol (BZAL) at the lowest haematocrit (26%) level and for the enniatins (ENNs) at the lowest volume (40 µL). The developed method was transferred to an LC-high resolution mass spectrometry instrument to determine phase II metabolites. Then, the DBS technique was applied in a proof-of-concept toxicokinetic study including a comparison with LC-MS/MS data from plasma obtained with conventional venous blood sampling. A strong correlation (r > 0.947) was observed between plasma and DBS concentrations. Finally, DBSs were also applied in a pilot exposure assessment study to test their applicability under field conditions.
| S-EPMC6784198 | biostudies-literature