Affinity Purification Coupled to Stable Isotope Dilution LC-MS/MS Analysis to Discover IgG4 Glycosylation Profiles for Autoimmune Pancreatitis.
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ABSTRACT:
SUBMITTER: Chen MX
PROVIDER: S-EPMC8584136 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as ?-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [(13)C10]?-carotene and 1 mg [(13)C10]retinyl acetate in human subjects over a 2 week period. A reverse phase C18 column and binary mobile phase solvent system separated ?-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537?321 and m/z 269?93 for respective [(12)C]?-carotene and [(12)C] retinoids; m/z 547?330 and m/z 274?98 for [(13)C10]?-carotene and [(13)C5] cleavage products; and m/z 279?100 for metabolites of [(13)C10]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [(13)C10]retinyl acetate with [(13)C10]?-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual's vitamin A status.
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Project description:The accurate measurement of androstenedione in human serum and plasma is required for steroid profiling to assure the appropriate diagnosis and differential diagnosis of hyperandrogenism. In this work, we introduce an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) candidate reference measurement procedure for the quantification of androstenedione in human serum and plasma. The performance of the procedure enables its use in the evaluation and standardization of routine assays and for the evaluation of patient samples to ensure the traceability of individual patient results. As the primary standard, a certified reference material from NMIA (National Measurement Institute, Australia) was used. Additionally, a quantitative nuclear magnetic resonance (qNMR) method was developed for the value assignment of the primary reference material, which ensures the direct traceability to SI units, as well as the independence from the availability of reference materials. 13C3-labeled androstenedione was used as the internal standard. The introduced method allows the measurement of androstenedione in the range of 0.05-12 ng/mL, and the assay imprecision was found to be <2% between 5 and 12 ng/mL, 3.5% at 1.5 ng/mL, and 5.2% at 0.05 ng/mL, with an accuracy of 95-105% for the serum and 91-103% for the plasma matrix. The transferability to a second laboratory was validated by method comparison based on 112 patient samples. The comparison of the results obtained from the presented method and an LC-MS/MS routine assay, using 150 native patient samples, showed a good correlation with a bias of the routine method of ≤4.0%.
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Project description:Autoimmune pancreatitis (AIP) is a peculiar type of pancreatitis of presumed autoimmune etiology. Many new clinical aspects of AIP have been clarified during the past 10 years, and AIP has become a distinct entity recognized worldwide. However, its precise pathogenesis or pathophysiology remains unclear. As AIP dramatically responds to steroid therapy, accurate diagnosis of AIP is necessary to avoid unnecessary surgery. Characteristic dense lymphoplasmacytic infiltration and fibrosis in the pancreas may prove to be the gold standard for diagnosis of AIP. However, since it is difficult to obtain sufficient pancreatic tissue, AIP should be diagnosed currently based on the characteristic radiological findings (irregular narrowing of the main pancreatic duct and enlargement of the pancreas) in combination with serological findings (elevation of serum gamma-globulin, IgG, or IgG4, along with the presence of autoantibodies), clinical findings (elderly male preponderance, fluctuating obstructive jaundice without pain, occasional extrapancreatic lesions, and favorable response to steroid therapy), and histopathological findings (dense infiltration of IgG4-positive plasma cells and T lymphocytes with fibrosis and obliterative phlebitis in various organs). It is apparent that elevation of serum IgG4 levels and infiltration of abundant IgG4-positive plasma cells into various organs are rather specific to AIP patients. We propose a new clinicopathological entity, "IgG4-related sclerosing disease", and suggest that AIP is a pancreatic lesion reflecting this systemic disease.
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Project description:Vitamin B6 comprises an important set of molecules tightly interwoven with the human amino acid, fatty acid, and carbohydrate metabolism. Analytical methods striving for the quantification of individual B6 vitamers so far mostly rely on methods based on HPLC in combination with fluorescence detection, but their application encounters multiple difficulties due to the chemical divergence of the single vitamers. The present study describes the development of a method based on LC-MS/MS and stable isotope dilution assay (SIDA) for the simultaneous quantification of five vitamers (PN, PL, PM, PMP, and PNG) of the B6 group in food samples. [13C3]-PN, [13C3]-PL, and [13C6]-PNG were applied as internal standards for the analysis of PN, PL, and PNG. PM and PMP were quantified via matrix-matched calibration referring to [13C3]-PN. The developed method was validated using starch matrix. The limits of detection and quantification ranged from 0.0028 to 0.02 mg/kg and from 0.0085 to 0.059 mg/kg, respectively, for all analytes. Calculated recoveries varied from 92 to 111%. Intra-injection precisions ranged from 0 to 9%, inter-day precisions from 4 to 10%, and intra-day precisions from 4 to 10%. A total of 14 plant-based food samples including fruits, vegetables, and cereals were examined for their content of vitamin B6 using the validated method. Furthermore, the first quantitation of PNG without enzymatic steps or divergent internal standards was undertaken utilizing LC-MS/MS and SIDA.
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Project description:The methionine cycle is a key pathway contributing to the regulation of human health, with well-established involvement in cardiovascular diseases and cognitive function. Changes in one-carbon cycle metabolites have also been associated with mild cognitive decline, vascular dementia, and Alzheimer's disease. Today, there is no single analytical method to monitor both metabolites and co-factors of the methionine cycle. To address this limitation, we here report for the first time a new method for the simultaneous quantitation of 17 metabolites in the methionine cycle, which are homocysteic acid, taurine, serine, cysteine, glycine, homocysteine, riboflavin, methionine, pyridoxine, cystathionine, pyridoxamine, S-adenosylhomocysteine, S-adenosylmethionine, betaine, choline, dimethylglycine, and 5-methyltetrahydrofolic acid. This multianalyte method, developed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), provides a highly accurate and precise quantitation of these 17 metabolites for both plasma and cerebrospinal fluid metabolite monitoring. The method requires a simple sample preparation, which, combined with a short chromatographic run time, ensures a high sample throughput. This analytical strategy will thus provide a novel metabolomics approach to be employed in large-scale observational and intervention studies. We expect such a robust method to be particularly relevant for broad and deep molecular phenotyping of individuals in relation to their nutritional requirements, health monitoring, and disease risk management.
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Project description:Various genotoxic carcinogens ubiquitously present in the human environment or respective reactive metabolites form adducts in DNA and proteins, which can be used as biomarkers of internal exposure. For example, the mass spectrometric determination of Val adducts at the N-termini of hemoglobin (Hb) peptide chains after cleavage by an Edman degradation has a long tradition in occupational medicine. We developed a novel isotope-dilution UHPLC-MS/MS method for the simultaneous quantification of Val adducts of eight genotoxic substances in Hb after cleavage with fluorescein-5-isothiocyanate (FIRE procedure™). The following adducts were included [sources in square brackets]: N-(2,3-dihydroxypropyl)-Val [glycidol], N-(2-carbamoylethyl)-Val [acrylamide], N-(2-carbamoyl-2-hydroxyethyl)-Val [glycidamide], N-((furan-2-yl)methyl)-Val [furfuryl alcohol], N-(trans-isoestragole-3'-yl)-Val [estragole/anethole], N-(3-ketopentyl)-Val [1-penten-3-one], N-(3-ketooctanyl)-Val [1-octene-3-one], and N-benzyl-Val [benzyl chloride], each of which was quantified with a specific isotope-labeled standard. The limits of quantification were between 0.014 and 3.6 pmol/g Hb (using 35 mg Hb per analysis); other validation parameters were satisfactory according to guidelines of the U.S. Food and Drug Administration. The quantification in erythrocyte samples of human adults (proof of principle) showed that the median levels of Hb adducts of acrylamide, glycidamide, and glycidol were found to be significantly lower in six non-smokers (25.9, 12.2, and 4.7 pmol/g Hb, respectively) compared to those of six smokers (69.0, 44.2, and 8.6 pmol/g Hb, respectively). In summary, the method surpasses former techniques of Hb adduct quantification due to its simplicity, sensitivity, and accuracy. It can be extended continuously with other Hb adducts and will be used in epidemiological studies on internal exposure to carcinogens.
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Project description:A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methodology has been developed to differentiate core- and antennary-fucosylated glycosylation of glycopeptides. Both the glycosylation sites (heterogeneity) and multiple possible glycan occupancy at each site (microheterogeneity) can be resolved via intact glycopeptide analysis. The serum glycoprotein alpha-1-antitrypsin (A1AT) which contains both core- and antennary-fucosylated glycosites was used in this study. Sialidase was used to remove the sialic acids in order to simplify the glycosylation microheterogeneity and to enhance the MS signal of glycopeptides with similar glycan structures. β1-3,4 galactosidase was used to differentiate core- and antennary-fucosylation. In-source dissociation was found to severely affect the identification and quantification of glycopeptides with low abundance glycan modification. The settings of the mass spectrometer were therefore optimized to minimize the in-source dissociation. A three-step mass spectrometry fragmentation strategy was used for glycopeptide identification, facilitated by pGlyco software annotation and manual checking. The collision energy used for initial glycopeptide fragmentation was found to be crucial for improved detection of oxonium ions and better selection of Y1 ion (peptide+GlcNAc). Structural assignments revealed that all three glycosylation sites of A1AT glycopeptides contain complex N-glycan structures: site Asn70 contains biantennary glycans without fucosylation; site Asn107 contains bi-, tri- and tetra-antennary glycans with both core- and antennary-fucosylation; site Asn271 contains bi- and tri-antennary glycans with both core- and antennary-fucosylation. The relative intensity of core- and antennary-fucosylation on Asn107 was similar to that of the A1AT protein indicating that the glycosylation level of Asn107 is much larger than the other two sites.
| S-EPMC6077116 | biostudies-literature
Project description:IntroductionPancreatic ductal adenocarcinoma (PDAC) is the fifth most common cause of cancer-related death in Europe with a 5-year survival rate of <5%. Chronic pancreatitis (CP) is a risk factor for PDAC development, but in the majority of cases malignancy is discovered too late for curative treatment. There is at present no reliable diagnostic marker for PDAC available.ObjectivesThe aim of the study was to identify single blood-based metabolites or a panel of metabolites discriminating PDAC and CP using liquid chromatography-mass spectrometry (LC-MS).MethodsA discovery cohort comprising PDAC (n = 44) and CP (n = 23) samples was analyzed by LC-MS followed by univariate (Student's t test) and multivariate (orthogonal partial least squares-discriminant analysis (OPLS-DA)) statistics. Discriminative metabolite features were subject to raw data examination and identification to ensure high feature quality. Their discriminatory power was then confirmed in an independent validation cohort including PDAC (n = 20) and CP (n = 31) samples.ResultsGlycocholic acid, N-palmitoyl glutamic acid and hexanoylcarnitine were identified as single markers discriminating PDAC and CP by univariate analysis. OPLS-DA resulted in a panel of five metabolites including the aforementioned three metabolites as well as phenylacetylglutamine (PAGN) and chenodeoxyglycocholate.ConclusionUsing LC-MS-based metabolomics we identified three single metabolites and a five-metabolite panel discriminating PDAC and CP in two independent cohorts. Although further study is needed in larger cohorts, the metabolites identified are potentially of use in PDAC diagnostics.
| S-EPMC5376388 | biostudies-literature
Project description:Recent studies have demonstrated that various DNA adducts can be detected in human tissues and fluids using liquid chromatography connected to tandem mass spectrometry (LC-MS/MS). However, the utility of a single DNA adduct as a biomarker in risk assessment is debatable because humans are exposed to many genotoxicants. We established a method to measure DNA adducts derived from 16 ubiquitous genotoxicants and developed an analytical technique for their simultaneous quantification by ultra performance liquid chromatography (UPLC)-MS/MS. Methods for the enrichment of the analytes from DNA hydrolysates and chromatographic separation preceding mass spectrometric analysis were optimized, and the resultant technique was used for the simultaneous analysis of the 16 DNA adducts in human lung biopsy specimens. Eleven adducts (formed by benzo[a]pyrene, 1-methylpyrene, 4-aminobiphenyl, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 1-methoxy-3-indolylmethylglucosinolate, 5-hydroxymethylfurfural, and malondialdehyde) were not detected in any tissue sample (limits of detection: 0.02-7.1 adducts/10(8) nucleosides). 3,N(4)-etheno-2'-deoxycytidine and 1,N(6)-etheno-2'-deoxyadenosine, formed from 2,3-epoxyaldehydes of endogenous lipid peroxidation products, were present in all subjects (16.9-115.3 and 27.2-179/10(8) nucleosides, respectively). The same was true for N(2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine, the major adduct of methyleugenol (1.7-23.7/10(8) nucleosides). A minor adduct of methyleugenol and two adducts of furfuryl alcohol were detected in several pulmonary specimens. Taken together, we developed a targeted approach for the simultaneous mass spectrometric analyses of 16 DNA adducts, which can be easily extended by adducts formed from other mutagens. The method allowed one to detect adducts of furfuryl alcohol and methyleugenol in samples of human lung.
| S-EPMC4287830 | biostudies-literature