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The analysis of alpha-1-antitrypsin glycosylation with direct LC-MS/MS.

ABSTRACT: 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. ?1-3,4 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.

SUBMITTER: Yin H

PROVIDER: S-EPMC6077116 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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The analysis of alpha-1-antitrypsin glycosylation with direct LC-MS/MS.

Yin Haidi H An Mingrui M So Pui-Kin PK Wong Melody Yee-Man MY Lubman David M DM Yao Zhongping Z

Electrophoresis 20180226 18

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 use ...[more]

PMID: 29405331

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Project description:BackgroundBisphenol A (BPA) is typically measured in urine using an indirect method that involves enzymatic deconjugation and extraction. In contrast, the direct method measures free and conjugated BPA concurrently and sums them to estimate urinary BPA concentrations. Statistical comparison of total BPA results using the direct and indirect methods is necessary to accurately interpret biomonitoring data for risk assessments.ObjectivesTo compare urinary BPA concentrations estimated from the indirect and direct methods in duplicate first trimester urine samples collected from 1879 pregnant women from the MIREC Study.MethodsFor the indirect method, we measured urinary BPA concentrations using GC-MS/MS. For the direct method, we summed free and conjugated BPA concentrations measured using LC-MS/MS. We evaluated deviation between the two methods using the Bland-Altman analysis in the total sample and stratified (1) by specific gravity and (2) at the limit of quantification (LOQ).ResultsMedian urinary BPA concentrations for the direct and indirect methods were 0.89 µg BPA equivalents/L and 0.81 µg/L respectively. Concentrations from the direct method were, on average, 8.6% (95% CI: 6.7%, 10.5%) higher than the indirect method in a Bland-Altman analysis. The percent differences between the two methods was 4.0% in urines with specific gravities < 1.02 (n = 1348, 72%) and 20.3% in urine with specific gravity ≥ 1.02. In values below the LOQ (n = 663, 35%), we observed smaller average percent deviation (4.8%) between the two methods but wider limits of agreement.DiscussionResults from this study, based on the largest statistically rigorous comparison of the direct and indirect methods of BPA measurement, contrast previous findings reporting that the indirect method underestimates total BPA exposure. The difference in urinary BPA concentrations we observed with the indirect and direct methods is unlikely to alter the interpretation of health outcome data.

Dataset Information

The analysis of alpha-1-antitrypsin glycosylation with direct LC-MS/MS.

Publications

The analysis of alpha-1-antitrypsin glycosylation with direct LC-MS/MS.

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