Dataset Information

Simultaneous and Rapid Detection of Multiple Epimers and Isomers of Aspartyl Residues in Lens Proteins Using an LC-MS-MRM Method.

ABSTRACT: Traditionally, studies of post translational modifications (PTMs) by mass analysis have been limited to modifications such as deamidation and oxidation that have a mass shift. Although Asp isomerization is an important PTM, the selective detection of Asp isomers by mass spectrometry was originally thought to be impossible due to the identical mass of the isomers. The recent development of an LC-MS-based method has facilitated rapid and accurate quantitative analysis of Asp isomers in long-lived proteins; however, because the quantification is based on the extracted ion chromatogram acquired by an MS1 scan, this methodology is not always efficient for detecting extremely low-abundance peptides in complex biological samples. In this paper, we evaluated Asp isomer-containing peptides of ?A-crystallin present in tryptic digests of human lens samples with different degrees of protein aggregation and different ages using LC coupled with multiple reaction monitoring (MRM). In a single analysis, the LC-MRM method enabled three tryptic peptides containing isomers of Asp58, Asp91/92, and Asp151 to be detected simultaneously. The extent of isomerization and epimerization of these specific Asp sites in ?A-crystallin increased with the progress of ?-crystallin aggregation. For the analysis of samples known to isomerize at specific Asp residues, MRM gives a more rapid, less laborious, and high-quality separation of Asp isomer-containing peptides relative to the previous MS1-based quantitative method.

SUBMITTER: Fujii N

PROVIDER: S-EPMC7594319 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Simultaneous and Rapid Detection of Multiple Epimers and Isomers of Aspartyl Residues in Lens Proteins Using an LC-MS-MRM Method.

Fujii Noriko N Takata Takumi T Kim Ingu I Matsubara Toshiya T

ACS omega 20201016 42

Traditionally, studies of post translational modifications (PTMs) by mass analysis have been limited to modifications such as deamidation and oxidation that have a mass shift. Although Asp isomerization is an important PTM, the selective detection of Asp isomers by mass spectrometry was originally thought to be impossible due to the identical mass of the isomers. The recent development of an LC-MS-based method has facilitated rapid and accurate quantitative analysis of Asp isomers in long-lived ...[more]

PMID: 33134726

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Project description:Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. BIOLOGICAL SIGNIFICANCE: We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and selective LC-MS-MRM method. Despite an overall increase in core fucosylation of many of the glycoproteins that we examined, core fucosylation changed in a protein- and site-specific manner. Moreover, increased and decreased fucosylation was observed on different N-glycopeptides of the same protein. Altered core fucosylation of N-glycopeptides might be used as an alternative serologic assay for the evaluation of fibrotic liver disease.

Dataset Information

Simultaneous and Rapid Detection of Multiple Epimers and Isomers of Aspartyl Residues in Lens Proteins Using an LC-MS-MRM Method.

Publications

Simultaneous and Rapid Detection of Multiple Epimers and Isomers of Aspartyl Residues in Lens Proteins Using an LC-MS-MRM Method.

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