Dataset Information

Comprehensive N-glycoproteomic profiling of humanized antibody expressed in transgenic mouse serum

ABSTRACT: This dataset reports a comprehensive N-glycoproteomic profiling of serum-derived humanized antibodies expressed in transgenic mice using an intact glycopeptide (IGP)–based LC–MS/MS workflow. The study aimed to identify and characterize site-specific N-glycosylation modifications of mouse serum proteins, with emphasis on antibody-related glycoforms. Following sample digestion and HILIC enrichment, glycopeptides were analyzed by high-resolution Orbitrap Astral mass spectrometry in data-dependent acquisition (DDA) mode. The identified N-linked glycopeptides were annotated for glycan composition, glycosylation heterogeneity, and functional classification through GO, KEGG, Reactome, and STRING analyses. The dataset provides an extensive resource for understanding host-dependent glycosylation patterns and their potential impact on antibody functionality.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Blood Cell, Blood Serum

SUBMITTER: Zhiyang Jiang

LAB HEAD: Jing Wang

PROVIDER: PXD070488 | Pride | 2025-12-08

REPOSITORIES: Pride

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Publications

In Vivo engineering of transgenic mice for systemic human neutralizing antibody production against staphylococcal enterotoxin B.

Jiang Zhiyang Z Jia Beichen B Hu Naijing N Zhang Mengmeng M Xiao He H Chen Guojiang G Yu Jijun J Li Xinying X Shen Beifen B Feng Jiannan J Wang Jing J

Frontiers in immunology 20251118

Transgenic animal bioreactors provide a complementary strategy to traditional mammalian cell culture systems for the production of therapeutic human monoclonal antibodies (mAbs). Here we present a CRISPR/Cas9-mediated breakthrough in creating two novel genetically engineered (GE) mouse models with species-specific chromosomal integration of human anti-staphylococcal enterotoxin B (SEB) mAb genes at either the ROSA26 or Hipp11 (H11) safe-harbor loci - evolutionarily conserved ...[more]

PMID: 41341583

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Project description:The glycosylation of antibody-based proteins is vital in conferring the right therapeutic outcomes of the patient. Despite this, significant infrastructure is required to analyse biologic glycosylation in various unit operations from biologic development, process development to QA/QC in bio-manufacturing. The simplified mass spectrometers offer ease of operation as well as the portability of method development across various operations. Furthermore, data analysis would need to have a degree of automation to relay information back to the manufacturing line. We set out to investigate the applicability of using a semiautomated data analysis workflow to investigate glycosylation in different biologic development test cases. The workflow involves data acquisition using a BioAccord LC-MS system with a data-analytical tool called GlycopeptideGraphMS along with Progenesis QI to semi-automate glycoproteomic characterisation and quantitation in complex LC-MS1 data of a glycoproteomic analysis. Data analysis which involved identifying glycopeptides and their quantitative glycosylation was performed in 30 minutes with minimal user intervention. To demonstrate the effectiveness of the antibody and biologic glycopeptide assignment in various scenarios akin to biologic development activities, we demonstrate the effectiveness in the filtering of IgG1 and IgG2 subclasses from human serum IgG as well as innovator drugs trastuzumab and adalimumab and glycoforms by virtue of their glycosylation pattern. We demonstrate a high correlation between conventional released glycan analysis with fluorescent tagging and glycopeptide assignment derived from GraphMS. GraphMS workflow was then used to monitor the glycoform of our in-house trastuzumab biosimilar produced in fed-batch cultures, and it offers valuable insights into the effect of culture media changes on bioprocess and biologic characterisation. The demonstrated utility of GraphMS to semi-automate quantitation and qualitative identification of glycopeptides proves to be an easy and scalable data analysis method that can complement emerging multi-attribute monitoring (MAM) analytical toolsets in bioprocess environments.

Project description:Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) in human serum is challenging due to the wide dynamic range of serum protein abundances, the lack of a complete serum N-Glycan database and the existence of non-specifically digested peptides and numerous modifications. In this regard, a spectral library search method was presented for serum N-linked intact glycopeptides identification with target-decoy and motif-specific false discovery rate (FDR) control. Low-abundant glycoproteins were firstly separated from high-abundant proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) column and a portion of the enriched N-linked intact glycopeptides were processed by N-Glycosidase F (PNGase F) to generate de-glycopeptides. Both N-linked intact glycopeptides and de-glycopeptides were analyzed by LC-MS/MS. From N-linked de-glycopeptides datasets, 764 N-linked glycoproteins, 1,699 N-linked glycosites and 3,328 unique N-linked de-glycopeptides were identified. The spectra of these N-linked de-glycopeptides were utilized for N-linked de-glycopeptides library construction and identification of N-linked intact glycopeptides. Four types of N-linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the N-linked de-glycopeptides, and two different N-Glycan mass databases including 27 modified N-Glycan masses and 712 unmodified N-Glycan masses were combined and utilized during spectral library search for identification of N-linked intact glycopeptides. In total, from the N-linked intact glycopeptides datasets, 526 N-linked glycoproteins, 1,036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-Glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level and N-linked de-glycopeptides level.

			Action	DRS
	XB03428B2PIIg_S1.pepXML	Pepxml
	XB03428B2PIIg_S1_1.raw	Raw
	XB03428B2PIIg_S2.pepXML	Pepxml
	XB03428B2PIIg_S2.raw	Raw
	XB03428B2PIIg_S3.pepXML	Pepxml

Dataset Information

Comprehensive N-glycoproteomic profiling of humanized antibody expressed in transgenic mouse serum

Dataset's files

Publications

<i>In Vivo</i> engineering of transgenic mice for systemic human neutralizing antibody production against staphylococcal enterotoxin B.

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Dataset Information

Comprehensive N-glycoproteomic profiling of humanized antibody expressed in transgenic mouse serum

Dataset's files

Publications

&lt;i&gt;In Vivo&lt;/i&gt; engineering of transgenic mice for systemic human neutralizing antibody production against staphylococcal enterotoxin B.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

<i>In Vivo</i> engineering of transgenic mice for systemic human neutralizing antibody production against staphylococcal enterotoxin B.