PXD067893-1

PXD067893 is an original dataset announced via ProteomeXchange.

Title	Rapid High-Throughput Antibody Analysis Using Microwave-Assisted Digestion
Description	Monoclonal antibodies (mAbs) are a major class of biotherapeutic proteins that have been extensively developed over the past decade, resulting in improved standards of care for multiple diseases. Regulatory agencies recommend a Quality by Design (QbD) approach for manufacturing therapeutic proteins, which necessitates a comprehensive understanding of the molecular attributes critical for safety, efficacy, and product quality. In this context, multi-attribute methods (MAMs) have emerged as powerful tools for monitoring critical quality attributes (CQAs) in biotherapeutics, leveraging high-resolution Orbitrap mass spectrometry and automated computational pipelines for identification of pre-established modifications using data-dependent acquisition (DDA). In this study, we describe the development of a 17-minute, mass spectrometry–based workflow capable of monitoring a broad panel of post-translational modifications (PTMs), including key CQAs such as oxidation and deamidation. We evaluated microwave-assisted digestion (MW) under different buffer systems and pH conditions, assessing sequence coverage, missed cleavages, and the occurrence of chemical artifacts. Analyses were performed using both DDA and data-independent acquisition (DIA), with raw data processed in MaxQuant (DDA, dependent-peptide search) and Spectronaut (DIA, PTM-probing search), which enabled PTM discovery without prior knowledge. Our results demonstrate that microwave-assisted digestion, combined with precise control of temperature and pH, provides a fast and reliable alternative for efficient digestion of complex biomolecules, achieving high sequence coverage while minimizing artificial PTM formation, especially deamidation. DIA outperformed DDA in terms of sequence coverage and the identification of both modified and unmodified peptides, highlighting its potential for comprehensive characterization of therapeutic antibodies. Among the tested buffers, sodium acetate in MW was the most effective in reducing deamidation and oxidation levels.
HostingRepository	PRIDE
AnnounceDate	2026-01-27
AnnouncementXML	Submission_2026-01-26_16:14:22.946.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hellen Valerio
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	dihydroxylated residue; (R)-5-oxo-1; S-carboxamidoethyl-L-cysteine; sulfated residue; myristoylated residue; acetylated residue; monohydroxylated residue; iodoacetic acid derivatized residue; deamidated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Ascend

Revision	Datetime	Status	ChangeLog Entry
0	2025-08-29 20:44:12	ID requested
⏵ 1	2026-01-26 16:14:23	announced

10.1016/j.jprot.2026.105601;

de Toledo TM, Valerio HP, Neto AMM, Mule SN, Dos Santos Donado PR, Pascale CBA, Palmisano G, Rapid high-throughput antibody analysis using microwave-assisted digestion. J Proteomics, 326():105601(2026) [pubmed]

submitter keyword: data independent acquisition, post translational modifications,antibodies, data dependent acquisition, microwave-assisted digestion

Giuseppe Palmisano
contact affiliation	Glycoproteomics Laboratory, Institute of Biomedical Sciences, University of São Paulo
contact email	palmisano.gp@usp.br
lab head
Hellen Valerio
contact affiliation	Instituto de Ciencias Biomedicas, Universidade de São Paulo
contact email	hellenpvalerio@gmail.com
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/01/PXD067893

PRIDE project URI

• PRIDE
  1. PXD067893
     1. Label: PRIDE project
     2. Name: Rapid High-Throughput Antibody Analysis Using Microwave-Assisted Digestion