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Middle-Down Mass Spectrometry Characterization of
Monoclonal Antibody Enabled by Electron-Activated Dissociation on
a Time-of-Flight Platform: A Case Study of Methionine Oxidation PTM
Identification and Quantification
Abdulafeez Akinloye (11453989)
Leigh Donnellan (20456138)
Parul Mittal (86140)
Clifford Young (458349)
Mariam Nassiri (25133535)
Mark R. Condina (2029189)
Nathan Edwards (1849612)
Alok Shah (5954273)
Peter Hoffmann (232366)
Biophysics
Biochemistry
Medicine
Physiology
Chemical Sciences not elsewhere classified
Sociology
Inorganic Chemistry
Infectious Diseases
Computational Biology
support formulation development
strong correlation (<
peptide mapping data
large intact molecules
control mab samples
analyzed via lc
methionine oxidation localization
monoclonal antibody enabled
translational information profiling
mass spectrometry characterization
broader sequence coverage
monoclonal antibody products
using methionine oxidation
specific oxidation localization
quantification characterizing post
hr </ sup
ms methods offer
rapidly characterize ptms
2 </ sup
antibody products
specific post
specific characterization
quantification using
oxidation levels
r </
translational modifications
sequence confirmation
subunits using
>< sup
widely adopted
simultaneous identification
poor gas
phase fragmentation
multiple attributes
molecular connectivity
m192 ),
m101 ),
locations confirmed
light chain
ides enzyme
high complexity
flight platform
artifact generation
activated dissociation
Characterizing
post-translational modifications (PTMs) in monoclonal
antibody products is critical for ensuring product quality attributes
understanding and monitoring. While the bottom-up approach is widely
adopted, it suffers from the loss of molecular connectivity and is
susceptible to artifact generation. Conversely, top-down approaches
face limitations due to the high complexity and poor gas-phase fragmentation
of large intact molecules to get site-specific post-translational
information profiling. Middle-down MS methods offer a complementary
middle-ground approach to both bottom-up and top-down, with broader
sequence coverage, and facilitate the simultaneous identification
of multiple attributes and proteoforms. In this study, we optimized
the electron-activated dissociation (EAD) approach for middle-down
sequencing of NISTmAb subunits and present a potential MS and MS/MS
strategy to rapidly characterize PTMs on antibodies, using methionine
oxidation as an example. Peroxide-stressed and control mAb samples
were digested and reduced into subunits using the IdeS enzyme and
analyzed via LC-MS, with the optimized MRM<sup>HR</sup> EAD method.
The resulting data enabled high-resolution subunit mass analysis,
sequence confirmation, and potential for site-specific oxidation localization.
Methionine oxidation localization in NISTmAb was achieved in light
chain (M4), Fd’ (M34, M101), and Fc/2 (M16, M122, M192), with
these locations confirmed with bottom-up peptide mapping data. Quantification
using the Fc/2 subunit enabled detection of oxidation levels up to
1% relative, with strong correlation (<i>R</i><sup>2</sup> > 0.99) between expected and observed values using MS1 data.
This
middle-down electron-activated dissociation (EAD) approach provides
a potential method for site-specific characterization of PTMs in antibody
products, offering valuable complementary mapping to support formulation
development and production.
2026-09-28T00:00:00Z
Text
Journal contribution
10.1021/acs.analchem.6c03432.s001
https://figshare.com/articles/journal_contribution/Middle-Down_Mass_Spectrometry_Characterization_of_Monoclonal_Antibody_Enabled_by_Electron-Activated_Dissociation_on_a_Time-of-Flight_Platform_A_Case_Study_of_Methionine_Oxidation_PTM_Identification_and_Quantification/34018734
CC BY-NC 4.0