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Related Concept Videos

Mass Spectrometry: Carboxylic Acid, Ester, and Amide Fragmentation01:01

Mass Spectrometry: Carboxylic Acid, Ester, and Amide Fragmentation

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The fragmentation patterns observed for compounds such as carboxylic acids, esters, and amides in the mass spectra include ⍺-cleavage and McLafferty rearrangement. Fragmentation by ⍺-cleavage preferentially occurs at the carbon-carbon bond at the ⍺-position next to the carboxylic group to generate a neutral radical and a cation. Long chain compounds with hydrogen at their γ-carbon undergo McLafferty rearrangement to give a radical cation and a neutral alkene.
For example, the...
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Mass Spectrometry: Overview01:19

Mass Spectrometry: Overview

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Mass spectrometry is an analytical technique used to determine the molecular mass and molecular formula of a compound. The basic principle of mass spectrometry is to generate ions from the analyte molecule and measure these ion abundances against their molecular mass. One common type of ionization, known as electron ionization or EI, bombards the analyte molecules in the gas phase with high-energy electron beams. The electron beams displace an electron from the molecule and leave behind a...
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Tandem Mass Spectrometry01:21

Tandem Mass Spectrometry

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Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and reduce chemical noise during analyte detection. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N2, Ar, He, etc., is called...
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Mass Spectrometry of Amines01:15

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In mass spectroscopy, amines undergo fragmentation to give parent ions with odd molecule weights. This observed mass spectrum follows the nitrogen rule; a molecule with an odd number of nitrogen atoms produces a molecular ion with an odd molecular weight. Amines undergo fragmentation through α cleavage, producing nitrogen-containing cations—iminium ions—and alkyl radicals. Mass spectra of aromatic and cyclic aliphatic amines exhibit strong molecular ion peaks, but acyclic...
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Mass Spectrometry: Isotope Effect01:13

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Most elements exist in nature as a mixture of isotopes. The isotopes differ in weight due to their respective number of neutrons. The molecular weight of a molecule is different depending on the specific isotope of its elements involved. As a result, the mass spectrum of the molecule exhibits peaks from the same fragment at multiple positions. The positions of these mass signals depend on the mass differences between isotopes. Furthermore, the intensity of these signals is dependent on the...
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Chemical Ionization (CI) Mass Spectrometry01:21

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The molecular ion peak of a molecule in the mass spectrum provides vital information for molecular identification. However, conventional electron impact ionization can lead to the rapid dissociation of some molecular ions before they reach the detector. A milder ionization method is required to increase the lifetime of such ionized analyte molecules. Chemical ionization (CI) is a gas-phase protonation reaction useful for mass-analyzing analyte molecules that are easily protonated to yield the...
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Updated: Jan 30, 2026

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Quantitative Mapping of NHS Ester-Protein Reactivity Using Native Top-Down Mass Spectrometry.

Jack L Bennett1,2, Olivia B Ramsay1,3, Corinne A Lutomski1,2

  • 1Kavli Institute for Nanoscience Discovery, University of Oxford, Dorothy Crowfoot Hodgkin Building, Oxford OX1 3QU, U.K.

Journal of the American Society for Mass Spectrometry
|January 28, 2026
PubMed
Summary

This study introduces a mass spectrometry method to measure how covalent ligands react with intact proteins. This allows for precise analysis of protein modification patterns, improving the design of therapeutics and probes.

Keywords:
NHS esterscovalent modificationelectrophile reactivityintact proteinsnative top-down mass spectrometryproteoform analysis

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Area of Science:

  • Biochemistry
  • Proteomics
  • Chemical Biology

Background:

  • Covalent ligands are crucial for protein labeling and modulation.
  • Current methods analyzing modifications often use peptide-centric readouts, obscuring intact proteoform details.
  • Understanding modification colocalization and stoichiometry on intact proteins is limited.

Purpose of the Study:

  • To develop a general native top-down mass spectrometry workflow for quantifying electrophile reactivity directly on intact proteins.
  • To provide proteoform-level resolution for modification analysis.
  • To enable better design of covalent therapeutics, bioconjugates, and activity-based probes.

Main Methods:

  • Utilized native top-down mass spectrometry.
  • Applied a deconvolution framework to analyze reactivity of NHS esters (model electrophiles) at primary amines.
  • Focused on promiscuous, multisite modification patterns on intact proteins.

Main Results:

  • Quantified differential electrophile reactivity directly on intact proteins.
  • Preserved full modification connectivity without denaturation or digestion artifacts.
  • Demonstrated a method applicable to electrophiles with unknown reactivity.

Conclusions:

  • The presented workflow offers a general platform for analyzing covalent modification patterns at the proteoform level.
  • This approach overcomes limitations of peptide-centric analyses.
  • It facilitates the rational design of targeted covalent drugs and chemical probes.