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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 signal-to-noise ratio for the analyte. 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 collision-induced...
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Mass Spectrometry: Molecular Fragmentation Overview01:20

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The ionization of a molecule into a molecular ion inside the mass spectrometer causes instability in the molecule's structure due to the loss of an electron. This eventually leads to the fragmentation or breaking of some bonds in the molecule. The fragmentation occurs predominantly at specific bonds to yield relatively stable fragments.
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Peptide Identification Using Tandem Mass Spectrometry01:33

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Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
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Mass Spectrometry: Carboxylic Acid, Ester, and Amide Fragmentation01:01

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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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MALDI-TOF Mass Spectrometry01:19

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Mass spectrometry is a powerful characterization technique that can identify and separate a wide variety of compounds ranging from chemical to biological entities, based on their mass-to-charge ratio (m/z). The instruments that allow this detection, known as mass spectrometers, have three components: an ion source, a mass analyzer, and a detector. These spectrometers differ based on the nature of their ion source and analyzers.
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Mass Spectrometry: Amine Fragmentation00:55

Mass Spectrometry: Amine Fragmentation

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Amines can be identified using mass spectroscopy based on their characteristic fragmentation patterns. The molecular ions of amines undergo fragmentation via ⍺-cleavage. The ⍺-cleavage of the carbon-carbon bonds in amines generates an alkyl radical and resonance-stabilized nitrogen-containing cation.
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Internal Fragments Generated from Different Top-Down Mass Spectrometry Fragmentation Methods Extend Protein Sequence

Muhammad A Zenaidee1, Benqian Wei1, Carter Lantz1

  • 1Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California 90095, United States.

Journal of the American Society for Mass Spectrometry
|June 8, 2021
PubMed
Summary

Top-down mass spectrometry (TD-MS) can now utilize internal fragments for enhanced protein sequencing. This method improves sequence coverage by 15-20%, enabling more reliable modification site localization.

Keywords:
CADUVPDelectron capture dissociationelectron ionization dissociationinternal fragmentstop-down mass spectrometry

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Top-down mass spectrometry (TD-MS) analyzes intact proteins, generating fragment ions correlated to the primary sequence.
  • Traditionally, internal fragments in TD-MS are avoided due to assignment ambiguities.
  • Existing methods primarily rely on terminal fragments (N- or C-terminus).

Purpose of the Study:

  • To demonstrate the utility and assignment of internal fragments in TD-MS.
  • To show that internal fragments provide valuable sequence information.
  • To increase the extent of protein primary sequence coverage in TD-MS analysis.

Main Methods:

  • Utilized collision-based, electron-based, and photon-based fragmentation methods in TD-MS.
  • Analyzed three model proteins: cytochrome c, myoglobin, and carbonic anhydrase II.
  • Included both terminal and internal fragment assignments for comprehensive analysis.

Main Results:

  • Internal fragments were successfully formed and assigned across different fragmentation techniques.
  • Inclusion of internal fragments increased protein sequence coverage by approximately 15-20%.
  • Achieved no less than 85% sequence coverage for the tested proteins, nearing complete coverage when combined with terminal fragments.

Conclusions:

  • Internal fragments are rich in sequence information and reliably assignable in TD-MS.
  • Integrating internal fragments significantly enhances protein sequence coverage.
  • This approach enables deeper protein sequence analysis and more reliable localization of modification sites.