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Related Concept Videos

Peptide Identification Using Tandem Mass Spectrometry01:33

Peptide Identification Using Tandem Mass Spectrometry

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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: 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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Mass Spectrometry: Overview01:19

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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 electrospray 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...
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Next-generation Sequencing03:00

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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Related Experiment Video

Updated: Aug 22, 2025

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
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Free Radical-Based Sequencing for Native Top-Down Mass Spectrometry.

Carolina Rojas Ramírez1, Rayan Murtada2, Jinshan Gao2

  • 1Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.

Journal of the American Society for Mass Spectrometry
|November 8, 2022
PubMed
Summary
This summary is machine-generated.

Free-radical-initiated peptide sequencing (FRIPS) with TEMPO reagents enhances native top-down proteomics. This method improves protein complex sequence coverage and fragment ion production without disrupting complex structure.

Keywords:
free-radical-initiated peptide sequencingnative mass spectrometrytop-down mass spectrometry

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Area of Science:

  • Proteomics and Mass Spectrometry
  • Biochemistry and Molecular Biology

Background:

  • Native top-down proteomics is crucial for identifying proteoforms and characterizing the structure of cellular protein complexes.
  • Tandem mass spectrometry (MS) faces challenges in native MS due to high analyte ion mass and low charge states, leading to low fragmentation efficiencies.
  • Current methods to improve sequence coverage often require specialized hardware and software.

Purpose of the Study:

  • To optimize free-radical-initiated peptide sequencing (FRIPS) labeling for native top-down sequencing experiments.
  • To evaluate the effectiveness of TEMPO-based FRIPS reagents in enhancing protein complex analysis.
  • To assess the impact of combining CID and FRIPS datasets on sequence coverage and fragment ion production.

Main Methods:

  • Optimization of TEMPO-based FRIPS labeling for intact protein complexes.
  • Application of FRIPS in conjunction with conventional Collision-Induced Dissociation (CID) experiments.
  • Analysis of protein complexes ranging from 36 to 106 kDa.

Main Results:

  • The FRIPS labeling approach successfully accessed intact complexes using TEMPO-based reagents without significant denaturation or assembly disruption.
  • Combining CID and FRIPS datasets resulted in sequence coverage improvements of up to 50% for protein complexes.
  • Fragment ion production increased by as much as 102% in these experiments.

Conclusions:

  • TEMPO-based FRIPS reagents significantly enhance sequence coverage in native top-down proteomics experiments.
  • This optimized FRIPS approach offers a valuable method for detailed characterization of protein complexes.
  • The integration of FRIPS with CID presents a powerful strategy for overcoming limitations in native MS-based protein analysis.