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Related Concept Videos

Mass Spectrum01:23

Mass Spectrum

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A mass spectrum is the graphical representation of the relative abundance of the charged fragments in an analyte plotted against their mass-to-charge ratio (m/z). The plot's x-axis represents the ratio of the mass of the charged fragment to the number of charges it carries. The y axis of the plot represents the relative abundance of each charged species. The relative abundance is calculated from the signal intensity of each charged species recorded at the detector. The most intense signal (the...
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Tandem Mass Spectrometry01:21

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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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An unknown compound can be established by identifying the molecular ion peak in the mass spectrum. The molecular ion peak is often weak or absent due to the predominance of fragmentation in high-energy electron beams. In such cases, a soft-energy electron beam can be used to scan the spectrum to enhance the intensity of the molecular ion peak. Additionally, chemical ionization, field ionization, and desorption ionization spectra are used to obtain a relatively intense molecular ion peak.To...
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Deconvolution01:20

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Deconvolution, also known as inverse filtering, is the process of extracting the impulse response from known input and output signals. This technique is vital in scenarios where the system's characteristics are unknown, and they must be inferred from the observable signals.
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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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The relative amounts of reactants and products represented in a balanced chemical equation are often referred to as stoichiometric amounts. However, in reality, the reactants are not always present in the stoichiometric amounts indicated by the balanced equation.
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Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation
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Limits for Resolving Isobaric Tandem Mass Tag Reporter Ions Using Phase-Constrained Spectrum Deconvolution.

Christian D Kelstrup1, Konstantin Aizikov2, Tanveer S Batth1

  • 1The Novo Nordisk Foundation Center for Protein Research , University of Copenhagen , 2200 Copenhagen , Denmark.

Journal of Proteome Research
|September 18, 2018
PubMed
Summary

Optimizing tandem mass tag (TMT) quantification requires balancing mass resolution and speed on Orbitrap instruments. We determined the minimum resolution needed for accurate TMT reporter ion separation, enhancing proteome depth.

Keywords:
OrbitrapSDMTMT multiplexingTMT10-plexphase-constrained spectrum deconvolutionquantitative proteomicsresolution requirementsignal processingsuper-FT resolutionΦ

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Area of Science:

  • Proteomics and Mass Spectrometry
  • Analytical Chemistry

Background:

  • Isobaric labeling techniques like tandem mass tags (TMT) are crucial for multiplexed proteome quantification.
  • Reporter ion quantification relies on fragment ions generated during tandem mass spectrometry.
  • Increasing multiplexing levels demands higher mass resolving power for TMT reporter ions, potentially conflicting with faster peptide sequencing on Orbitrap instruments.

Purpose of the Study:

  • To define the lower mass resolution limits for resolving TMT reporter ions with small mass differences (0.0063 Da).
  • To guide optimal Orbitrap instrument settings for highly multiplexed TMT-based proteomics experiments.
  • To balance proteome depth and quantification accuracy/precision.

Main Methods:

  • Utilized an ultra-high-field Orbitrap mass spectrometer.
  • Investigated TMT reporter ion separation at varying transient acquisition times (64 ms and 32 ms).
  • Employed phase-constrained spectrum deconvolution for data processing.

Main Results:

  • Established the minimum resolving power required for separating TMT reporter ions with 0.0063 Da mass differences.
  • Demonstrated that a 64 ms transient acquisition time provides sufficient resolution for TMT reporter ions with up to 16-fold ratio changes.
  • Showed that a 32 ms transient with phase-constrained deconvolution increased identifications by over 50% with >99% quantification, albeit with minor impacts on precision and accuracy.

Conclusions:

  • Optimal Orbitrap resolution settings for TMT quantification depend on reporter ion ratios and desired proteome depth.
  • Faster acquisition (32 ms transient) with advanced processing can enhance proteomic coverage while maintaining high quantification rates.
  • These findings provide practical guidance for optimizing TMT-based proteomics experiments on Orbitrap mass spectrometers.