Dissociation Technique Technology Overview - US (2024)

Dissociation Technique Technology Overview - US (1)

What is molecular dissociation?

Molecular dissociation, which is also called fragmentation, enables more complete sequence and structural information to be obtained from the mass spectra of analyte ions. Tandem mass spectrometry or MS/MS is performed by isolating a precursor ion, imparting internal energy causing fragmentation and measuring the resulting product ions. Sequential or multistage dissociation may be applied to particularly large or complex molecules such as proteins and lipids.

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Why are fragmentation techniques important in mass spectrometry (MS)?

To better understand the molecular structure, tandem mass spectrometry is performed to convert the precursor ion into product ions prior to mass spectral acquisition. Tandem mass spectral analysis provide a molecular "fingerprint" that can help a scientist to determine its molecular structure. In diagnostics and applied laboratories, several fragmentation stages may be used to confirm the identity of a compound. For example, MS^3 is performed by first isolating the precursor ion, dissociating it, isolating an MS^2 product ion, and dissociating it and measuring the resulting MS^3 product ions.

Which molecular dissociation processes are used?

Over the years, various MS dissociation techniques have been developed. Most of these techniques are dependent on the configuration of the mass spectrometer. Some techniques are coupled with distinct ionization processes.

  • Collision-induced dissociation (CID) or Collisionally-activated dissociation (CAD):
    CID or CAD refers to the process whereby the ions kinetic energy has been increased prior to colliding with neutral molecules converting kinetic energy to internal energy that can induce fragmentation.

CID is associated with ion trap activation that is performed by applying an on-resonance RF for a user-defined duration. The increased kinetic energy of the activated ion undergoes successive collisions with neutral helium slowly building internal energy until fragmentation occurs. The resulting product ions are no longer in resonance with the applied RF and thus "cooled" to prevent secondary fragmentation. Generally, the lowest energy fragmentation pathways are primarily accessed.

Triple quadrupole (QQQ) mass spectrometers use CID to fragment analyte ions. The first Q (Q1) of the triple quadrupole typically consists of a mass filter, which selects a precursor ion for HCD in the collision cell (Q2) where the remaining precursor and all subsequent product ions enter Q3, Q3 then filters subsequent ion transmission based on mass to charge ratios (m/z) where discreet ions exit Q3 for detection. A triple quadrupole mass spectrometer can acquire a full scan MS/MS spectrum by scanning Q3 across a user-defined m/z range or Q3 can be set to filter a narrow m/z range corresponding to a specific product ion representing selected reaction monitoring (SRN).

  • Higher-energy collisional dissociation (HCD): HCD is a CID technique associated with Thermo Scientific Orbitrap instruments. Voltage offsets between components increase the kinetic energy of the precursor ion and disscociated in the ion routing multipole cell from collisions with nitrogen molecules. After which they are transferred back to the C-trap and then injected into the Orbitrap mass analyzer for detection. Figure 1 provides a generic schematic of the HCD cell, C-trap and Orbitrap mass analyzer. Because HCD generates and traps fragment ions of low m/z in the cell, it is useful for detecting protein modifications such as phosphotyrosine, and for tandem mass tag (TMT) experiments [1].
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Figure 1. The Ion-Routing Multipole or HCD located inside the Thermo Scientific Mass Spectrometer fragments ions prior to their separation and analysis.

  • Because HCD is able to resolve ions of lower molecular masses, it is useful for detecting protein modifications such as phosphotyrosine, and for stable isotope labeling by amino acids in cell culture (SILAC) experiments [1].HCD can also be performed on Tribrid mass spectrometers as it also has the ion routing multipole. The Tribrid mass spectrometer is different in that the ion routing multipole can direct product ions into either the dual pressure linear ion trap or Orbitrap mass analyzer. Performing HCD outside of the linear ion trap enables detection of low-mass product ions that can be missed using on-resonance CID.
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Figure 2. Data generated using CID and HCD technologies can be combined to provide better sequence coverage of proteins and peptides.

  • Electron transfer dissociation (ETD): ETD induces fragmentation by transferring electrons from a singly charged anion to higher charge state (e.g., +3) cationic molecules, including large molecules such as peptides and whole proteins. Because ETD induces fragmentation of the amide (N-C ) bonds along the peptide/protein backbone, complementary c- and z-type fragment ions, are generated. It is preferentially used in metabolomics and biopharmaceutical applications to analyze and preserve labile post-translational modifications (PTMs) [2].
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Figure 3. ETD transfers electrons to higher charge state cationic molecules, inducing fragmentation along peptide amide bonds.

In contrast, CID-induced fragmentation of peptides/proteins often results in complementary N-terminal b- and C-terminal y-type ions [3] as well as cleavage of the posttranslational modification (PTM) reducing the ability to determine the modification site. Because peptide sequence information from ETD and CID spectra are complementary, the two dissociation technologies are often toggled to improve sequence coverage and increase protein ID confidence.

ETD has also been coupled to legacy ion trap and hybrid ion trap-Orbitrap mass analyzers, using a nano ESI source that receives a supply of reagent and a high voltage discharge pin that generates the electrons.

  • Electron capture dissociation (ECD): Like ETD, ECD induces fragmentation of higher charge state cationic molecules. While ECD is used primarily in Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, there are applications for it within quadrupole ion trap mass spectrometers [4, 5].
  • Ultraviolet photodissociation (UVPD): Unique to Thermo Scientific Tribrid mass spectrometers, UVPD differs from CID and HCD in that it utilizes photons generated from a laser source to increase the internal energy of a selected precursor ion until there is sufficient internal energy present to overcome the barrier to dissociation. The UVPD laser is embedded in the mass spectrometer chassis and the fragments can be generated in the linear ion trap for subsequent detection by either the ion trap or Orbitrap mass analyzer.

Precursor ions are confined within an ion trap and irradiated with UV light, which may take the form of laser pulses. Photons are directly absorbed by target molecules depending on their UV absorption profile. Once a sufficiently excited state is reached, the barrier for dissociation is overcome and fragmentation is induced.

To avoid continued dissociation of the product ions, RF excitation is applied to the mass range not overlapping with the precursor m/z value to expand the ion motion out of the path of the laser beam.

UVPD, when conducted in an ion trap, is not limited by inherent low-mass cut-off during the detection of product ions, which is a shortcoming of CID. For this reason, UVPD is often combined with other molecular dissociations, completing the mass spectral profile.

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Figure 4. Fragmentation of luteolin 8-C-glycoside (Orientin) by UVPD and HCD produces more complete mass spectra and unique compound fragmentations.

Why are there multiple molecular dissociation techniques?

Needs to analyze different types of molecules and the various MS technologies available, have led to the development and use of different dissociation techniques. When using MS for structural elucidation, scientists typically start with well-established dissociation techniques such as CID, HCD, and ETD. Each of these techniques are well characterized for large- or small-molecule applications and are supported by software to predict fragmentation and reference mass spectral libraries to evaluate the MS data generated. In addition, each dissociation method can generate slightly different product ions that can provide complementary information to help characterize compounds. When traditional fragmentation methods don’t lead to unambiguous characterization of compounds, UVPD offers a complementary technique.

References

  1. Olsen JV, Macek B, Lange O et al (2007) Higher-energy C-trap dissociation for peptide modification analysis Nat Methods (4): 709-712. PubMed
  2. Han H, Xia Y, McLuckey SA (2007) Ion Trap Collisional Activation of c and z• Ions Formed via Gas-Phase Ion/Ion Electron Transfer Dissociation J Proteome Res 6(8): 3062-3069. PMC
  3. Creese AJ, Cooper HJ (2007) Liquid Chromatography Electron Capture Dissociation Tandem Mass Spectrometry (LC-ECD-MS/MS) versus Liquid Chromatography Collision-induced Dissociation Tandem Mass Spectrometry (LC-CID-MS/MS) for the Identification of Proteins J Am Soc Mass Spectrom 18(5): 891–897. PMC
  4. Sleno L, Volmer DA (2004) Ion activation methods for tandem mass spectrometry J. Mass Spectrom. 39: 1091-1112. PubMed
  5. Zhurov KO,Fornelli L,Wodrich MD et al (2013) Principles of electron capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis Chem Soc Rev 42: 5014-5030. PubMed

Mass Spectrometry Resource Library

Learn more about how dissociations elucidate molecular information at our library of applications notes, scientific posters, webinars and more.

Related products

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Related Resources

  • Mass Spec Analysis Technical Handbooks
  • Case study: Nano ESI increases peptide/protein identification
  • The “ETD-like” Fragmentation of Small Molecules

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