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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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.
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.
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).
In contrast, CID-induced fragmentation of peptides/proteins often results in complementary N-terminal b- and C-terminal y-type ions  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.
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.
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.
Learn more about how dissociations elucidate molecular information at our library of applications notes, scientific posters, webinars and more.