Molecular Dissociation Technology Overview

Why is molecular dissociation required during mass spectrometry?

To better understand the structural composition of a molecule, and especially a larger molecule such as a peptide, a dissociation technique is used to break it up (i.e., induce fragmentation) into its smaller constituents prior to mass spec. As a result, the one large m/z peak that would have resulted from mass spectral analysis of the molecule instead becomes a set of two, three or more peaks. These additional peaks act as the molecule's "fingerprint", helping to confirm characteristics such as molecular groups and tags, oxidation states and degradation products.

In diagnostics and applied laboratories, several sequential dissociation processes may be used to confirm the identity of a compound. For example, biological samples being tested for cocaine metabolites typically undergo several rounds of molecular dissociation in forensic toxicology laboratories.

Which molecular dissociation processes are used?

Over the years, various mass spectrometry dissociation techniques have developed. Most of these techniques rely on a specific mass analyzer. Some techniques are coupled with distinct ionization processes.

• Collision-induced dissociation (CID) or Collisionally-activated dissociation (CAD): Often used in conjunction with GC-MS, CID, which is also called CAD, refers to the process whereby a an ion and a neutral species (e.g., helium, nitrogen, argon) collide, and the energy of this collision transfer to the internal energy of the ion, resulting in bond breakage and fragmentation. These fragments are then accelerated towards the mass analyzer. The molecular and/or atomic fragments then exit the collision cell and are analyzed by a detector.
  
  Triple quadrupole (QQQ) mass spectrometers use CID to fragment samples into their base components. The first Q (Q1) of the triple quadrupole typically consists of a mass filter, which selects a given ion and accelerates it towards the collision cell (Q2) of the spectrometer. Here, the ion is fragmented. After it exits the Q2, the mass ranges of the fragments are scanned by the third quadrupole (Q3).

• Higher-energy C-trap dissociation (HCD): HCD refers to a CID variation that uses a higher RF voltage to retain fragment ions in the C-trap. The HCD cell is used to fragment the ions, after which they are accelerated into, cooled down and stored inside of the C-trap. Ions are then injected into and separated inside the Orbitrap based on their rotational frequency differences. The schematic shown below features an HCD cell, C-trap and Orbitrap mass analyzer.

• 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].

• Electron transfer dissociation (ETD): ETD induces fragmentation by transferring electrons 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_α) bonds along the peptide/protein backbone, producing complementary c- and z-type fragment ions, it is preferentially used in metabolomics and biopharmaceutical applications to analyze and preserve labile post-translational modifications (PTMs) [2].

• In contrast, CID-induced fragmentation of peptides/proteins often induces cleavage of the PTMs (e.g., phosphorylations), resulting in complementary N-terminal b- and C-terminal y-type ions [3]. However, 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 is frequently coupled to 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 by transforming their electric potential energy to kinetic energy. 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): Also referred to as infrared multiphoton dissociation (IRMPD), this dissociation technology takes advantage of the energy of incident photons to raise the internal energy of target molecules.
  
  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 overfragmentation arising from further dissociation of the product ions, an excitation field is established within the ion trap such that the product ions, but not the precursor ions, are kinetically excited to trajectories that extend outside of the irradiated region.
  
  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 and HCD. For this reason, UVPD is often combined with other molecular dissociations, completing the mass spectral profile.

Why are there multiple molecular dissociation techniques?

Different molecule sizes and ionization states, as well as available technologies such as mass analyzers, have led to different dissociation processes to best induce fragmentation. Smaller molecules can often be successfully fragmented via CID, while larger molecules such as peptides and proteins are better suited to analysis by ETD. In fields such as proteomics, several dissociation methods must be used to characterize complex proteins that contain extensive branching and linkages.

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