Mass Spectrometry

Use of ECD and ETD Fragmentation Mechanisms for Peptide Sequencing

In the previous instalment of MS Solutions, I described some of the problems encountered with collisionally-activated dissociation (CAD) of peptides when used in MS/MS sequencing experiments. We noted that the number and type of structurally significant ions produced is dependent on the sequence of amino acid residues. Some sequences, most notably those containing a a C-terminal arginine, result in very little in the way of useful MS/MS spectra.

Recently, two new techniques have arisen which result in more complete fragmentation with better predictability for multiple-charge precursor ions. The techniques are electron capture dissociation (ECD) and electron transfer dissociation (ETD). The mechanism of dissociation in both cases is the same, the difference being in the source of the electrons.

MS Solutions #18: Use of ECD and ETD Fragmentation Mechanisms for Peptide Sequencing-1
Figure 1: Sequence of the example peptide showing the location of phosphoserine residues and the expected m/z values for single-charge b, c, y, and z ions.

   The mechanism for ETD and ECD involves electron capture neutralization of a charge site on a
multiple-charge ion resulting in formation of a radical cation:

[M+n H]+n + e– → [M+n H]+(n-1)• → c and z product ions

   Cleavage occurs at the amine bond proximal to the radical site, i.e. to the bond between the peptide bond nitrogen and the α-carbon along the peptide backbone. Referring to Figure 1 in my earlier MS Solutions article “Peptide Sequencing with Electrospray LC/MS Part 1” we see that this bond cleavage produces c and z product ions.

   Advantages of ETD and ECD vs conventional CAD for peptide sequencing:

  • ETD/ECD produces a predictable, homologous series of c and z ions as the principle products (secondary reactions form a and y ions). CAD generally forms b and y ions but without the same degree of predictability or homogeneity.
  • Fragmentation by ECD/ETD is sequence independent. We have seen in previous MS Soutions that this is not true for CAD.
  • Post-translational modifications (PTMs) are preserved with ECD/ETD but are frequently lost in CAD fragmentation.
  • ETD/ECD fragmentation efficiencies allow sequencing of longer peptides (20-30 residues), whereas CAD fragmentation is limited to 20 or fewer residues.

   The ECD technique is used in conjunction with an electrospray interface and a fourier-transform ion cyclotron resonance (FTICR, also called a magnetic ion trap), mass spectrometer. The source of electrons is a simple heater filament. ETD is specifically designed to work with ESI/quadrupole ion-trap instrumentation (both 2D or linear ion traps, and traditional 3D traps may be used). In ETD, odd-electron anions (e.g., C14H9– •, C14H11– •) are injected into the trap and electron transfer to the multiply-charged analyte species takes place via ion-ion reactions.

   These techniques may be combined with traditional CAD. ETD used in conjunction with CAD (known as ETcaD), can provide nearly 90% sequence coverage in peptide sequencing by MS. The combination of ECD and CAD (called AI-ECD or “activated ion- electron capture dissociation”), is employed in so-called “top-down” sequencing for intact protein precursors as large as 45 KDa.

   The determination of the type and location of post-translational modifications (PTMs) of proteins is an important elements of proteomic analysis. PTMs change the biological activity of proteins and nearly all mammalian proteins are modified in some way between translation and site of action. Phosphorylation (addition of HPO3 to S, T, and Y residues), is a common PTM. The example shown here compares conventional MS/MS using CAD fragmentation with electron-transfer dissociation (ETD) for the eight-residue phosphorylated peptide ERpSLpSRER*. A triple-charge precursor ion is selected for these experiments. In Figure 1 we see the phosphorylation sites on the peptide and the predicted masses for single-charge b, c, x, and y ions. 

MS Solutions #18: Use of ECD and ETD Fragmentation Mechanisms for Peptide Sequencing-2
Figure 2: CAD experiment on the peptide ERpSLpSRER using a +3 precursor ion.

   In the CAD experiment in Figure 2, very little useful information is obtained. The principle products are, as expected, multiple-charge variants of the precursor ion with the loss of H3PO4 from the phosphoserine residues. No structurally significant ions are observed. The ETD experiment in Figure 3, however, yields (1) a complete series of c and z ions which allow a complete sequence determination and (2) the phosphorylation PTMs are preserved and phosphoserine can be observed as a neutral-loss which permits an exact determination of the number and location of phosphorylation sites on the peptide. Note also that charge reduction by electron capture and subsequent loss of hydrogen from the +3 percursor results in intact [M+H]+1 and [M+2H]+2 peptide ions.

MS Solutions #18: Use of ECD and ETD Fragmentation Mechanisms for Peptide Sequencing-3
Figure 3: ECD experiement on the peptide ERpSLpSRER using a +3 precursor ion.

   ECD requires purchase of a linear ion-trap–FTICR hybrid mass spectrometer and associated ECD options. These are very expensive instruments which also require a very well-trained operator. But for those applications requiring predictable fragmentation with super-high resolving power and exact mass determinations (e.g., top-down sequencing of very large, intact protein ions), the investment is well worth it. Recently, ECD has become available on linear ion-trap–Orbitrap hybrid instruments as well. These instruments are somewhat less expensive and easier to use than those hybrids containing an FTICRMS.

   ETD can be had as an option for 3D (traditional) and linear ion-trap mass spectrometers and represents a lower level of investment than ECD but, of course, without the high performance of the FTICR or Orbitrap instrument.

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