One of the more popular forms of fragmentation, collision-induced dissociation (CID, or collision-activated dissociation, CAD), uses energetic collisions to cause an immediate, single fragmentation. Briefly, the precursor ions selected in the MS1 phase are accelerated by electrostatic pulses and forced to collide with a large neutral target gas, such as helium or argon. When the precursor ions hit these curtain gasses, the weakest bond in the peptide breaks and creates smaller, fragmented ions. The amount of energy transferred is a function of the energy of the ion (Eion), the mass of the collision gas (mgas), and the mass of the ion (mion). For a single collision, the center of mass collision energy (Ecom) is represented by the following equation:
Ecom = Eion × (mgas / (mgas + mion))
As Ecom increases, the number of fragmentations increases because the internal ion energy increases. These spectra created by peptide fragments are usually visualized by plotting
each ion’s mass-to-charge ratio (m/z; x-axis) against the relative ion intensity (y-axis) 62
(Figure 1.3).
For protein analysis, one can expect MS/MS fragmentation to break peptides in predictable ways. In fact, there are a limited number of types of ions that one would expect to see. A common nomenclature for the types of ions from peptide fragmentation is described below (Figure 1.4).
The notation of an a, b, or c ion indicates that during the cleavage, the charge was on the fragment with the N-terminus. Conversely, the notation of an x, y, or z ion indicates that the charge was on the fragment with the C-terminus. The most common types of ions, b and y, represent a cleavage between the carboxyl and amide group. For each amino acid at position i within a peptide sequence of length L, the N-terminus ions can be thought of having a relationship with a paired C-terminus ion using the equation:
C(i) = L + 1 – N(i)
where N(i) represents the position of the N-terminus ion, defined by N(i) = i, and C(i) is the complementary C-terminus ion for a given amino acid position i.
Depending on the type of fragmentation method used, one is more likely to see different types of N- or C-terminus ions.
The mass difference between each peptide’s adjacent fragment ions represents a single amino acid, so one could manually take the differences between all of the peaks in the MS/MS scan, compare the mass differences to the masses of amino acids, and stitch the peptide sequence back together. Some complications in this process may arise due to the presence of different ion types, or additional peaks that represent noise or chemical additions that are not from the peptide fragmentation. Several computational algorithms have been developed to avoid these noise peaks and determine the best peptide-spectrum match (PSM).
Chromatogram
Survey Scan (MS1)
Fragmentation Scan (MS/MS)
Figure 1.3. Illustration of data collected in a tandem mass spectrometry run.
(A) The chromatogram reflects the separation of peptides by liquid chromatography, graphing the total collected intensity (TIC; y-axis) by time (minutes; x-axis). (B) The survey scan details which individual ions (m/z values) are observed. Their most abundant peaks are selected for fragmentation (MS/MS). (C) The fragment ions in an MS/MS scan can be used to sequence the peptide.
(A)
(B)
Figure 1.4. Illustration of collision induced fragmentation of a polypeptide.
A peptide backbone with four amino acid residues (R) and the types of fragment ions generated in a CID MS/MS spectrum. If the charge is retained on the N-terminal side, the fragment ion is classified as either a, b, or c. If the charge is retained on the C- terminal side, the ion type is x, y, or z.
N
N
O
O
O
R
1
R
2
R
3
N
O
OH
R
4
H
2N
x 3 y3 z3 x 2 y2 z2 x 1 y1 z1 a3 b3 c3 a2 b2 c2 a1 b1 c1To aid in this process, most algorithms do not work from spectral information to infer peptide sequences, but actually work in the opposite order. That is, because peptides fragment in predictable ways, in silico fragmentation can generate the ion series that is expected to be found in the MS/MS spectra from each peptide sequence. In CID, peptides generally
fragment along the peptide bonds (as opposed to backbone bond cleavages) to generate b and y ions. Figure 1.5 illustrates the b and y ion series predicted from the peptide sequence and which peaks in the spectra correspond to the expected m/z values, thus contributing to the peptide’s identification.
Energetic collisions often cleave off amino acids’ post-translational modifications, so one would not typically expect to see the addition of a phosphoryl group in the masses of the ions generated from CID. This “invisibility” of modifications is not strictly true for electron transfer dissociation (ETD or electron capture dissociation, ECD). ETD is similar in concept to CID except that it typically causes more cleavages than CID and different backbone cleavages. It requires low energy electrons and long reaction times, fragmenting at the most labile bonds. Because of the low-energy added, modifications typically stay on the amino acids so one can expect to see their addition to the m/z values for the ions. For example, if a peptide had a phosphorylated serine, one would expect the masses of the ETD ions that include the modified residue to be shifted at least the mass of a phosphorylation (79.979 Da), and thus greater than the corresponding ions CID would generate for the same peptide. However, even for unmodified peptides, one might not expect to see the same types of ions present for CID and ETD fragmentations. Whereas CID favors b and y ions, ETD more commonly produces c and z ions for certain peptides. Thus, the two techniques are complementary to each other. In fact, recent studies have favored the implementation of a decision-tree instrument setting that makes a decision about which fragmentation technique to use for a particular MS scan based on the charge and m/z ratio of the ion, whether CID or ETD is most likely to yield a better distribution of ions.
b b(+2) y y(+2) 187.087 94.047 1 W 15 1779.848 890.428 288.134 144.571 2 T 14 1593.769 797.388 359.171 180.089 3 A 13 1492.721 746.864 472.255 236.631 4 L 12 1421.684 711.346 529.277 265.142 5 G 11 1308.6 654.804 660.317 330.662 6 M 10 1251.579 626.293 773.401 387.204 7 L 9 1120.538 560.773 870.454 435.731 8 P 8 1007.454 504.231 1007.513 504.26 9 H 7 910.401 455.704 1136.556 568.782 10 E 6 773.342 387.175 1251.583 626.295 11 D 5 644.3 322.654 1348.635 674.821 12 P 4 529.273 265.14 1476.694 738.851 13 Q 3 432.22 216.614 1605.737 803.372 14 E 2 304.162 152.584 1761.838 881.422 15 R 1 175.119 88.063
Figure 1.5. A peptide-spectrum match.
(A) Peptide WTALGMLPHEDPQER (+2) matches 24 peaks in the MS/MS spectrum. Observed peaks that match the peptide’s b and y ions are highlighted in purple and blue. (B) The observed m/z values from the spectrum that matched the expected m/z values for the peptide are listed in the table in bold.
The most recently adopted fragmentation technique, higher energy collision dissociation (HCD), is most noteworthy for overcoming the limitation of CID fragmentation known as the “one-third rule,” that is, the loss of mass ions less than 1/3 the parent ion mass. Whereas CID converts kinetic energy to internal, mostly vibrational energy that affect the weakest bonds, HCD uses a beam-type energy that results in fragment ions with higher levels of energy, allowing for not only more primary, but also secondary dissociation to occur. The increased energy also reduces rearrangement reactions and increases the reproducibility of HCD fragmentation spectra of the same peptide. Furthermore, HCD and CID differ in where they physically occur within the instrument. HCD activates ions in the collision cell at the far side of the instrument, requiring ions to pass through the C- trap before they are analyzed by the orbitrap. In total, then, HCD is able to achieve high resolution and high accuracy for both the precursor and fragment scans. The tradeoff for these desirable figures of merit result in diminished sensitivity compared to CID as well as slower duty cycle. However, instruments that have this extra HCD collision cell also include a new device, termed an S-lens, that is touted to improve total ion current by 10- fold, arguably minimizing the disadvantages to HCD fragmentation. In tandem mass spectrometry analyses using HCD for fragmentation, one could either send the ions to the orbitrap or LTQ for measurements. Detection of an LTQ measurement requires the ions to be accelerated towards the curved surface of a dynode cup which then directs the ions to an electron multiplier, which amplifies the signal so that the ion current leaving the detector is an amplified intensity or signal. On the other hand, detection of ions in an orbitrap involves a broadband image current detection and fast Fourier transformation algorithm, which converts the frequency of each orbiting ion into a m/z signal. Because the orbitrap is much closer, sending ions there would take a shorter transmission time and would most likely result in a higher yield, not to mention very high resolution (~100k) and highly accurate mass measurements (<1 part per million). However, the MS/MS scans themselves would be much slower than if the ions were sent to the LTQ. Depending on the goal of the experiment, one analytical strategy may be more appropriate than the other.