Formation of y + 10 and y + 11 Ions in the Collision-Induced Dissociation of Peptide Ions
Tandem mass spectra of peptide ions, acquired in shotgun proteomic studies of selected proteins, tissues, and organisms, commonly include prominent peaks that cannot be assigned to the known fragmentation product ions (y, b, a, neutral losses). In many cases these persist even when creating consensus spectra for inclusion in spectral libraries, where it is important to determine whether these peaks represent new fragmentation paths or arise from impurities. Using spectra from libraries and synthesized peptides, we investigate a class of fragment ions corresponding to yn-1 + 10 and yn-1 + 11, where n is the number of amino acid residues in the peptide. These 10 and 11 Da differences in mass of the y ion were ascribed before to the masses of [+ CO – H2O] and [+ CO – NH3], respectively. The mechanism is suggested to involve dissociation of the N-terminal residue at the CH-CO bond following loss of H2O or NH3. MS3 spectra of these ions show that the location of the additional 10 or 11 Da is at the N-terminal residue. The yn-1 + 10 ion is most often found in peptides with N-terminal proline, asparagine, and histidine, and also with serine and threonine in the adjacent position. The yn-1 + 11 ion is observed predominantly with histidine and asparagine at the N-terminus, but also occurs with asparagine in positions two through four. The intensities of the yn-1 + 10 ions decrease with increasing peptide length. These data for yn-1 + 10 and yn-1 + 11 ion formation may be used to improve peptide identification from tandem mass spectra.
Key wordsPeptide CID Fragmentation y + 10 y + 11
High-throughput proteomics experiments generate large sets of tandem mass (MS/MS) spectra and require the use of a sequence search algorithm [1, 2], or a reference library [3–5], to match peptide sequences to the fragmentation patterns. Upon fragmentation in low energy collision-induced dissociation (CID), peptide ions typically form a, b, and y ions, and ions from losses of neutral molecules such as H2O and NH3. Other types of fragment ions have been reported, which originate from unusual neutral losses [6–8] or peptide sequence scrambling [9–11]. While several groups have studied peptide fragmentation based on trends from large sets of spectra [12–15], studies of single peptides [16–21], or computer modeling of fragmentation [22, 23], reliable prediction of major product ions and their intensities during CID is not yet possible. However, the accuracy of peptide identifications from tandem mass (MS/MS) spectra using search algorithms or reference libraries relies on the ability to provide accurate models or representative spectra for peptide fragmentation patterns [24–26].
The National Institute of Standards and Technology (NIST) peptide library of MS/MS spectra currently contains over 900,000 ion trap (IT) MS/MS spectra generated from the tryptic digests of samples from many organisms including humans, yeast, mice, and fruit flies . Before being included in the library, spectra undergo quality control to ensure that the correct peptide sequence is assigned to a high quality spectrum . A major factor used in assessing the quality of a spectrum is the fraction of intensity for the major product ions arising from known fragmentation pathways . While evaluating spectra, many are found that contain one or more significant peaks, which are not assigned to the customary y, b, a, and neutral loss product ions. During examination of unassigned peaks, it was noticed that many peaks had m/z values corresponding to yn-1 + 10 and yn-1 + 11, formed from peptides with n amino acid residues. Such processes have been observed with a few synthetic peptides and ascribed to yn-1 + CO-H2O and yn-1 + CO-NH3, respectively . In this study, we expand the observations to thousands of peptide ion MS/MS spectra from the NIST library and use this vast statistical information to characterize this type of fragmentation in an effort to develop rules that can be used to predict the formation of yn-1 + 10 and yn-1 + 11 ions.
2.1 Sample Preparation and LC-MS/MS Analysis
Tryptic tripeptides were synthesized in mixtures as described previously  and analyzed by high performance liquid chromatography with electrospray ionization tandem mass spectrometry using an ion trap (LTQ; ThermoFisher Scientific, Waltham, MA, USA), or a quadrupole time-of-flight (QTOF, model 6530; Agilent, Santa Clara, CA, USA) instrument. Peptides with longer sequences were synthesized individually and the peptide MRFA was purchased from Sigma Aldrich (St. Louis, MO, USA). Tandem mass spectra for selected peptide ions were acquired at 20 different collision voltages in a triple quadrupole mass spectrometer (QQQ, Micromass Quattro Micro; Waters Corp., Milford, MA, USA), as described previously , and peak intensities were plotted as a function of collision voltage. Fragmentation was also examined by MS3 experiments in the IT mass spectrometer.
2.2 Database Analysis
The NIST peptide library of MS/MS spectra from IT instruments was used to investigate the distribution and characteristics of peptide spectra with y + 10 and y + 11 fragment ions. The libraries are available for download without charge at http://peptide.nist.gov/. Programs written in-house were used to perform the statistical survey of the MS2 spectra having major y + 10 or y + 11 ions, in peptides with n amino acids, at positions n-1, n-2, or n-3. Peaks were assigned as y + 10 or y + 11 only if the observed m/z was within ±0.4 mass units of the expected value and no other assignment could be made. Peaks that may have been 13C isotopic peaks of assigned fragments were excluded. Spectra containing y + 10 or y + 11 peaks were then analyzed to determine the intensity of the ion relative to the largest peak in the spectrum and its dependence on adjacent amino acids. Analyses also examined y + 6, y + 8, and y + 12 ions to assess the magnitude of random peaks in the vicinity of the peaks of interest.
3.1 Observations on y + 10 and y + 11 Fragment Ions in the NIST Peptide Library
Following analysis of selected peptide MS/MS spectra in the NIST library, many peaks were identified that could be assigned as yn-1 + 10 or, less commonly, as yn-1 + 11 ions, formed from peptide ions with n amino acid residues. Spectra having peaks at yn-1 + 8 were also identified to serve as markers for random (noise) peaks. To better distinguish clear fragmentation products from spurious peaks, the fraction of peptide spectra having these peaks with intensity >5% (of the largest peak in the spectrum) were counted. These studies showed that yn-1 + 10 and yn-1 + 11 ions were generated at levels clearly above the random yn-1 + 8 peaks. Several peptide MS/MS spectra contained peaks ascribable to yn-2 + 10 and yn-3 + 10 ions, but the number of such spectra were too small to provide an assessment of their contribution.
3.2 Location of the Additional 10 Da
Experiments with synthetic peptides show that the yn-1 + 10 ions are formed during MS/MS and MS3. To determine the location of the addition in m/z, MS3 spectra were measured for the [y6 + 10]2+ ion from the triply charged albumin peptide LCVLHEK (Figure S12a) and the [y5 + 10]+ ions from the singly charged synthetic peptide PSFLYK (Figure S12b). These MS3 spectra show that all b and a ions present have an m/z increased by 10 Da but all y ions are lacking this additional 10 Da. This confirms the previous conclusion  that the N-terminal residue of the peptide is involved in the formation of the yn-1 + 10 ion.
3.3 Influence of Peptide Length
3.4 Influence of Peptide Sequence
To assess the validity of the small circles in Figure 3a, we created similar plots for [yn-1 + 8]+ ions (figure not shown, data are in the Supplementary Material Spreadsheet). In Figure 3a, there are 129 points with values > 0, with an average value of 7.5 %, but for the [yn-1 + 8]+ ions there are only 27 points with values > 0 and their average value was only 0.8 %. Of all the yn-1 + 8 points, only 11 have values greater than 10 % of the corresponding values for the yn-1 + 10 points. This shows that the amount of noise in Figure 3a is small. Data were also collected for yn-1 + 6 and yn-1 + 12 peaks (not shown) to serve as additional background noise for comparison with the data in the other plots in Figure 3 and the comparison affirms the validity of those plots.
The intensity of [yn-1 + 10]+ ions depends on the second residue as well. This is particularly so for doubly charged peptides with N-terminal P, where the highest values are predominantly associated with S and T in the second position (Figure 3a). N-terminal N generates significant [yn-1 + 10]+ ions with a number of different residues in the second position (most often with A, C, E, F, L, M, W, and Y) and, in contrast with P, it shows low intensities for S and T in the second position. N-terminal H generates significant [yn-1 + 10]+ with fewer amino acids in the second position (D, R, S, T) compared to N. When S or T residues are in the second position (Figure 3a), P is the N-terminal residue with the most intense [yn-1 + 10]+ ions, but F, I, and L also show high values. It should be pointed out with regards to N-terminal P that such peptides are less likely to be produced during tryptic digestion because of the specificity of trypsin. However, peptide ions with N-terminal P are frequently formed by in-source fragmentation and are expected to exhibit the same behavior as if produced by protonation of a peptide during ESI. We have demonstrated this similarity by synthesizing the peptide LLPHEFYAK and showing that the MS3 spectrum of its y9 ion, PHEFYAK, is identical with the MS/MS spectrum of the synthetic peptide PHEFYAK (data not shown).
Figure 3b shows the results for doubly charged peptide ions producing intense [yn-1 + 11]+ ions. The data for [yn-1 + 11]+ have been coarsely corrected for contribution from [yn-1 + 10]+ isotopic peaks by subtracting from the former peak the intensity of the latter. It is clear that N in the second position shows the largest values in this figure and indicates that the loss of NH3, necessary for formation of [yn-1 + 11]+ ions, takes place from this residue. Among the N-terminal residues facilitating this process, H appears to have the strongest effect. It is possible that this basic residue helps localize the second proton of the doubly charged peptide at the N-terminus and, thus, facilitates loss of NH3 from the adjacent asparagine. It is also possible that H at this location increases the likelihood that the yn-1 + 11 fragment ion is formed with a single rather than double charge (note that the figure is for singly charged product ions only). An example of a spectrum of a peptide with terminal HN showing a [yn-1 + 11]+ ion is in Figure 1d. While Figure 3b shows data for N in the second position, there were also spectra found to contain yn-1 + 11 ions when N is located in position 3 (at about the same level) and position 4 (at about 30% of that level) (sample spectra are shown in Figures S5–S7).
Plots similar to those in Figure 3a and b were created for several other combinations of peptide charge and yn-1 + 10 product ion charge. Of these, we show in Figure 3c the results for doubly charged peptides producing doubly charged yn-1 + 10 ions. Although both Figure 3a and c are for doubly charged peptide precursor ions, there is a profound difference between the sequence dependence for formation of singly (Figure 3a) versus doubly (Figure 3c) charged yn-1 + 10 ions. In both figures, the N-terminal residues most frequently associated with intense yn-1 + 10 ions are N and P. However, the highest intensities for amino acid residues paired with N-terminal N and P are different for doubly charged peptide ions. The highest values in Figure 3c are for [yn-1 + 10]2+ ions are formed from peptides with N-terminal PH and NK. Peptide sequences starting with PH also give exceptionally high intensity [yn-1 + 10]2+ peaks (see Figure 1b for an example). The basic residues H and K attract the second proton and increase the likelihood that the yn-1 + 10 ion produced is doubly charged. It is not clear, however, why the effects of H and K are not the same with both P and N at the N-terminus.
For triply charged peptides forming doubly charged yn-1 + 10 ions (Figure 3d), the formation of [yn-1 + 10]2+ follows the same general trends as in Figure 3a (doubly charged peptides with singly charged yn-1 +10 ions). The highest intensity of the corresponding yn-1 + 10 ions in both plots are associated with N-terminal N and P. The effects of S and T in second position are greater in Figure 3d than in 3a and occur with many more N-terminal residues (A, B, F, H, I, K, L, M, N, N, S, V, W, and Y).
3.5 Results with Different Mass Spectrometers
While the results discussed above were obtained with ion trap (IT) mass spectrometers, synthetic peptides were analyzed with IT as well as with QQQ and QTOF mass spectrometers. In the latter two instruments the MS/MS spectra were recorded at increasing collision energies, which show the progression of fragmentation, resulting in MS/MS spectra with different relative peak intensities. The spectra obtained with all three instruments show mostly the same peaks but the relative intensities vary with collision energy; in general a spectrum taken at one of the intermediate collision energies was found to be very similar to that taken with the IT instrument. For the following discussion of the intensities of the yn-1 + 10 ions, we consider the maximal intensities, i.e., those obtained at the collision energy, which gave the highest value.
Similarly, other amino acids may have their own specific effects on the mechanism of yn-1 + 10 formation. For example, histidine residues at the N-terminal or second position may have enhanced y + 10 formation due to the ability to localize a proton and/or to form a six-membered ring (Scheme S2). When asparagine is in the second position, the side chain may attack the Ccarbonyl and lose either H2O or NH3 forming the experimentally observed yn-1 + 10 and yn-1 + 11 ions, respectively (Scheme S3). These mechanisms and structures are speculative and require further studies for confirmation.
In conclusion, the identification and characterization of the yn-1 + 10 and yn-1 + 11 peptide ions in the NIST peptide MS/MS library show trends in the data that can be used to create fragmentation rules that may be applied in sequence search algorithms to improve peptide identification. The specificity of the amino acid sequence at the N-terminus for forming intense yn-1 + 10 and yn-1 + 11 peaks can be used in de novo sequencing to determine the position of residues when this cannot be directly derived from the spectrum. These rules may also be used to improve the confidence of peak assignments and inclusion of peptide MS/MS spectra in reference libraries.
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