Statistical characterization of ion trap tandem mass spectra from doubly charged tryptic peptides - PubMed (original) (raw)

Statistical characterization of ion trap tandem mass spectra from doubly charged tryptic peptides

David L Tabb et al. Anal Chem. 2003.

Abstract

Collision-induced dissociation (CID) is a common ion activation technique used to energize mass-selected peptide ions during tandem mass spectrometry. Characteristic fragment ions form from the cleavage of amide bonds within a peptide undergoing CID, allowing the inference of its amino acid sequence. The statistical characterization of these fragment ions is essential for improving peptide identification algorithms and for understanding the complex reactions taking place during CID. An examination of 1465 ion trap spectra from doubly charged tryptic peptides reveals several trends important to understanding this fragmentation process. While less abundant than y ions, b ions are present in sufficient numbers to aid sequencing algorithms. Fragment ions exhibit a characteristic series-specific relationship between their masses and intensities. Each residue influences fragmentation at adjacent amide bonds, with Pro quantifiably enhancing cleavage at its N-terminal amide bond and His increasing the formation of b ions at its C-terminal amide bond. Fragment ions corresponding to a formal loss of ammonia appear preferentially in peptides containing Gln and Asn. These trends are partially responsible for the complexity of peptide tandem mass spectra.

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Figures

Figure 1

Figure 1

Peptide bond cleavage. Low-energy CID primarily cleaves peptide bonds, resulting in b ions (which contain the N-terminus and the atoms to the left of the dotted line) and y ions (which contain the C-terminus and the atoms to the right of the dotted line). b ions (pictured) generally take on an oxazolone structure, which may subsequently fragment to produce smaller b ions or lose carbon monoxide to form a ions. The remaining possible backbone ions (c, x, z) do not typically form under low-energy conditions.

Figure 2

Figure 2

Residue frequencies and content. The peptides included in this analysis have a somewhat modified composition with respect to the sequence database by which they were identified. The residues identified most often among the peptides had alkyl side chains. The six rarest residues in the identified peptides were the same as those seen least often in the sequence database. Cysteine was present in only 134 peptides, and Met and Trp were each present in 178 peptides.

Figure 3

Figure 3

Mass spectrum of the sequence AVDDFLLSLDGTANK. The y5 ion corresponds to the intensity of the median y ion for all spectra in this analysis. The base peak in the above spectrum, which was identified as y8, embodies 9% of the spectrum's total intensity. Identified b and y ions account for 32.4% of this spectrum's total intensity.

Figure 4

Figure 4

Intensity distributions of ion species. Small peaks are more common than intense ones for each series, but the more intense the peak, the more likely it is to represent an ion from the y series. The distribution of peak intensities extends beyond the most intense category in this graph: 31% of y peaks are more intense than 2% of the spectrum's summed intensity, as compared to 9% of b peaks, 3% of a peaks, and 1% of background (x) peaks.

Figure 5

Figure 5

Fragment ion peak height versus relative mass. Peak intensities are related to the relative masses of the fragment ions they represent. The horizontal axis gives masses of fragments as a proportion of precursor mass. The bar shows the intensity of the median peak for the collection of ions in a particular relative mass bin, with a line extending above and below to show the 75th and 25th percentile intensities. Missing peaks are assigned intensities of zero. The y series shows a distinct peak at ∼60% of precursor mass, while b peaks crest at 45%.

Figure 6

Figure 6

Unusual fragmentation of Pro. Because Pro's side chain forms a ring to its nitrogen, attack on its carbonyl carbon by the preceding carbonyl oxygen would result in a strained 5–5 bicyclic ring. Cleavage to its C-terminus is reduced, and cleavage to its N-terminus is encouraged, yielding a large differential between the fragment ion peaks adjacent the residue.

Figure 7

Figure 7

Ratio of intensity differences to intensity sums for individual residues. N-bias measures the extent to which each residue directs local fragmentation. The statistic measures the difference between the N-terminal peak intensity and the C-terminal peak intensity, normalizing this difference by the sum of the two peak intensities. Residues with N-biases of greater absolute value impact local fragmentation to a greater extent. The median bias for each residue is marked by the line across each box, and the upper and lower edges of the boxes represent the 75th and 25th percentiles, respectively. The most distinctive bias toward N-terminal fragmentation is that of Pro. A smaller N-bias appears for Gly and Ser. Hydrophobics Ile, Leu, and Val show a bias toward C-terminal cleavage in y ions, and His shows a pronounced bias toward C-terminal cleavage in b ions.

Figure 8

Figure 8

Histidine cleavage. Histidine can form unusual b ions. In normal fragment ion formation, the carbonyl N-terminal to a residue attacks the carbonyl at the residue's C-terminus, resulting in an intermediate that produces a b ion with a single, five-membered ring. The side chain of His may short-circuit that process by attacking its own carbonyl, yielding a b ion with a double ring structure.

Figure 9

Figure 9

Composition ratio versus the residues in b-17 and y-17 ions. Fragment ions that exhibit prominent loss of ammonia are more likely to contain Asn or Gln than fragment ions in general. Ammonia loss is enhanced by His for y ions, but His suppresses the loss for b ions. Neutral loss of ammonia may be diminished by the presence of Pro and Met. The vertical axis shows the ratio between the sequence composition of the ions displaying intense loss peaks and the sequence composition of fragment ions from the appropriate series.

Figure 10

Figure 10

Composition ratio versus the residues in b-18 and y-18 ions. Fragment ions that exhibit prominent loss of water are more likely to contain Ser, Thr, or Glu. As seen in ammonia loss, His appears to enhance loss in y ions but diminish it in b ions. The mass accuracy of the ion trap, combined with the window for identifying peaks in DaughterDB, may result in the misidentification of some ammonia loss peaks as water loss peaks, thus resulting in Asn and Gln's ranking above.

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