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Comparative Study
. 2007 Apr 10;104(15):6140-5.
doi: 10.1073/pnas.0701130104. Epub 2007 Apr 2.

Protein identification by spectral networks analysis

Nuno Bandeira  1 , Dekel Tsur, Ari Frank, Pavel A Pevzner
Affiliations
Comparative Study

Protein identification by spectral networks analysis

Nuno Bandeira et al. Proc Natl Acad Sci U S A. .

Abstract

Advances in tandem mass spectrometry (MS/MS) steadily increase the rate of generation of MS/MS spectra. As a result, the existing approaches that compare spectra against databases are already facing a bottleneck, particularly when interpreting spectra of modified peptides. Here we explore a concept that allows one to perform an MS/MS database search without ever comparing a spectrum against a database. We propose to take advantage of spectral pairs, which are pairs of spectra obtained from overlapping (often nontryptic) peptides or from unmodified and modified versions of the same peptide. Having a spectrum of a modified peptide paired with a spectrum of an unmodified peptide allows one to separate the prefix and suffix ladders, to greatly reduce the number of noise peaks, and to generate a small number of peptide reconstructions that are likely to contain the correct one. The MS/MS database search is thus reduced to extremely fast pattern-matching (rather than time-consuming matching of spectra against databases). In addition to speed, our approach provides a unique paradigm for identifying posttranslational modifications by means of spectral networks analysis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Spectral network constructed by aligning spectra from overlapping peptides. (Left) Spectral network for 945 spectra representing different peptides from the fragment IVDLQRSPMGRKQGGTLDDLEEQARELYRRLREK of the human IKKβ protein. The spectral network is constructed without any knowledge of the peptide annotations. Each of 117 vertices in the spectral network corresponds either to a single MS/MS spectrum or to a consensus spectrum of multiple MS/MS spectra from the same peptide (derived by clustering). Two vertices are connected by an edge whenever the corresponding spectra form a spectral pair. (Center) A subnetwork of the entire spectral network spanning the fragment KQGGTLDDLEEQAREL (shown by red vertices Left). (Right) Paired peptides found by analyzing the Center spectral subnetwork with our paired spectra detection procedure.
Fig. 2.
Fig. 2.
Discovery of modifications by using spectral networks. (a) Histogram of absolute parent mass differences for all detected spectral pairs on the IKKβ data set; the y axis represents the number of spectral pairs with a given difference in parent mass. For clarity, we only show the mass range 1–100 Da. The peaks at masses 71, 87, and 99 Da correspond to amino acid masses, and the peaks at masses 14, 16, 18, 22, 28, 32, and 53 Da correspond to known modifications that were also found by Tsur et al. (7) using blind database search. The peak at mass 34 Da corresponds to a putative modification that remains unexplained to date. (b) Modification network for peptide MDVTIQHPWFK from the Lens data set. The shaded node was annotated as peptide + 42MDVTIQHPWFK by database search of the tag VTIQHP; the remaining nodes were annotated by iterative propagation. On each propagation, the source peptide annotation is combined with the modification determined by the spectral product to yield a new peptide annotation (different modifications are shown as edges with different colors).
Fig. 3.
Fig. 3.
Spectral products for terminal and internal modifications. (a) Spectral product for the theoretical spectra of the peptides TETMA and TETMAFR (all points at the intersections between the vertical and horizontal lines). The blue circles correspond to matching b ions in the two spectra; the red circles correspond to matching y ions. The blue and red circles are located on the blue and red diagonals. (b) Spectral product for uninterpreted spectra of the peptides TETMA and TETMAFR. The two diagonals in the spectral product matrix still reveal the points at which peaks from the spectrum at the top match peaks from the spectrum on the left. (c) Spectra S1,2b and S1,2y defined by the blue and red diagonals. (d) Spectral product for uninterpreted spectra with one internal modification. The top spectrum corresponds to an unmodified peptide, and the left-side spectrum corresponds to a modified peptide. In these cases it is not appropriate to construct Si,jb/Si,jy by simply selecting peaks on the diagonals. (e) The algorithm described in the text allows for modifications to occur in the middle of the peptide and separates the overlapping series of b and y ions (blue and red diagonals, respectively). The peaks selected from each spectrum by the blue/red diagonals are shown in the corresponding color.
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