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Review
. 2012 Sep;11(9):550-7.
doi: 10.1074/mcp.R112.018556. Epub 2012 May 17.

Peptide identification by tandem mass spectrometry with alternate fragmentation modes

Adrian Guthals  1 , Nuno Bandeira
Affiliations
Review

Peptide identification by tandem mass spectrometry with alternate fragmentation modes

Adrian Guthals et al. Mol Cell Proteomics. 2012 Sep.

Abstract

The high-throughput nature of proteomics mass spectrometry is enabled by a productive combination of data acquisition protocols and the computational tools used to interpret the resulting spectra. One of the key components in mainstream protocols is the generation of tandem mass (MS/MS) spectra by peptide fragmentation using collision induced dissociation, the approach currently used in the large majority of proteomics experiments to routinely identify hundreds to thousands of proteins from single mass spectrometry runs. Complementary to these, alternative peptide fragmentation methods such as electron capture/transfer dissociation and higher-energy collision dissociation have consistently achieved significant improvements in the identification of certain classes of peptides, proteins, and post-translational modifications. Recognizing these advantages, mass spectrometry instruments now conveniently support fine-tuned methods that automatically alternate between peptide fragmentation modes for either different types of peptides or for acquisition of multiple MS/MS spectra from each peptide. But although these developments have the potential to substantially improve peptide identification, their routine application requires corresponding adjustments to the software tools and procedures used for automated downstream processing. This review discusses the computational implications of alternative and alternate modes of MS/MS peptide fragmentation and addresses some practical aspects of using such protocols for identification of peptides and post-translational modifications.

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Figures

Fig. 1.
Fig. 1.
Complementary fragmentation in CID and ETD for peptide TAAANAAAGAAENAFRAP. CID (top) and ETD (bottom) spectra were separately identified against this C-terminal tryptic peptide at 1% FDR. Enough ions were separately detected in each spectrum to identify the peptide (65% of breaks in CID, 53% in ETD). But combining the two yields full coverage of all possible breaks, thus giving higher confidence to breaks observed in both spectra and possibly enabling full-length de novo sequencing. See Fig. 2 and supplementary Table S1 for evidence of CID/ETD complementarity over all identified spectra.
Fig. 2.
Fig. 2.
Ion statistics for alternative peptide fragmentation modes. (Left) Peptide MS2 ion statistics for alternative fragmentation modes - This shows the percentage of breaks observed by each ion type over all identified MS2 spectra with precursor charge 2 or 3 for each fragmentation method. z° corresponds to peaks at offset +H from z ions (94). Ions were counted if observed peak masses were within 20 ppm of expected ion masses. The “noise” ion corresponded to offset b+0.5, which was counted to show the level of noise in each type of MS2 spectra. (Right) Peptide break statistics for combinations of alternative fragmentation modes—peptide breaks were counted for all unique peptides identified by all three fragmentation modes. The six columns show the percentage of breaks detected by each fragmentation mode and combination of fragmentation modes per precursor charge state. In CID and HCD spectra, the presence of breaks was indicated by the presence of b or y ions. For ETD, c, z°, or z°+H ions indicated the presence of a break. Multiply charged ions (up to the spectrum's precursor charge) were also considered in each spectrum. Prior to this analysis, peak filtering was applied all CID, HCD, and ETD spectra such that each peak was retained only if its intensity was ranked fifth or higher over all neighboring peaks in a ±56 Da radius. If a peptide was identified by more than one CID, HCD, or ETD spectrum, a single representative spectrum was randomly chosen for each fragmentation mode.
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