Effectiveness of CID HCD and ETD with FT MS/MS for degradomic-peptidomic analysis: comparison of peptide identification methods

摘要

We report on the effectiveness of CID, HCD, and ETD for LC-FT MS/MS analysis of peptides using a tandem linear ion trap-Orbitrap mass spectrometer. A range of software tools and analysis parameters were employed to explore the use of CID, HCD, and ETD to identify peptides isolated from human blood plasma without the use of specific “enzyme rules”. In the evaluation of an FDR-controlled SEQUEST scoring method, the use of accurate masses for fragments increased the numbers of identified peptides (by ~50%) compared to the use of conventional low accuracy fragment mass information, and CID provided the largest contribution to the identified peptide datasets compared to HCD and ETD. The FDR-controlled Mascot scoring method provided significantly fewer peptide identifications than with SEQUEST (by 1.3–2.3 fold) at the same confidence levels, and CID, HCD, and ETD provided similar contributions to identified peptides. Evaluation of de novo sequencing and the UStags method for more intense fragment ions revealed that HCD afforded more sequence consecutive residues (e.g., ≥7 amino acids) than either CID or ETD. Both the FDR-controlled SEQUEST and Mascot scoring methods provided peptide datasets that were affected by the decoy database and mass tolerances applied (e.g., the identical peptides between the datasets could be limited to ~70%), while the UStags method provided the most consistent peptide datasets (>90% overlap) with extremely low (near zero) numbers of false positive identifications. The m/z ranges in which CID, HCD, and ETD contributed the largest number of peptide identifications were substantially overlapping. This work suggests that the three peptide ion fragmentation methods are complementary, and that maximizing the number of peptide identifications benefits significantly from a careful match with the informatics tools and methods applied. These results also suggest that the decoy strategy may inaccurately estimate identification FDRs.