A hierarchical MS2/MS3 database search algorithm for automated analysis of phosphopeptide tandem mass spectra

Proteomics. 2009 Apr;9(7):1763-70. doi: 10.1002/pmic.200800282.


A novel hierarchical MS(2)/MS(3) database search algorithm has been developed to analyze MS(2)/MS(3) phosphopeptides proteomic data. The algorithm is incorporated in an automated database search program, MassMatrix. The algorithm matches experimental MS(2) spectra against a supplied protein database to determine candidate peptide matches. It then matches the corresponding experimental MS(3) spectra against those candidate peptide matches. The MS(2) and MS(3) spectra are used in concert to arrive at peptide matches with overall higher confidence rather than combining MS(2) and MS(3) data searched separately. Receiver operating characteristic analysis showed that hierarchical MS(2)/MS(3) database searches with MassMatrix had better sensitivity and specificity than the two-stage MS(2)/MS(3) database searches obtained with MassMatrix, MASCOT, and X!Tandem. A greater number of true peptide matches at a given false rate were identified by use of this new algorithm for data collected on both LCQ and LTQ-FTICR mass spectrometers. The additional MS(3) spectral data also improved the overall reliability and the number of true positives (TPs) due to the fact that the TPs of the MS(2)/MS(3) search results had higher scores than those of the MS(2).

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Algorithms*
  • Animals
  • Caseins / chemistry
  • Cattle
  • Data Interpretation, Statistical
  • Phosphopeptides / chemistry*
  • Proteomics / methods*
  • ROC Curve
  • Sensitivity and Specificity
  • Software*
  • Tandem Mass Spectrometry*


  • Caseins
  • Phosphopeptides