Identification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to μ LC-ESI tandem mass spectrometry

Hanjo Lim, Jimmy Eng, John R. Yates, Sandra L. Tollaksen, Carol S. Giometti, James F. Holden, Michael W.W. Adams, Claudia I. Reich, Gary J. Olsen, Lara G. Hays

Research output: Contribution to journalArticlepeer-review


A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and μLC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by μLC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTipC18 and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using μLC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. μLC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by μLC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for ∼70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

Original languageEnglish (US)
Pages (from-to)957-970
Number of pages14
JournalJournal of the American Society for Mass Spectrometry
Issue number9
StatePublished - Sep 2003

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

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