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What Method to use for Protein Identification Between LC-MS/MS, MALDI-TOF and Edman Sequencing?

LC-MS/MS: Nano HPLC coupled to an ion-trap or a QqTOF mass spectrometer. Analysis of proteolytic fragments.

  • High sensitivity (10 fmols)
  • Tandem mass spectrometry yields sequence dependent data
  • Several proteins can often be identified from a single sample
  • Possible to get very high confidence identifications due to sequence dependence of data
  • Exact, or high homology protein sequence must be present in database

MALDI-TOF Based Peptide Mass Mapping: Mass determination of proteolytic fragments.

  • High sensitivity
  • Relatively quick analysis
  • Relatively inexpensive
  • Requires relatively pure samples
  • More ambiguity in identifications due to lack of true sequence dependence of data
  • Exact, or high homology protein sequence must be present in database

Edman Seqeuncing: Chemical sequence determination of intact, unblocked proteins or proteolytic fragments

  • Moderate sensitivity
  • Gives true de novo sequence data
  • Generally not affected by PTMs
  • Works with intact proteins, provided N-terminus is not blocked
  • Slowest of the three methods
  • Relatively expensive
  • Requires relatively pure samples (no more than one or two minor contaminants)
  • Analysis of proteolytic fragments requires chromatographic pre-fractionation

In general, the LC-MS/MS method will yield the highest confidence identifications and is the method of choice, provided the expected protein sequence or a highly homologous sequence is in the searched database. If you are dealing with an organism with a relatively small genome, MALDI-TOF peptide mass mapping analysis may be sufficient for your needs. If you are dealing with an organism whose genome is not known or you wish to obtain N-terminal sequence information on an intact protein, Edman sequencing analysis is probably your best choice since it yields sequence information.

Both Edman sequencing and LC-MS/MS are useful for looking at protein modifications such as phosphorylations, acetylations, methylations, etc. However, due to the relatively low resolution characteristics of our ion-trap instrument, unambiguous assignments of modification sites can be difficult with this instrument. Edman sequencing can offer more unambiguous identification of modification sites, provided that suitable standards for the modified amino acids are available. Edman sequencing also suffers from the limitations listed above.