A peptide centric (bottom up) approach using nanoLC-MS/MS and multiple modes of fragmentation (CID, HCD, ECD) to identify the amino acid sequences of peptides.
Samples from solution or polyacrylamide gels are digested using trypsin (alternative enzymes available); peptides are purified, concentrated, and loaded onto a nanoLC C18 analytical column. Multiply charged peptides are selected by abundance for sequencing by the mass spectrometer. Multiple fragmentation modes can be used -- for more detailed information please discuss advantages with Ryan Leib. For solution samples, in NO instances can we work with samples containing NP40, TritonX, Tween, PEG and many other detergents/surfactants. Samples containing these detergents/surfactants must be run on a 1D PAGE gel -- for further discussion contact the lab. In some special circumstances we can work with SDS containing samples -- for more detailed information on this workflow contact Ryan Leib or Kratika Singhal. The amount of time the mass spectrometer spends interrogating the sample can be adjusted to sample complexity; in many instances we can identify >500 proteins in 1hr acquisition time. The MS data is converted to the necessary format and database searched (Sequest and/or Byonic) against the appropriate database specified by the user, uploaded into Scaffold, and electronically sent to the user. The most recent Scaffold viewer can be downloaded, free of charge, from the Proteome Software website at: http://www.proteomesoftware.com/products/free-viewer/