De Novo Peptide Sequencing by Nanoelectrospray Tandem Mass Spectrometry Using Triple Quadrupole and Quadrupole/Time-of-Flight Instruments

Recent developments in technology and instrumentation have made mass spectrometry the method of choice for the identification of gel-separated proteins using rapidly growing sequence databases (1). Proteins with a full-length sequence present in a database can be identified with high certainty and high throughput using the accurate masses obtained by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry peptide mapping (2). Simple protein mixtures can also be deciphered by MALDI peptide mapping (3) and the entire identification process, starting from in-gel digestion (4) and finishing with acquisition of mass spectra and database search, can be automated (5). Only 1–3% of a total digest are consumed for MALDI analysis even if the protein of interest is present on a gel in a subpicomole amount. If no conclusive identification is achieved by MALDI peptide mapping, the remaining protein digest can be analyzed by nanoelectrospray tandem mass spectrometry (Nano ESI-MS/MS) (6). Nano ESI-MS/MS produces data that allow highly specific database searches so that proteins that are only partially present in a database, or relevant clones in an EST database, can be identified (7). It is important to point out that there is no need to determine the complete sequence of peptides in order to search a database-a short sequence stretch consisting of three to four amino acid residues provides enough search specificity when combined with the mass of the intact peptide and the masses of corresponding fragment ions in a peptide sequence tag (8) (see Subheading 3.4.).