Two different Methods in Protein Identification by Mass Spectrometry

There are tow major methods that are widely used for protein identification by mass spectrometry: MALDI-TOF based protein fingerprinting and LC-MS/MS based peptide sequencing. In the MALDI-TOF based protein fingerprinting method, a sample is digested with certain proteolytic enzyme (usually trypsin) and one MS spectrum is acquired which generates the massed of all peptides (or MH+), and these masses are used as a fingerprint to search proteins in a database. The hits are ranked according certain scoring method. A candidate protein that contains more proteolytic peptides, which can match measured masses, has a higher score. A Protein identified often is the protein of highest score. In contrast, protein identification by LC-MS/MS peptide sequencing is following different principle: typically, the peptide mixture from a proteolytic digestion (usually trypsin digestion) is separated a HPLC (often with 75 uM ID column with column flow rate less than 1ul/min). A Tandem mass spectrometer is on-line coupled with HPLC so peptides elute from HPLC column is fragmented by a process called collision-induced dissociation (CID) and a MS/MS spectra is acquired for each fragmented peptide (there are often several thousands MS/MS spectra from each sample). Each MS/MS spectrum (corresponding a specific peptide sequence) is used to search protein database for matched peptides. A Protein hit is identified often by multiple independently sequenced peptides from the same protein. Protein identification based on MALDI-TOF MS fingerprinting has some significant advantages over Nano-LC-MS/MS based method. The first advantage is its speed: Acquiring a MALDI-TOF MS data often takes less than one minutes. Subsequent database search often takes less than three minutes, and analysis of search result is often rapid and straightforward. In contrast, Nano LC-MS/MS data acquisition typically takes 1-2 hours with several thousands MS/MS spectra generated, and subsequent database search may take several hours on a PC, and analyzing search result could take even longer time. The Second advantage is its easiness: operating a MALDI-TOF mass spectrometer is a much easier task than operating a Nano LC-MS/MS system.