Direct Analysis of Proteins in Mixtures

Abstract

Tandem mass spectrometry is a powerful mixture analysis technique suitable for sequence analysis of peptides (1). A tandem mass spectrometer uses two stages of analysis to generate structurally informative fragmentation. The first stage involves separation of an ion from all the other ions that may be entering the mass spectrometer analyser. Ion isolation can be accomplished by separating an ion in time or in space; these processes are used by ion trap instruments or by triple quadmpole instruments, respectively (2–4). The isolated ion is then subjected to ion activation using energetic gas-phase collisions. In the second analysis stage, the mass-to-charge ratio (m/z) values of the fragmentation products are determined. This method is used for protein sequence analysis by first creating a collection of peptides using site-specific enzymatic or chemical proteolysis (1). The collection of peptides is introduced into the mass spectrometer through a separation technique (liquid chromatography [LC] or capillary electrophoresis [CE]) or by batch infusion and finally ionized using electrospray ionization (5–7). Computer control of the data acquisition process allows highly efficient acquisition of these tandem mass spectra as well as unassisted operation of the mass spectrometer (8,9). The resulting tandem mass spectra can reveal the amino acid sequence of peptides by interpretation, or, with the recent expansion of sequence databases, the tandem mass spectra can be used to search protein and nucleotide sequence databases directly to identify the amino acid sequence represented by the spectrum (l,8,10,11). Because tandem mass spectra can be acquired quickly and selectively on individual peptides present, the identities of proteins in a mixture can be determined (10,12,13).