Fig. A.16 Characterization of multiprotein complexes by mass on differences in their mass to charge (m/z) ratio by subjecting spectrometry. Following immunoprecipitation of a multiprotein them under high vacuum to a magnetic field. A detector collects complex using antibodies specific for one component of the information on the signal intensity for each peptide ion and displays complex, the individual proteins are separated by gel electrophoresis. the information as a histogram. This histogram, usually referred to An individual band representing one protein is isolated and digested as a spectrum, is compared with a database containing potential with a protease such as trypsin. The digested protein sample is cleavage sites for the proteolytic enzyme used in all known protein injected into the mass spectrometer, which ionizes the peptides, sequences, allowing for identification (ID) of the protein in the sample. transfers them to the gas phase, and then separates them based ratio of each ion, and a detector collects information on the signal intensity for each ion and displays the data as a histogram (Fig. A.16). This histogram, usually referred to as a spectrum, can be compared with a database containing potential cleavage sites (for the proteolytic enzyme used) in all known protein sequences. Due to the precision of these measurements, and the information derived from multiple peptides comprising the initial protein, the spectrum can often be unambiguously assigned to a unique protein in the database.
Modern multidimensional mass spectrometers (MS/MS) allow peptide ions to be sequenced as well as analyzed by their mass. In these instruments, peptide ions separated in one sector are fragmented in a second sector by collision with other molecules (often an inert gas such as N2), with the resultant fragments separated in a third section (Fig. A.17). Fragmentation occurs primarily in the
Fig. A.17 Determining the amino acid sequence of a peptide by multidimensional mass spectrometry (MS/MS). Multidimensional mass spectrometers (MS/MS) consist of two mass spectrometers linked in tandem but with an interceding middle sector that fragments ions. In the first sector, the first mass spectrometer separates peptide ions, as shown in Fig. A.16. Each peptide ion from this first separation is then fragmented in the middle sector of the apparatus by collision with other molecules (often an inert gas such as N2). Since fragmentation occurs primarily in the peptide backbone, a mixture of fragments is generated in which the fragments each differ by one amino acid residue. The resultant fragments are then separated in the second mass spectrometer, the final sector. The sequence of the peptide can be read directly from the second mass spectrum. The order of amino acid residues in the peptide can be deduced because of the exquisite precision of the measurements of each ion together with knowledge of the exact mass of each possible amino acid residue.
peptide backbone, allowing the sequence of the peptide to be read directly from the mass spectrum of the mixture of fragments. In place of gel electrophoresis, a liquid chromatograph can be employed upstream of the mass analyzers (LC-MS/MS), to provide additional separation of peptides prior to mass separation and allow a very complex mixture of thousands of peptides to be sequenced in a single run. It was this latter technique that played an essential role in the early studies that identified the repertoire of peptides bound to MHC molecules on the surface of antigen-presenting cells (see Chapter 6).
A-16 Isolation of peripheral blood lymphocytes by density-gradient fractionation.