The Tools of Proteomics

Abstract

Publisher Summary The chapter focuses on the tools used in proteomics. Mass spectrometry (MS)-based methods have become the primary technology used to identify proteins. The sensitivity of MS instrumentation allows for the identification of proteins below the 1-pmol level and in many cases in the femtomole or even the attomole range. The mass-measurement accuracy afforded with current MS technology, routinely to less than 50 parts per million (ppm) and better, provides more confident protein identifications. Additional sequence information provided by tandem MS (MS/MS) approaches improves the level of confidence of the identification. The major technological advances associated with MS are described in this chapter along with the MS-based methods related to the analysis of proteins separated by one- and two-dimensional gel electrophoresis. Non-polyacrylamide gel electrophoresis (PAGE)-based analyses are also addressed; however, separation methods such as two-dimensional liquid chromatography have specific advantages over the PAGE approach. The developments can be divided into a few general categories, including ionization methods, analyzers, and sample processing. A vast majority of protein identifications made using MS data is accomplished through a comparison with known protein sequences found in a variety of different databases populated with either protein or DNA sequence information. The chapter focuses on the most commonly used software for identifying proteins both through peptide mapping and fragmentation. Peptide mapping is the simplest method for identifying a protein. The second common method to identify a protein through characterization at the peptide level is by tandem MS, which produces fragmentation spectra. In addition, individual laboratories have also designed tools to mine their own data in an attempt to maximize the amount of information retrievable from a proteomic analysis.