Identification and characterization of DNA-binding proteins by mass spectrometry.
Mass spectrometry is the most sensitive and specific analytical technique available for protein identification and quantification. Over the past 10 years, by the use of mass spectrometric techniques hundreds of previously unknown proteins have been identified as DNA-binding proteins that are involved in the regulation of gene expression, replication, or DNA repair. Beyond this task, the applications of mass spectrometry cover all aspects from sequence and modification analysis to protein structure, dynamics, and interactions. In particular, two new, complementary ionization techniques have made this possible: matrix-assisted laser desorption/ionization and electrospray ionization. Their combination with different mass-over-charge analyzers and ion fragmentation techniques, as well as specific enzymatic or chemical reactions and other analytical techniques, has led to the development of a broad repertoire of mass spectrometric methods that are now available for the identification and detailed characterization of DNA-binding proteins. These techniques, how they work, what their requirements and limitations are, and selected examples that document their performance are described and discussed in this chapter.
Nordhoff
Eckhard
E
Department Lehrach, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany. nordhoff@molgen.mpg.de
Lehrach
Hans
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D041961:Deuterium Exchange Measurement / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D011485:Protein Binding; D020539:Sequence Analysis, Protein / Q000379:methods*
D004268:DNA-Binding Proteins; D004247:DNA