Quantitative ESI-TOF analysis of macromolecular assembly kinetics.
The Escherichia coli small (30S) ribosomal subunit is a particularly well-characterized model system for studying in vitro self-assembly. A previously developed pulse-chase monitored by quantitative mass spectrometry (PC/QMS) approach to measuring kinetics of in vitro 30S assembly suffered from poor signal-to-noise and was unable to observe some ribosomal proteins. We have developed an improved LC-MS based method using quantitative ESI-TOF analysis of isotope-labeled tryptic peptides. Binding rates for 18 of the 20 ribosomal proteins are reported, and exchange of proteins S2 and S21 between bound and unbound states prevented measurement of their binding kinetics. Multiphasic kinetics of 3' domain proteins S7 and S9 are reported, which support an assembly mechanism that utilizes multiple parallel pathways. This quantitative ESI-TOF approach should be widely applicable to study the assembly of other macromolecular complexes and to quantitative proteomics experiments in general.
Bunner
Anne E
AE
Department of Molecular Biology, Scripps Research Institute, 10550 North Torrey Pines Rd, La Jolla, California 92037, USA.
Trauger
Sunia A
SA
Siuzdak
Gary
G
Williamson
James R
JR
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism*; D021122:Protein Subunits; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D046911:Macromolecular Substances; D021122:Protein Subunits; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins