Detection and selective dissociation of intact ribosomes in a mass spectrometer.
Intact Escherichia coli ribosomes have been projected into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques. Species with mass/charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the stability of intact ribosomes was investigated and found to increase as a result of cross-linking ribosomal proteins to the rRNA. By lowering the Mg(2+) concentration in solutions containing ribosomes the particles were found to dissociate into 30S and 50S subunits. The resolution of the charge states in the spectrum of the 30S subunit enabled its mass to be determined as 852,187 +/- 3,918 Da, a value within 0.6% of that calculated from the individual proteins and the 16S RNA. Further dissociation into smaller macromolecular complexes and then individual proteins could be induced by subjecting the particles to increasingly energetic gas phase collisions. The ease with which proteins dissociated from the intact species was found to be related to their known interactions in the ribosome particle. The results show that emerging mass spectrometric techniques can be used to characterize a fully functional biological assembly as well as its isolated components.
Rostom
A A
AA
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QT, United Kingdom.
Fucini
P
P
Benjamin
D R
DR
Juenemann
R
R
Nierhaus
K H
KH
Hartl
F U
FU
Dobson
C M
CM
Robinson
C V
CV
D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000648:ultrastructure*; D008274:Magnesium / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D012335:RNA, Ribosomal / Q000737:chemistry; D012269:Ribosomal Proteins / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure*
D003432:Cross-Linking Reagents; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D008274:Magnesium